RU2394569C2 - Application of phthalide derivatives - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: claimed invention relates to cemical-pharmaceutical industry and deals with phthalide derivatives, their application in production of sensibilisers or means of reversion of anti-tumour medications. Phthalide derivatives according to claimed invention can overcome or reduce polyrestance to medications, 5-30 times enhance growth inhibition caused by chemical therapeutic medications as well as considerably promote death of tumour cells caused by medications.

EFFECT: said phthalide derivatives considerably enhance sensitivity of tumour cells irrespective of anti-tumour medication type.

10 cl, 6 ex, 6 tbl , 4 dwg

Description

The invention relates to the use of phthalide derivatives (RA). Monomers and dimers of such derivatives can enhance the sensitivity of drug-resistant tumor cells to a chemotherapeutic drug.

State of the art

Polyresistance (MDR) means that tumor cells have cross-resistance to various anti-cancer drugs with different chemical structures, functions and mechanisms of action. A clinical study shows that some solid tumors, such as colon cancer, kidney cancer, hepatocarcinoma, non-small cell lung cancer, glioma, Kaposi’s sarcoma, and prostate cancer, are usually drug resistant, probably even when they are only diagnosed, and thus belong to tumors with internal MDR. In such tumor cells, as a rule, there are various resistance mechanisms that jointly provoke broad resistance to chemotherapeutic drugs. At the same time, some tumors, such as acute myeloid leukemia, acute lymphocytic leukemia, multiple myeloma, lymphoma, breast cancer, ovarian cancer and other tumors, are usually susceptible to chemotherapeutic agents in the initial stage of treatment, but gradually during treatment become drug resistant, especially during the treatment of relapse, when drug resistance increases significantly. This type of drug resistance is called acquired MDR. The therapeutic effect may decrease markedly after resistance to drugs has been established. MDR leads to poor therapeutic effect, poor prognosis, and easy relapse and metastasis of the tumor with conventional chemotherapy. Approximately 90% of deaths among cancer patients are associated with inherent or acquired drug resistance.

The mechanisms of the appearance of MDR have been extensively investigated. In general, it was found that MDR is regulated simultaneously by many factors and many mechanisms, including, mainly, the outflow of chemotherapeutic drugs due to ABC transport proteins, enhanced expression of antiapoptotic proteins, a change in the metabolic pathway and type of regulation, and an increase in the detoxification system.

Therefore, at the modern level of technology, a chemotherapeutic sensitizer with a tangible effect is urgently needed.

Disclosure of invention

The aim of the present invention are chemotherapeutic sensitizers with tangible effectiveness and their use in the preparation of sensitizers or reversing agents for anti-cancer chemotherapy. These sensitizers are phthalide or its derivatives of formula (I) (including monomers and dimers).

In a first aspect, the present invention relates to the use of a phthalide or a derivative of formula (I) thereof in the preparation of a sensitizer or anti-tumor drug reversing agent.

In the formula (I)

R1 is hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C1-C8 alkoxyl, C1-C4 carboxyl or halogen;

R2 is hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C1-C8 alkoxyl, C1-C4 carboxyl or halogen, or R2 is absent;

R3 and R4 are independently hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C1-C8 alkoxyl or halogen;

R5 and R8 are hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C1-C8 alkoxyl, C1-C4 carboxyl, phenyl, aryl, aralkyl, 5-6 membered heterocyclyl containing 1-2 a nitrogen atom, or halogen;

R6 and R7 are independently hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C4 carboxyl or halogen; or R6 and R7 combine together to form a 5-7 membered ring;

where alkyl, alkenyl, alkynyl, cycloalkyl, alkoxyl, phenyl, aryl, aralkyl and heterocyclyl are substituted with 0-3 substituents selected from the group consisting of C1-C3 alkyl, hydroxyl and halogen.

In another preferred embodiment, said derivative is a dihydro derivative or tetrahydro derivative of a phthalide of formula (I) or a dimer of a phthalide of formula (I), a dihydro derivative or tetrahydro derivative thereof.

Preferably, the structure of said phthalide or its derivative is selected from

where in the above formulas, R1-R8 have the meanings indicated above.

In another preferred embodiment, the structure of said phthalide or derivative thereof is selected from

Preferably, said antitumor drugs are selected from among adriamycin; vincristine; taxol, cisplatin; actinomycin; bleomycin; busulfan; capecitabine; carboplatin; carmustine; chlorambucil; cyclophosphamide; cytarabine; daunorubicin; epirubicin; etopla; Vepeside; etoposide; fludarabine; fluorouracil; gemcitabine; trastuzumab; hydroxycarbamide; idarubicin; ifosfamide; irinotecan; lomustine; melphalan; mercaptopurine; methotrexate; mitomycin; mitoxantrone; dihydroxyanthraquinonel; oxaliplatin; procabazine; methylhydrazine; mabters; a steroid; streptozocin; docetaxel; thioguanine; thiotepa; ice breaks; tespamine; raltitrexeda; topotecan; threosulfan; uracil; vinblastine; vinca alkaloid; vindesine; vinorelbine; gleevec; hydroxycamptothecin and their derivatives or mixtures.

In another preferred embodiment, said antitumor drugs are used to treat tumors selected from the group consisting of non-small cell lung cancer, prostate cancer, colon cancer, hepatocarcinoma, leukemia, myeloma, lymphoma, breast cancer, ovarian cancer, stomach cancer, esophageal cancer colon cancer or sarcoma.

In a second aspect, the present invention relates to a pharmaceutical composition that contains (a) an effective amount of a phthalide or a derivative of formula (I), (b) a safe and effective amount of an antitumor drug, and (c) a pharmaceutically acceptable carrier.

Preferably, said antitumor drug is selected from the group consisting of adriamycin; vincristine; taxol; cisplatin; actinomycin; bleomycin; busulfan; capecitabine; carboplatin; carmustine; chlorambucil; cyclophosphamide; cytarabine; daunorubicin; epirubicin; etopla; Vepeside; etoposide; fludarabine; fluorouracil; gemcitabine; trastuzumab; hydroxycarbamide; idarubicin; ifosfamide; irinotecan; lomustine; melphalan; mercaptopurine; methotrexate; mitomycin; mitoxantrone; dihydroxyanthraquinonel; oxaliplatin; procabazine; methylhydrazine; mabters; a steroid; streptozocin; docetaxel; thioguanine; thiotepa; ice breaks; tespamine; raltitrexeda; topotecan; threosulfan; uracil; vinblastine; vinca alkaloid; vindesine; vinorelbine; gleevec; hydroxycamptothecin and their derivatives or mixtures.

In a third aspect, the present invention relates to the use of a phthalide or its derivative of formula (I) in the preparation of a composition for over-expression of P-gp or Bcl-2 or overactivity of glyoxylase I.

In a fourth aspect, the present invention relates to a method for treating a tumor, comprising administering to a mammal in need of such treatment a safe and effective amount of phthalide or a derivative of formula (I) thereof.

Preferably, said method also includes administering at least one of the above antitumor drugs.

Description of drawings

Figure 1 shows a chromatogram of phthalide derivatives.

Figure 2 shows the results of Western blot analysis of Bcl-2 expression.

Figure 3 shows the enhancing effect of RA derivatives on Adr-induced cell death among MCF-7 / Adr cells.

Figure 4 shows the potentiating effect of RA derivatives in combination with Adr on Adr-induced apoptosis of MCF-7 / Adr cells, as measured by FACS.

The implementation of the invention

As a result of a wide and intensive study, the authors of the present invention found that the phthalide and its derivatives of formula (I) are effective sensitizers of antitumor chemotherapeutic agents. Based on this sensitizing effect, phthalide and its derivatives can effectively invert the multiresistance of tumors to antitumor drugs. On this basis, the present invention has been completed.

The term “alkyl”, as used herein, refers to a linear or branched saturated aliphatic hydrocarbon group containing 1-8 (preferably 1-6) carbon atoms. Said alkyl may be branched, and examples thereof are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, hexyl, etc. The term "alkenyl" refers to a linear or branched hydrocarbon group containing at least one C — C double bond and 2-8 (preferably 2-6) carbon atoms. The term "alkynyl" refers to a linear or branched hydrocarbon group containing at least one CC triple bond and 2-8 (preferably 2-6) carbon atoms.

The term “aryl” as used herein refers to an aromatic system, which may be a mononuclear cycle or a polynuclear aryl cycle, which is already fused or combined so that at least a portion of the fused or bound rings can form a conjugated aromatic system . The aryl group includes, but is not limited to, phenyl, naphthyl, and tetralyl.

The term “cycloalkyl”, as used herein, refers to a cycloalkyl group with 3-8 carbon atoms, such as cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl, etc.

The term “alkoxyl” refers to an alkoxyl with 1-8 carbon atoms, such as methoxyl, ethoxyl, propoxyl, butoxyl, pentyloxyl, hexoxyl, etc.

The term “heterocyclyl” refers to a stable 4-7-membered (preferably 5-6-membered) monocyclic or polycyclic heterocyclyl. The specified heterocyclyl may be saturated, partially unsaturated or unsaturated and consists of carbon atoms and 1-4 heteroatoms selected from among the atoms N, O and S. The atoms N and S can be oxidized. Heterocyclyl may also include any polycycle, and any of the above heterocyclyl may condense with the aryl ring.

"Substituted aryl" or "substituted heterocyclyl" means an aryl group or heterocyclyl which is substituted with a substituent selected from the group consisting of halogen, CN, OH, NO ₂ , amino, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxyl, aryloxy, substituted alkoxyl, alkylcarbonyl, alkylcarboxyl, alkylamino or arylsulfonyl. Preferably, the substituent is halogen, C1-C4 alkyl, alkyl or hydroxyl.

The term “halogen” as used herein refers to an element of the halogen family and denotes F, O, Br or I.

Active ingredients

The active ingredients of the present invention are phthalide derivatives of formula (I).

The term “phthalide derivative” as used herein refers to a phthalide of formula (I) when the phthalide derivative monomer may contain a hydrogen atom attached to phenyl. The term also includes dimers or polymers (preferably dimers) of a phthalide of formula (I) or their hydrogenated products.

The term “dihydro derivative” as used herein means that two hydrogen atoms are attached to adjacent carbon atoms corresponding to a double bond in the phenyl ring of the phthalide. The term “tetrahydro derivative” means that four hydrogen atoms are attached to adjacent carbon atoms corresponding to double bonds in the phenyl ring. For example, a compound of formula (k) 'is a dihydro derivative of a compound of formula (k); and a compound of formula (k) ”is a tetrahydro derivative of a compound of formula (k).

The phthalide derivatives of the present invention can be artificially synthesized or isolated from a natural source. For example, the phthalide monomer and / or phthalide dimer of formula (I) can be distinguished by extraction from angelica or cactus vaginalis (ligusticus wallichii franchet) from among umbrella plants.

Pharmaceutical composition

The present invention also relates to a pharmaceutical composition which comprises a phthalide derivative of the formula (I) (monomer or dimer) as an active ingredient and one or more pharmaceutically acceptable carriers or excipients (such as a solvent, diluent). A pharmaceutically acceptable carrier that can be used in the present invention includes conventional solid and liquid carriers as long as they satisfy the properties of the active ingredient and the desired specific route of administration. For example, solid carriers include starch, lactose, CaHPO ₄ , microcrystalline cellulose, etc., and liquid carriers include sterile water, polyethylene glycol, etc.

The pharmaceutical composition of the present invention can be obtained in various conventional forms, such as a tablet, capsule, dispersible powder, granule, suspension, syrup (containing, for example, about 10-50% sugar) and elixir (containing about 20-50% ethanol). The form of the pharmaceutical composition may also be a sterile solution or suspension for injection (approximately 0.05-5% of a suspending agent in an isotonic medium) for parenteral administration. For example, the pharmaceutical composition may contain 0.01-99.9 wt.%, Preferably 2.5-90 wt.%, More preferably 5-60 wt.%, Of the active ingredient mixed with a carrier.

In another preferred embodiment, the pharmaceutical composition also comprises an antitumor drug. For example, the pharmaceutical composition contains (a) 0.01-99.9 wt.% (Preferably 0.1-90 wt.%) Of a phthalide derivative monomer or dimer; (b) 0.01-99.9 wt.% (preferably 0.1-90 wt.%) of an antitumor drug; (c) a pharmaceutically acceptable carrier. Typically, the mass ratio of component (a) and component (b) is 1: 100-100: 1, preferably 10: 1-1: 10.

The pharmaceutical composition may also include other additives, such as a pigment, preservative and antioxidant, etc.

The effective dosage of the active ingredient may vary depending on the route of administration and the severity of the disease being treated. However, the effect will be satisfactory when a phthalide derivative monomer or dimer is administered at a dosage of about 0.05-500 mg / kg body weight (preferably 0.1-100 mg / kg body weight) per day. Preferably, 2-4 separate doses may be administered per day or administered in a gradual release form.

Method of treatment

The present invention also relates to a method of treatment comprising the step of administering a safe and effective amount of a phthalide derivative monomer or dimer to a mammal in need of such treatment. Preferably, said method also includes the step of co-administering other antitumor drugs (such as, for example, an antitumor drug as a substrate for P-gp) or other treatment methods (such as chemotherapy).

Various tumors can be treated by administering a phthalide derivative monomer or dimer alone or in combination with other agents. Typical examples include, but are not limited to, non-small cell lung cancer, prostate cancer, colon cancer, hepatocarcinoma, leukemia, myeloma, lymphoma, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, colon cancer, sarcoma, etc. .

The method of introducing a monomer or dimer of a phthalide derivative is not particularly limited. They can be administered orally, intravenously, intramuscularly, locally, intratumor or subcutaneous, etc. Preferably, they are administered orally, intravenously or intratumorally.

The main advantages of the present invention are as follows.

All monomers or dimers of the phthalide derivatives of the present invention can, over a wide range, increase the sensitivity of tumor cells to antitumor drugs and reverse drug resistance (MDR), thereby effectively increasing the ability of antitumor drugs to kill tumor cells. Experiments show that they have a better reversing effect than verapamil (VER, P-gp inhibitor) when combined with antitumor agents such as adriamycin (Adr) to kill drug-resistant tumor cells. When they are administered with vincristine (VCR), taxol or cisplantin (DDP), the reversal effect is 2-5 times higher than verapamil. Such derivatives can reduce the resistance of tumor cells to drugs by 5-30 times and significantly increase the apoptosis of tumor cells induced by many chemotherapeutic drugs.

The present invention will be further illustrated by the following examples. It should be borne in mind that these examples are intended only to illustrate the invention, and not to limit the scope of the invention. In the following examples, experiments, unless otherwise indicated, are carried out under generally accepted conditions or under conditions specified by the manufacturer.

Example 1. Extraction of derivatives of phthalide from gamovnik vaginalis and angelica

Dried and crushed 3 kg of medicinal raw materials (cinnamon vaginalis or angelica) and sieved through a 20 mesh sieve. The resulting powder was extracted 3 times with a double volume (v / w) of chloroform. Chloroform is removed from the extract and the remaining solution is condensed to an extractum. The extractum is then extracted with n-hexane and methanol. The methanol phase is re-passed through a G254 silica gel chromatography column, n-hexane and acetonitrile (in a volume ratio of 9: 1 to 5: 5) are used as eluent, and compounds S1-S5 corresponding to peaks 8, 12, 7, 14 are obtained , 3 in figure 1, respectively. The n-hexane phase was re-passed through a chromatography column with silica gel G254, with ligroin and ethyl acetate (in a ratio of 9: 1 to one ethyl acetate) as eluent, and compounds S6-S8 corresponding to peaks 16, 18, 17 in figure 1 were obtained, respectively. Each of these monomers is dissolved in a suitable polar liquid and recrystallized by supersaturation at room temperature 2-3 times. The resulting crystalline substances identify NMR.

Compounds S1-S5 are monomers of derivatives of PA, and compounds S6-S8 are dimers of derivatives of PA.

S1:

¹ H-NMR (CDCl ₃ ) δ 4.96 (1H, dd, J = 6.5, 4.0, H-3), 2.45 (4H, m, H-4, H-5) 5, 88 (1H, dt, J = 10.0, 3.5, H-6), 6.16 (1H, dt, J = 10.0, 1.0, H-7), 1.51 (1H, m, H-8a), 1.85 (1H, m, H-8b), 1.36 (4H, m, H-9, H-10), 0.87 (3H, t, J = 7.5 , H-11).

S2:

¹ H-NMR (CDCl ₃ ) δ 5.52 (1H, dd, J = 8.0, 3.2, H-3), 7.33 (1H, dd, J = 7.5, 1.4, H-5), 7.31 (1H, t, J = 7.5, H-6), 7.10 (1H, dd, J = 7.5, 1.4, H-7), 2, 31 1.73 (each 1Н, m, Н-8), 1.37 (4Н, m, Н-9, Н-10), 0.89 (3Н, t, J = 7, l, Н-11) 9.59 (1H, s, 4-OH).

¹³ C-NMR (CDCl ₃ ) δ 171.2 (s, C-1), 80.9 (d, C-3), 136.1 (s, C-3a), 152.4 (s, C- 4), 120.2 (d, C-5), 130.3 (d, C-6), 115.9 (d, C-7), 127.8 (s, C-7a), 32.3 (t, C-8), 26.9 (t, C-9), 22.4 (t, C-10), 13.9 (q, C-11).

S3:

¹ H-NMR δ 2.59 (2H, m, H-4), 2.06, 2.14 (each 1H, m, H-5), 4.33 (1 H, ddd, J = 5.5 , 3.5, 2.5, H-6), 4.61 (1H, brigth, J = 2.5, H-7), 5.36 (1H, t, J = 8.0, H-8 ), 2.38 (2H, m, J = 8.0, 7.5, H-9), 1.58 (2H, m, J = 7.5, 7.5, H-10), 0, 96 (3H, t, J = 7.5, H-11).

S4:

¹ H-NMR (CDCl ₃ ) δ 2.47 (2H, m, H-4), 2.57 (2H, t, J = 13.5, H-5), 5.96 (1H, dt, J = 9.5, 4.0, H-6), 6.24 (1H, dt, J = 9.5, 1.5, H-7), 5.19 (1H, t, J = 8.0 , H-8), 2.33 (2H, q, J = 7.5, H-9), 1.47 (2H, m, H-10), 0.92 (3H, t, J = 7, 5, H-11).

S5:

¹ H-NMR (CDCl ₃ ) δ 2.01, 1.91 (2H, m, H-4); 1.92, 1.67 (2H, m, H-5); 3.46 (1H, q, H-5); 3.77 (1H, d, H-7); 5.13 (1H, t, H-8); 1.96 (2H, m, H-9); 1.37 (2H, m, H-10); 0.96 (3H, t, H-11); 2.0 (1H, m, 6-OH, 7-OH).

S6:

¹ H-NMR (CDCl ₃ ) δ 2.02, 2.08 (each 1H, m, H-4), 1.54, 1.91 (each 1H, m, H-5), 2.55 (1H , t, J = 7.8, H-6), 3.25 (1H, d, J = 8.9, H-7), 5.7 (1H, t, J = 7.8, H-8 ), 2.29 (2H, q, J = 7.3, H-9), 1.46 (2H, m, H-10), 0.93 (3H, t, J = 7.3, H- 11), 1.4, 2.03 (1H, m, H-4 '), 1.30, 1.88 (1H, m, H-5'), 2.99 (2H, m, H-6 '), 7.36 (1H, d, J = 6.6, H-7'), 5.0 (1H, t, J = 7.6, H-8 '), 2.18 (2H, m , H-9 '), 1.45 (2H, m, H-9'), 0.92 (3H, t, J = 7.3).

¹³ C-NMR (CDCl ₃ ) δ 168.4 (C-1), 148.1 (C-3), 155.0 (C-3a), 19.8 (C-4), 29.0 (C -5), 38.4 (C-6), 41.6 (a, C-7), 126.6 (C-7a), 112.1 (C-8), 28.0 (C-9) , 22.3 (C-10), 13.9 (b, C-11), 164.9 (C-1 '), 150.5 (C-3'), 47.6 (C-3'a ), 31.1 (C-4 '), 25.8 (C-5'), 41.5 (a, C-6 '), 142.0 (C-7'), 134.3 (C- 7'a), 108.8 (C-8 '), 27.5 (C-9'), 22.3 (C-10 '), 13.8 (b, C-11').

S7:

¹ H-NMR (CDCl ₃ ) δ 2.02, 2.57 (each 1H, m, H-4), 2.02, 2.17 (each 1H, m, H-5), 2.55 (1H m, H-6), 3.47 (1H, d, J = 7.3, H-7), 5.21 (1H, t, J = 7.8, H-8), 2.33 ( 2H, m, H-9), 1.50 (2H, m, H-10), 0.95 (3H, t, J = 7.6, H-11), 2.58, 2.74 (each 1H, m, H-4 '), 2.47, 2.75 (each 1H, m, H-5'), 5.93 (1H, dt, J = 9.6, 4.1, H-6 '), 6.17 (1H, dt, J = 9.6, 1.8, H-7'), 2.94 (1H, q, J = 7.8, H-8 '), 1.45 (2H, m, H-9 '), 1.14 (2H, m, H-10'), 0.87 (3H, t, J = 7.6, H-11 ').

¹³ C-NMR (CDCl ³ ) δ 168.5 (C-1), 149.2 (C-3), 154.6 (C-3a), 19.6 (C-4), 26.2 (C -5), 35.0 (s, C-6), 44.0 (C-7), 122.3 (s, C-7a), 112.2 (C-8), 28.0 (C- 9), 22.4 (C-10), 13.9 (C-11), 170.3 (C-1 '), 92.0 (C-3'), 160.1 (C-3'a ), 21.0 (s, C-4 '), 20.7 (s, C-5'), 138.7 (C-6 '), 117.0 (C-7'), 122.5 ( d, C-7'a), 32.3 (s, C-8 '), 20.0 (s, C-9'), 22.6 (C-10 '), 14.1 (C-11 ').

S8:

¹ H-NMR (CDCl ₃ ) δ 4.56 (1H, m, H-3), 1.98, 2.08 (each 1H, m, H-4), 1.53, 1.90 (each 1H , m, H-5), 2.54 (1H, m, H-6), 3.18 (1H, d, J = 8.9, H-7), 1.38, 1.70 (each 1H m, Н-8), 1.26 (2Н, m, Н-9), 1.45 (2Н, m, Н-10), 0.93 (3Н, t, J = 7.34, Н- 11), 1.40, 2.03 (each 1H, m, H-4 '), 2.30, 1.87 (each 1H, m, H-5'), 2.97 (1H, m, H -6 '), 7.33 (1H, d, J = 6.6, H-7'), 4.98 (1H, t, J = 7.3), 2.18 (2H, q, J = 7.8, H-9 '), 1.44 (2H, m, H-10'), 0.93 (3H, t, J = 7.3, H-11 ').

¹³ C-NMR (CDCl ₃ ) δ 165.0 (C-1), 82.4 (C-3), 47.3 (C-3a), 30.9 (C-4), 25.7 (C -5), 41.6 (C-6), 141.9 (C-7), 134.5 (C-7a), 32.2 (C-8), 26.5 (C-9), 22 3 (C-10), 13.7 (C-11), 171.9 (C-1 '), 150.5 (C-2'), 168.1 (C-3a), 22.4 ( C-4 '), 28.8 (C-5'), 38.3 (C-6 '), 41.7 (C-7'), 127.1 (C-7a '), 108.6 ( C-8 '), 27.4 (C-9'), 22.2 (C-10 '), 13.9 (C-11').

Monomers or dimers of derivatives of PA S1-S8, obtained as described above, are used in subsequent experiments. Compounds S1-S5 are monomers of derivatives of PA, and compounds S6-S8 are dimers of derivatives of PA.

Example 2. Determination of resistance of cell lines to drugs

The following cell lines are used: K562 (human chronic myeloid leukemia cell line) and K562 / Adr (cell line with resistance to chemotherapeutic agents such as adriamycin caused by prolonged exposure to a low dosage of adriamycin); KB (cell line of the epithelium of the human oral cavity) and KBv200 (cell line with resistance to chemotherapeutics such as vincristine, caused by prolonged exposure to a low dosage of vincristine); MCF-7 (human breast cancer cell line) and MCF-7 / Adr (cell line with resistance to chemotherapeutic agents such as adriamycin caused by prolonged exposure to a low dosage of adriamycin). All of the above cell lines are purchased at the Hematology Institute of the Chinese Medical Academy.

The drug-resistant K562 / Adr cell line is characterized mainly by a combination of several resistant mechanisms, including increased expression of proteins of the P-gp and Bcl-2 families and enhanced glyoxylase I activity, etc. K562 / Adr is 20 times more resistant to adriamycin than K562, and cross-resistant to vincristine, daunomycin, mitoxantrone, taxol, etc.

The drug-resistant KBv200 cell line is also characterized mainly by a combination of several resistant mechanisms, including increased expression of proteins of the P-gp and Bcl-2 families and increased glyoxylase I activity, etc. KBv200 is more than 100 times resistant to vincristine compared to KB and cross-resistant to vincristine, daunomycin, taxol, etc.

Similarly, the drug-resistant cell line MCF-7 / Adr is also characterized mainly by a combination of several resistance mechanisms, including increased expression of proteins of the P-gp and Bcl-2 families and enhanced glyoxylase I activity, etc.

The half-inhibitory concentration (IC ₅₀ ) was calculated using GraphPad Prism software (San Diego, CA). Drug response is analyzed using a Sigmoldal nonlinear regression model. The following reagents and devices are used.

Monomer and dimer RA solution: the compound is dissolved in DMSO to obtain a working solution of 5 mg / ml, and then RPMI 1640 medium is used to obtain a working solution. Adriamycin (Adr), Vincristine (VIN), Taxol, Cisplatin (DDP), RPMI 1640, MTT, cow fetal serum, culture plate, carbon dioxide medical incubator, fermentometer, high performance liquid phase chromatograph (HPLC), fluorescence activated cell sorter (FACS), P-gp fluorescence antibody UIU2 kit (purchased from Immunotech A Coulter Company, )

1. P-gp expression as determined by FACS

Cells in the logarithmic growth phase are harvested and washed twice with PBS. The expression of P-gp is determined by FACS according to the instructions for the set of UIU2 antibodies to P-gp fluorescence. 10,000 cells per sample are counted.

results

K562: a positive degree of P-gp expression is 0.22%; K562 / Adr: positive P-gp expression is 83.6%.

KB: positive expression of P-gp is 0.37%; KBv200: positive P-gp expression is 76.3%.

MCF-7: positive P-gp expression is 0.36%; MCF-7 / Adr: positive P-gp expression is 83.4%.

The results show that the main mechanism of drug resistance of all tested cell lines can be associated with high expression of P-gp.

2. Measurement of glyoxylase I activity (GLO1)

Cells were frozen-thawed in PBS containing 1 mM PMSF, sonicated, centrifuged at 12,000 for 20 minutes, and then the supernatant was collected. To determine GLO1, a mixture of 7.9 mM MG, 1 mM glutathione, 14.6 mM MgSO ₄ and 182 mM imidazole-HCl (pH 7.0) was used. The OD value is determined at 240 nm. The results are shown in table 1.

Table 1 Cell line K562 K562 / Adr KB KBv200 Mcf-7 MCF-7 / Adr OD value 0.22 1.21 0.31 1.47 0.16 1.46

The results show that one of the mechanisms of drug resistance of the tested cell lines is associated with increased GLO1 activity.

3. Determination of expression of Bcl-2

Cells are lysed in an NP-40 cell lysis solution and the ICA is quantified. Proteins are separated in a 1.2% SDS-PAGE gel and transferred to cellulose nitrate membranes when the gel is semi-dry. Primary antibodies (Sigma) against Bcl-2 are added to the membranes and imaging is performed.

The results are shown in Figure 2. The results show that one of the drug resistance mechanisms of the tested cell lines is associated with high expression of Bcl-2.

2. Determination of the resistance of drug-resistant cell lines to traditional chemotherapeutic agents

Cells are harvested in a logarithmic growth phase and added to RPMI-1640 medium (containing 10% of fetal bovine serum) and a cell suspension is obtained. Then the cell suspension is seeded in a 96-well plate in an amount of 100 μl per well (1 × 10 ⁴ cells / well). Adriamycin, vincristine, taxol, cisplatin in different concentrations are added directly to the semi-suspension cells (K562 and K562 / Adr). Adhesive cells are cultured for 24 hours after inoculation, so that the cells can completely adhere, and then add the above funds in various concentrations. For each concentration gradient, there are six parallel repeat wells. The medium is added to each well to a final volume of 200 μl per well. Cells were cultured at 37 ° C, saturation humidity and 5% CO ₂ for 68 hours. Add 50 μl MTT (2 mg / ml) per well. Centrifuge the 96-well plate and remove the supernatant. Then add 120 μl of DMSO per well and process it with vibration on a vibration device. The OD value is measured at a wavelength of 590 nm using a fermentometer after the crystalline substance has completely dissolved.

Calculation Method:

IC ₅₀ = drug concentration when the degree of inhibition is 50%.

Drug Resistance Factor (RF) = IC ₅₀ drug resistant cells / IC ₅₀ sensitive cells.

The results of the determination of drug resistance of drug resistant cell lines in relation to traditional chemotherapeutic agents are shown in table 2 (IC ₅₀ : μg / ml).

The results show that all tested drug resistant cell lines have cross-resistance to traditional chemotherapeutic agents, i.e. polyresistance.

Example 3. Evaluation of the cytological effect of derivatives of RA - the ability to inhibit the growth of cell lines resistant to drugs

Methods Inoculate 1 × 10 ³ cells in a 96-well plate. RA is added directly to K562 / Adr, and to KBv200 and MCF-7 / Adr after adherence. The concentration of drugs is 0, 0.1, 0.5, 1.5, 10, 20, 40, 80, 160 μg / ml. MTT was determined according to the method in Example 2 and an IC50 was calculated. The experiment is repeated three times.

The results of determining the inhibitory effect of RA on the growth of drug resistant cells are shown in Table 3 (IC ₅₀ (μg / ml)).

The results show that derivatives of RA at a concentration of about 10 μg / ml do not have a noticeable cytotoxic effect.

Example 4. Evaluation of the cytological effect of derivatives of RA - sensitizing effect on traditional chemotherapeutic drugs

Cells are harvested in a logarithmic growth phase and added to RPMI 1640 medium (containing 10% fetal bovine serum) to obtain a cell suspension. The cell suspension is added to a 96-well plate at 100 μl per well, and the number of cells per well is 2 × 10 ³ (suspension cells) or 1 × 10 ³ (adhesive cells). After inoculation, suspension cells are adapted for half an hour on the plate before drug addition, and adhesive cells are cultured to adhere. Antitumor agents are added to the control groups of cells (Ctrl), but without RA. The concentration of the drug of each RA group is 10 μg / ml. The concentration of chemotherapeutic agents increases from 0.001 μg / ml to 20 μg / ml. For each concentration gradient, there are six parallel repeat wells. RPMI 1640 is added to a final volume of 200 μl per well. Cells were cultured at 37 ° C, saturation humidity and 5% CO ₂ for 68 hours. MTT was determined and IC ₅₀ was calculated as in Example 4.

Resistance Factor Formula:

Resistance Factor (RF) = IC ₅₀ without sensitizer / IC ₅₀ with sensitizer

The results of determining the sensitizing effect of RA (10 μg / ml) on K562 / Adr cells are shown in table 4.

The results of determining the sensitizing effect of RA (10 μg / ml) on KBv200 cells are shown in table 5.

The results of determining the sensitizing effect of RA (10 μg / ml) on MCF-7 / Adr cells are shown in table 6.

Example 5. Evaluation of the cytological effect of derivatives of RA - sensitizing effect in the case of cell death caused by chemotherapeutic drugs

Cell inoculation and drug induction are carried out as in Example 2. After forty-eight hours, 5 μl of trypan blue (4 mg / ml) is added to each well. After five minutes, the cells are checked under a microscope. Five hundred cells per well are counted. Blue stained cells are dead cells. The cell survival rate is calculated.

Cell survival rate (%) = [1-dead cells (blue) / 500] · 100

The sensitizing effect of the derivatives of RA in the case of cell death caused by adriamycin is shown in Table 3. The concentration of each compound S1-S8 is 10 μg / ml. The concentration of Adr is 2.5 μg / ml. S1 + -S8 + represents the combined use of S (10 μg / ml) and Adr (2.5 μg / ml). The results show that derivatives of RA can significantly enhance cell death caused by Adr.

Example 6. Determination of FACS sensitizing effect of derivatives of RA on apoptosis caused by chemotherapeutic drugs

MCF-7 / Adr cells are inoculated onto a 6-well plate (1 × 10 ⁶ cells / well). Cells were cultured for 24 hours, and then RA and chemotherapeutic drugs were added as described above. Cells are cultured for another 72 hours. Cells are hydrolyzed with 0.25% trisine and harvested. The cell pellet is fixed with pre-chilled 70% ethanol and stored at 4 ° C. Samples were washed with PBS 3 times, hydrolyzed with RNase (1 g / ml) for 15 minutes, stained with propidium iodide (PI, 50 mg / l) for 30 minutes, then measured using a fluorescence activated cell sorter and the percentage of dead cells was calculated.

The results are shown in figure 4. The concentration of Adr is 2.5 μg / ml. The concentration of RA is 10 μg / ml. S1 + -S8 + represents the combined use of S (10 μg / ml) and Adr (2.5 μg / ml). The results show that derivatives of RA can significantly sensitize cell death caused by Adr.

All documents cited herein are incorporated by reference. In addition, it should be borne in mind that in the above instructions, specialists in this field can make various changes or modifications according to the invention, but all such equivalent forms are included in the scope of the invention defined by the claims appended to the application.

Claims (10)

1. The use of phthalide or its derivative of formula (I) upon receipt of a sensitizer or means of reversing antitumor drugs

,
where R1 is hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C1-C8 alkoxyl, C1-C4 carboxyl or halogen;
R2 is hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C1-C8 alkoxyl, C1-C4 carboxyl or halogen, or R2 is absent;
R3 and R4 are independently hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C1-C8 alkoxyl or halogen;
R5 and R8 are hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C1-C8 alkoxyl, C1-C4 carboxyl, phenyl, aryl, aralkyl, 5-6 membered heterocyclyl containing 1-2 nitrogen atoms, or halogen;
R6 and R7 are independently hydrogen, hydroxyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C4 carboxyl or halogen; or R6 and R7 combine together to form a 5-7 membered ring;
where alkyl, alenyl, alkynyl, cycloalkyl, alkoxyl, phenyl, aryl, aralkyl and heterocyclyl are substituted with 0-3 substituents selected from the group consisting of C1-C3 alkyl, hydroxyl and halogen. 2. The use according to claim 1, wherein said derivative is a dihydro derivative or a tetrahydro derivative of the formula (I) or a phthalide dimer of the formula (I) or its dihydro derivative or a tetrahydro derivative. 3. The use according to claim 1, where the structure of the specified phthalide or its derivative is selected from among

where R1-R8 have the meanings indicated in claim 1. 4. The use according to claim 1, where the structure of the specified phthalide or its derivative is selected from among

. 5. The use according to claim 1, where these anticancer drugs are selected from among adriamycin; vincristine; taxol; cisplatin; actinomycin; bleomycin; busulfan; capecitabine; carboplatin; carmustine; chlorambucil; cyclophosphamide; cytarabine; daunorubicin; epirubicin; etopla; Vepeside; etoposide; fludarabine; fluorouracil; gemcitabine; trastuzumab; hydroxycarbamide; idarubicin; ifosfamide; irinotecan; lomustine; melphalan; mercaptopurine; methotrexate; mitomycin; mitoxantrone; dihydroxyanthraquinonel; oxaliplatin; procabazine; methylhydrazine; mabters; a steroid; streptozocin; docetaxel; thioguanine; thiotepa; ice breaks; tespamine; raltitrexeda; topotecan; threosulfan; uracil; vinblastine; vinca alkaloid; vindesine; vinorelbine; gleevec; hydroxycamptothecin and their derivatives or mixtures. 6. The use according to claim 1, where these anticancer drugs are used to treat tumors selected from the group consisting of non-small cell lung cancer, prostate cancer, colon cancer, hepatocarcinoma, leukemia, myeloma, lymphoma, breast cancer, ovarian cancer, stomach cancer, cancer of the esophagus, cancer of the colon or sarcoma. 7. A pharmaceutical composition comprising (a) an effective amount of a phthalide or a derivative of formula (I), (b) a safe and effective amount of an antitumor drug, and (c) a pharmaceutically acceptable carrier;
where the specified antitumor drug is selected from among adriamycin; vincristine; taxol; cisplatin; actinomycin; bleomycin; busulfan; capecitabine; carboplatin; carmustine; chlorambucil; cyclophosphamide; cytarabine; daunorubicin; epirubicin; etopla; Vepeside; etoposide; fludarabine; fluorouracil; gemcitabine; trastuzumab; hydroxycarbamide; idarubicin; ifosfamide; irinotecan; lomustine; melphalan; mercaptopurine; methotrexate; mitomycin; mitoxantrone; dihydroxyanthraquinonel; oxaliplatin; procabazine; methylhydrazine; mabters; a steroid; streptozocin; docetaxel; thioguanine; thiotepa; ice breaks; tespamine; raltitrexeda; topotecan; threosulfan; uracil; vinblastine; alkaloid vinca; vindesine; vinorelbine; gleevec; hydroxycamptothecin and their derivatives or mixtures. 8. The use of a phthalide derivative of formula (I) in the preparation of a composition that attenuates the overexpression of P-gp or Bc1-2 or the overactivity of glyoxylase I. 9. A method for treating tumors, comprising administering to a mammal in need of such treatment a safe and effective amount of phthalide or a derivative of formula (I) thereof. 10. The method according to claim 9, also comprising administering at least one antitumor drug selected from among adriamycin; vincristine; taxol; cisplatin; actinomycin; bleomycin ;: busulfan; capecitabine; carboplatin; carmustine; chlorambucil; cyclophosphamide; cytarabine; daunorubicin; epirubicin; etopla; Vepeside; etoposide; fludarabine; fluorouracil; gemcitabine; trastuzumab; hydroxycarbamide; idarubicin; ifosfamide; irinotecan; lomustine; melphalan; mercaptopurine; methotrexate; mitomycin; mitoxantrone; dihydroxyanthraquinonel; oxaliplatin; procabazine; methylhydrazine; mabters; a steroid; streptozocin; docetaxel; thioguanine; thiotepa; ice breaks; tespamine; raltitrexeda; topotecan; threosulfan; uracil; vinblastine; vinca alkaloid; vindesine; vinorelbine; gleevec; hydroxycamptothecin and their derivatives or mixtures.

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