WO2004039797A1

WO2004039797A1 - A special kind of indole compounds, their preparation, and their use in treatment and prevention of those disease such as cancer

Info

Publication number: WO2004039797A1
Application number: PCT/CN2003/000901
Authority: WO
Inventors: Jingcai Cheng; Longgui Wang
Original assignee: Jingcai Cheng; Longgui Wang
Priority date: 2002-10-29
Filing date: 2003-10-27
Publication date: 2004-05-13
Also published as: CN1199946C; AU2003280544A1; CN1424312A

Abstract

The present invention provides a series of bisindole derivatives, which may be used as CDKs (Cyclin-Dependent Kinases) inhibitors, their pharmaceutical acceptable salts, their production as well as their uses. These bisindole derivatives are capable of inhibiting the activity of Cyclin-Dependent Kinases specially, blocking the cell’s proliferation, evocating cell’s apoptosis, thus effectively inhibiting the growth of various of tumor cells, including those of carcinoma of colon, prostate, mammary and neuroblastoma. The present invention also provides the pharmaceutical composition of these bisindole derivatives.

Description

Specific indole compound and preparation method

And its application in the treatment and prevention of diseases such as cancer

The present invention belongs to the field of medicinal chemistry, and relates to a class of indole heterocyclic compounds JN-2518 series, its preparation method and medical use. This series of compounds can specifically inhibit cell cycle kinase activity and can be used to treat and prevent various diseases such as malignant tumors in humans and animals. Background technique

Due to advances in epidemiology and further improvements in early diagnosis and treatment methods, the treatment and prevention of cancer has made remarkable achievements in the past ten years. According to statistics from the American Cancer Society, since 1999, the incidence of tumors in the United States has shown a slight decline. Nevertheless, in the United States, tumors are still one of the major diseases that threaten people's lives. In China, the incidence of malignant tumors continues to rise due to environmental pollution and people's bad living habits. Cancer is more of a threat to people. Despite the continuous maturity of surgery and radiation therapy and the emergence of new treatment methods such as gene therapy, no method has yet replaced chemotherapy. On the other hand, due to the existing anticancer drugs, although they have a certain effect, they are mostly cytotoxic drugs with severe toxic and side effects. Therefore, the discovery of new high-efficiency and low-toxicity anti-cancer drugs is not only challenging, but also an urgent task that cannot be avoided.

Tumor cells proliferate indefinitely because they lose the regulatory function of cell cycle operation. A typical cell cycle can be divided into Gl, S, G2, and M phases (see Figure 1). The G1 phase, also known as the interstitial phase (GAP) or pre-synthesis of DNA. For mammalian cells, the G1 phase is extremely important. This phase provides a variety of proteins for DNA replication. Among them, the restriction point (or R point) is an important link that controls the cell's progress and directly determines the cell's destination, that is, the cell: or leaves the cell cycle and enters the stationary phase (G.), such as normal renewable tissues and cancer cells Under the action of chemotherapeutic drugs, it enters the stationary phase to avoid the effects of drugs; or enters apoptosis, such as cells whose DNA is damaged and cannot be repaired, enters apoptosis under the action of tumor suppressor gene P53; It leaves the cell cycle, which is the main way for normal cell tissues to mature and mature; or it crosses the limit point R and continues to enter the S, G2, and M phases repeatedly without limit proliferation (such as cancer cells).

During the cell cycle, the most critical protein components that control the entire cell engine are cell cycle factor-dependent cell cycle kinases (hereinafter referred to as cell cycle kinases or cdks), cell cycle factors (Cyclins), and corresponding endogenous inhibitory proteins (cdkl ). To date, at least 15 cell cycle factors, 9 Cdks and two families of endogenous inhibitory proteins have been discovered. They are active at different stages of the cell cycle.

A typical cell cycle kinase has an enzyme active center region composed of more than 300 amino acids, and a site for binding to cell cycle factors and endogenous inhibitory proteins. Cyclokinase must be combined with the corresponding cell cycle factor to form a Cyclin / Cdks complex to have kinase activity, and if the complex is bound to the endogenous inhibitory protein cdkl, the kinase activity disappear. Cyclin / Cdks complex is formed at a specific stage of the cell cycle, and its activity is essential for the continuous operation of the cell cycle. Studies have shown that almost all tumor cells have a variety of cell cycle kinase abnormalities, or that cell cycle factor is highly expressed, or that cell cycle kinase is highly activated, or that endogenous inhibitory proteins are missing. For example, CyclinD / Cdk4 / 6 is abnormal in up to 85% of breast cancer patients. Therefore, inhibition of cell cycle kinases can effectively prevent cell proliferation (but not kill cells). Furthermore, it can promote cell differentiation and maturity, or promote cell apoptosis to achieve the effect of treating various tumors. It can be considered that cell cycle kinase inhibitors are a new class of broad-spectrum anticancer drugs. At the same time, because they inhibit cytostatic rather than cytotoxic, these drugs should have lower toxic and side effects. Based on this, finding cell cycle kinase inhibitors has become a new strategy for the development of anticancer drugs. These drugs either act directly on the catalytic activity centers of cell cycle kinases or indirectly on the cell cycle kinase information pathway. Flavopidol, developed by the National Cancer Institute (NCI), is currently in clinical trials. Although the compound needs to be improved due to its own chemical stability and selectivity, the clinical prospects are still exciting.

Therefore, there is an urgent need in the art to develop compounds that are effective in treating cancer, especially compounds that inhibit cell cycle kinases. Summary of the Invention

It is an object of the present invention to provide a class of effective cell cycle kinase inhibitors.

Another object of the present invention is to provide the preparation and use of the inhibitor. In a first aspect of the invention, there is provided an indole compound of formula I or a pharmaceutically acceptable salt thereof,

among them:

!! ^ Is ^ C1-C6 fluorenyl, CI- C6 fluorenyl, —C ₂ H ₄ 0H, or — C¾0H;

R ₂ is H, CI-C6 alkyl, CI-C6 alkoxy, a C ₂ H ₄ 0H, or —CH ₂ 0H;

Is 11, or a halogen atom, or a S0 ₃ H;

R ₄ is H, or a halogen atom, or -S0 ₃ H.

In a preferred example, and are 11.

In a preferred example, at least one of and is H or a C¾0H.

In a preferred example, the compound is a mixture of isomers, and its structural formula is:

In a second aspect of the present invention, a pharmaceutical composition is provided, which contains a safe and effective amount of an indole compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Preferably, the dosage form of the composition is a tablet, a pill, a capsule, a paste, a cream, a patch, a powder, an injection, a spray, an implant, a suppository, a drop, or a fat emulsion.

In another preferred example, the pharmaceutically acceptable salt is sulfate, hydrochloride, phosphate, formate, acetate, butyrate, succinate, tartrate, amino acid salt, or Its combination.

In a third aspect of the invention, there is provided the use of an indole compound of formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating tumors and inhibiting cell cycle kinase activity.

In a fourth aspect of the present invention, a method for preparing a compound of formula I is provided, comprising the steps of: (a) the compound of formula II and formula III are condensed under reflux in toluene to obtain a compound of formula IV;

(b) a compound of formula IV is reacted with R ₂ I, wherein R ₂ is a CI-C6 alkyl group, thereby forming a compound of formula V

(c) The compound of formula V is protected under acidic conditions to obtain a compound of formula VI:

Among them! 1.C1-C6 alkyl, CI-C6 alkoxy, -C ₂ H.

(d) reacting a compound of formula VI with hydroxylamine to obtain a compound of formula I:

The definitions of R ₂ , and in each structural formula are as described above.

In a preferred example, it is 11, R ₂ is a C0H, and R ₃ and R ₄ are H. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Cell cycle distribution, regulatory mechanisms, key regulatory proteins and their roles;

Figure 2: Effect of JN-2518 on hormone-dependent prostate cancer cell LNCAP-concentration dependence and comparison with other drugs;

Figure 3: Effect of JN-2518 and other chemotherapeutic drugs on primary cultured B1011 prostate cancer ascites metastatic cancer cells and their comparison;

Figure 4: JW-2518 inhibits cell cycle kinase;

Figure 5: JN-2518 induces PARP protein degradation;

Figure 6: JI ^ -2518 induces the formation of DNA ladders in LNCAP cells (A) and N2A cells (B). detailed description

After extensive and intensive research, the inventors have synthesized a new class of specific indole compounds. This compound is a cell cycle kinase inhibitor, which can specifically inhibit cell cycle kinase, block cell proliferation, and induce apoptosis, so it can be used for the treatment and prevention of various diseases such as malignant tumors in humans and animals. The present invention has been completed on this basis. Definition

The term "combination of the present invention ¾""JN-2518 series of compounds" is used interchangeably and refers to an indole compound having Formula I or a pharmaceutically acceptable salt thereof. ¾ The term includes resolved isomers ( (Formulas IA and I-B) And unresolved isomer mixture:

(I-A) (I-B)

The term "JN-2518" or "Product 1" refers to a compound of the formula (including resolved isomers and unresolved mixtures of isomers), or a pharmaceutically acceptable salt thereof:

The compounds of the present invention can be made into various salts, such as inorganic salts (such as sulfates, hydrochlorides, and phosphates), organic acid salts (such as formate, acetate, butyrate, succinate, and tartaric acid). Salt, etc.), amino acid salts, various single salts, double salts, and all kinds of salts suitable for pharmaceutical applications. Preparation

The compound of the present invention can be prepared by the following method, which includes:

(a) condensation of a compound of formula II and formula III under reflux in toluene to obtain a compound of formula IV;

(b) A compound of formula IV is reacted with R ₂ I, wherein is (1-06 alkyl) to form a compound of formula V (isomer mixture)

(c) The compound of formula V is protected under acidic conditions to obtain a compound of formula VI (isomer mixture):

Where ^ CI-C6 alkyl, C1-C6 alkoxy, one C ₂ H ₄ 0H, one CH ₂ 0H;

(d) reacting a compound of formula VI with hydroxylamine to obtain a compound of formula I (isomer mixture):

Wherein, in the formulas Rl, R _2, and the definitions of Formula I defined.

A reaction scheme for preparing compound I (isomer mixture) is as follows:

For JN-2518 (1, isomer mixture), it can be prepared by the following methods:

Using 2-ketoindole (3,0xindole) and indolinedione (4, Isatin) as the basic raw materials, the compound 5 after the nitrogen acetylation reaction and the chlorinated intermediate 6 of indolinedone in toluene Condensation 7. The nitrogen-ethylated product 8 (isomer mixture) is protected under acidic conditions to obtain a compound (acetyl group) to obtain compound 9 (isomer mixture). The chemical reaction formula is:

After the compound 9 (isomer mixture) is obtained, it is reacted with hydroxylamine to obtain the product.

Structure mixture), its chemical reaction formula is:

Pharmaceutical composition and mode of administration

The compounds of the present invention (including resolved isomers and unresolved isomer mixtures) can specifically inhibit cell cycle kinase activity, induce apoptosis, and are used to treat and prevent diseases such as malignant tumors.

The tumors referred to include various leukemias and various solid tumors.

The invention also provides, in another aspect of the invention, a pharmaceutical composition. The pharmaceutical composition of the present invention comprises an effective amount of a pharmaceutically acceptable salt of a compound of formula I of the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient.

In use, it can be a single drug treatment or a combination treatment. The combination therapy can be combined with other chemotherapy, combined with Chinese herbal medicine, or combined with surgery, radiotherapy, immunotherapy, hormone therapy, gene therapy, and so on. The combination therapy can be a simultaneous treatment or a different treatment order.

The other diseases mentioned mainly include, but are not limited to, senile dementia, psoriasis, cardiovascular disease, glomerulonephritis, inflammation and AIDS.

The JN-2518 series of compounds are used for medicine, and can be used in any route and method. That is, oral, injection, inhalation, transdermal absorption, implantation, cavity, mucosa, and intravenous drip.

JN-2518 series compounds are used as medicines, and can be made into tablets, pills, capsules, ointments, creams, patches, powders, injections, sprays, implants, suppositories, drops or fat emulsions. Dosage form.

According to the present invention, studies on human cancer cells through the MTT and SRB methods show that JN-2518 has a good inhibitory effect on breast cancer, colon cancer, hormone-dependent and hormone-independent prostate cancer, and neuroblastoma. It is particularly important that JK-2518 has a good inhibitory effect on clinically helpless hormone-independent prostate cancer.

According to the present invention, the study of ascites metastatic cancer cells in patients with advanced prostate cancer by the MTT method shows that JN-2518 still has resistance to the first-line excellent anticancer drugs paclitaxel, daunorubicin, and isovinblastin, etc. Good curative effect.

According to the present invention, the comparison of tumor inhibition rates of rat W256 breast cancer shows that JN-2518 has significant anticancer activity on W256, and the anticancer activity is directly proportional to the administered dose. According to the present invention, by studying the cell cycle kinase activity of HUVEC and LNCAP, JN-2518 can selectively inhibit the cell cycle kinase activity.

The present invention confirmed that JN-2518 can induce apoptosis of cancer cells through research on the degradation of PARP protein and the formation of DNA ladder bands.

JN-2518 is a brand-new chemical structure compound. Because it is a specific cell cycle kinase inhibitor, this patent directly uses JN-2518 to treat and prevent animal tumors. The animals mentioned here mainly refer to humans, but are not limited to humans. They also apply to poultry, etc. At the same time, recent studies have shown that many other diseases are also related to cell cycle kinase abnormalities, so JN-2518 can also be used to treat senile dementia, psoriasis, cardiovascular system diseases, and glomerulonephritis. The examples given below are just to prove the utility of the patent, not to try to limit the scope of application of the invention.

It should be noted that during the treatment of tumor or other diseases, JN-2518 can be treated as a single drug or in combination with two, three or more drugs, administered simultaneously, or administered in a different order. The combination therapy mentioned here can be combined with other western medicine and Chinese herbal medicine, or combined with hormone therapy, radiation therapy, immunotherapy, chemotherapy, cryotherapy and gene combination therapy.

The invention provides pharmaceutical products for the treatment of tumors and other diseases. These products contain effective doses of -2518 and all suitable additives used in the pharmaceutical industry, as well as various suitable routes of administration.

The skillful pharmaceutical production process involves the use of non-toxic pharmaceutical industry-accepted prodrugs as raw materials, and JN-2518 is synthesized in a variety of ways. The skilled pharmaceutical production process also includes the use of solvents accepted by the pharmaceutical industry as a solvent for JN-2518, such as water, mineral oil, edible oil, dimethyl sulfoxide, and the like.

JN-2518 can be made into oral, topical, inhaled, anal and other dosage forms. Use non-toxic materials accepted by the pharmaceutical industry as carriers, additives, excipients. It is pointed out again that the mode of administration may be a combination. Dosage forms include: tablets, capsules, elixirs, lozenges, oral syrups or all other suitable dosage forms.

JN-2518 can be formulated into systemic dosage forms, including: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, spinal injection, intrathecal injection, or any other injection and infusion. In addition, in addition to the effective dose of NJ-2518, the JN-2518 preparation also contains all other non-toxic substances that are available in pharmacy. Includes one or more carriers, diluents, adjuvants, and other medicinal ingredients if needed.

JN-2518 can be made into any form of oral dosage form, including: tablets, lozenges, soft and hard capsules, water-containing agents, elixirs, oral suspensions, powders, emulsifiers, etc. Oral dosage forms may contain one or more sweeteners, flavors, colorants and preservatives to ensure perfect color and ease of use.

The excipients used for JN-2518 tablets are degradable diluents such as calcium carbonate, sodium carbonate, calcium lactate, calcium phosphate, sodium phosphate; granules: corn starch, alginic acid; binder : Starch, gelatin or gum arabic; lubricant: stearic acid, magnesium stearate or talc. Coated or uncoated tablets are made using all known techniques. Coating dosage forms include enteric coatings and sustained release dosage forms. The material for the sustained release dosage form may be a monoglyceride or a diglyceride.

M-2518 capsules can be hard capsules or soft capsules. The material used for the hard capsule can be calcium carbonate, calcium phosphate or kaolin. Materials for soft capsules such as water, oils include peanut oil, olive oil or liquid paraffin. JN-2518 oral aqueous suspension can be formulated with an effective dose of JN-2518 and a suitable diluent. Its diluent suspension may be sodium methylcellulose or methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth, acacia gum. Dispersants or humectants include: natural phospholipids, such as lecithin, or condensates of olefin oxides and fatty acids, such as 17 fluorenyl ethylene alkanol, or partially esterified condensation products of ethylene and fatty acids, or hexitols such as Polyoxyethylene sorbitol monooleate, or the product of partial fatty condensation of ethylene oxide and fatty acids, and glycolic anhydride, such as polyethylene sorbitol methyl ester. JN-2518 oral aqueous suspensions may also contain one or more preservatives, such as ethyl para or o-hydroxybenzene benzoin salt; and one or more sweeteners, such as sucrose or saccharin.

-2518 oil suspension can be used to suspend an effective dose of JN-2518 in vegetable oils, such as soybean oil, peanut oil, olive oil, sesame oil, cocoa butter and other vegetable oils, or mineral oils such as liquid paraffin. The preparation may also contain diluents such as beeswax, wax, or cetyl alcohol, the sweeteners described above, and flavoring agents and stabilizers such as the antioxidant vitamin C (ascorbic acid).

Using the dispersants, wetting agents, suspensions, preservatives, flavoring agents, sweeteners, and coloring agents described above, JN-2518 can also be made into oil-in-water formulations.

JN-2518 is made of oil-in-water emulsifier. The oil phase can be a vegetable oil as described above, such as soybean oil, olive oil, peanut oil, and the like. It can also be liquid paraffin, mineral oil, or a mixture of the two. Emulsifiers can be natural emulsifiers, such as acacia gum or tragacanth, natural phospholipids such as soybean phospholipids, lecithin, fatty acids, hexitol or anhydride lipids or hemilipids, such as sorbitan monoesters. It can also be a man-made chemical material, ethylene oxide half-fat condensate, such as polyethylene sorbitol monoester. JN-2518 oil-in-water emulsion preparations may also contain sweeteners and flavoring agents.

JN-2518's syrups and elixirs can be sweetened, such as propanol, propylene glycol, sorbitol or sucrose. The formulation may also contain wetting agents, preservatives, flavoring agents, and color additives.

JN-2518's injection can be sterile injection or oily suspension. And all pharmacologically applicable dispersants, wetting agents or suspensions can be used. The preparation can be a sterile clear liquid, using all pharmacologically applicable diluents, such as 1,3-butanediol, water, Ringer's solution, or physiological saline. In addition, sterile fats and oils can also be used as solvents or suspensions. It can also be synthetic mono- or di-glycerides, fatty acids such as oleic acid and the like.

JN-2518 can also be made into suppositories, such as anal suppositories. Suppositories can be composed of a certain amount of J-2518 and corresponding non-irritating excipients. The excipient is solid at normal temperature, but liquefies at anal body temperature to release the drug. Suitable excipients include cocoa butter and polyethylene glycols.

The systemic dosage form of JN-2518 can be a suspension type or a dissolution type according to the concentration of the carrier used. For ease of use or to ease patient suffering, preservatives, buffers and local anesthetics can be added.

JN-2518 can also be used as a veterinary medicine. For the convenience of animals, preparations containing JN-2518 can be added to animal feed or drinking water. It can also be made into convenient dosage forms as food or drink for animals.

For humans, in the treatment of the above diseases, the dosage range of JN-2518 is initially set at O. Olmg to 15 mg per kilogram of body weight, or about 0.6 mg to 900 mg per day per 60 kilograms of body weight. Depending on the mode of administration, JN-2518 Can be made into a single dose or multiple doses. The dosage unit is initially set to contain 1 mg to 500 mg of the active ingredient. The number of daily doses depends on the disease. In most cases, no more than four times a day. It should be noted that the dosage may depend on a variety of factors, such as age, weight, health status, gender, diet, as well as the timing, route, condition, and combination of drugs used. Therefore, the final dosing regimen should be determined by the doctor on a case-by-case basis.

The priority compounds mentioned in this patent have ideal pharmacological properties, including but not limited to the following aspects: superior oral bioavailability, low toxicity, low plasma protein binding rate, and ideal half-life in vivo and in vivo. Although these compounds can cross the blood-brain barrier, which is an advantage for treating central nervous system diseases, lower brain blood concentrations are more desirable for treating peripheral diseases. Compared with the background technology, the main beneficial effects are as follows:

Experiments have shown that 2518 can bind to the ATP binding site of cell cycle kinase. Since the two functional groups of the compound can form more hydrogen bonds with the ATP binding site of the enzyme, they have greater affinity than ATP, and can compete with ATP more effectively, thereby inhibiting the activity of the enzyme. The compound can inhibit cell cycle kinase activity mediated by cytokines A, B and D, inhibit cell proliferation, and induce apoptosis. It has a strong inhibitory effect on a variety of human tumor cells, with half the effective inhibitory concentration between 1-9 μM. It inhibited rat experimental tumor W256 by 70%. Compared with Flavopiridol developed by the National Cancer Research Center, JN-2518 has the following characteristics-

1. Flavopiridol is a flavonoid with relatively unstable physical and chemical properties. Must be administered intravenously. This is extremely inconvenient for patients who require long-term administration. JN-2518 has stable physical and chemical properties, and is not only effective orally, but also can be made into a variety of dosage forms.

2. Due to its unstable physical and chemical properties, Flavopiridol is easily metabolized and inactivated in the body. Therefore its in vitro activity cannot reflect in vivo effects. m-2518 is not.

3. Flavopiridol not only inhibits cell cycle kinases, but also protein kinase C (PKC), protein kinase A (PKA) and many other protein kinases that are essential for maintaining normal cell functions, so its selectivity is poor and side effects are numerous. And experiments show that JN-2518 is a specific cell cycle kinase inhibitor.

4. Flavopiridol has toxic and side effects of flavonoids, especially severe gastrointestinal irritation. JN-2518 parental compound does not have this toxic effect, and other side effects are slight.

A new compound with a new structure, JN-2518, is a promising new broad-spectrum anticancer drug. The present invention will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing conditions Conditions recommended by the manufacturer. Example 1: Chemical synthesis and structural confirmation of JN-2518 (I) Materials and methods

1. Reagents: 2-ketoindolin (Oxindole), indolin dione (Isatin), iodoethane, phosphorus pentachloride, hydroxylamine hydrochloride, acetic anhydride and toluene were purchased from Sigma-Aldrich Chemical Reagent Company .

2. Method: 2-ketoindole (3, Oxindole) was refluxed in acetic anhydride to obtain nitrogen acetylation product 5. The chlorinated intermediate 6 obtained by the reaction of indolin dione (4, Isatin) and phosphorus pentachloride in a benzene solution was immediately refluxed with compound 5 in anhydrous toluene for 2 hours. The obtained solid was filtered with toluene and ethanol in that order. Wash, recrystallize in dimethylamide to obtain compound 7, and then react with iodoethane under alkaline conditions to obtain nitrogen hydrocarbonation reaction product 8 (isomer mixture), nitrogen ethylation product 8 in Under acidic conditions, the protecting group (acetyl group) is deprotected to obtain compound 9 (isomeric mixture), and finally reacted with hydroxylamine hydrochloride in 20% ethanol to obtain product 1 (isomeric mixture).

(2) Results:

The structure of Product 1 was confirmed by mass spectrometry, infrared, and the like. Mass spectrometry (MS) was measured with an HP1100LC / MSD mass spectrometer. The mass spectrometry results for compound 1 were: ESI-MS: 328.2 [M + Na] ⁺ , 306.2 {M + H} ⁺ , 304.2 2 [M- H ]-, Compound 1: C ₁₈ H ₁₅ N ₃ 0 ₂ (M-305. 3).

The structure of product 1 was further confirmed by nuclear magnetic resonance. The results show that product 1 is a mixture of isomers and contains the following two isomers:

(I-A) (I-B) Example 2: Inhibitory effect of JN-2518 on human cancer cells

The following are examples of tumor treatment with JN-2518 and other preferred compounds (see Figure 2). It should be understood that the compounds of the invention are not only useful for treating tumors.

(I) Materials and methods

Reagent: JN-2518 and a series of compounds prepared in Example 1. The structure was confirmed by mass spectrometry, nuclear magnetic resonance, and infrared, and purified by high pressure liquid chromatography (HPLC). The purity is> 98.0%. JN-2518 is a dark red crystalline powder with a molecular weight of 305. During the experiment, a solution was prepared with DMS0 and stored at -20 ° C. Retinoic acid, daunorubicin, and paclitaxel were purchased from Sigma Chemical Reagent Company, USA. NS389, a novel cyclooxygenase II inhibitor, was purchased from the Biomol. Research Lab. Other chemical reagents used in the experiment were purchased from Sigma Chemical Reagent Company of the United States unless otherwise specified.

Cell culture: human cancer cell lines, breast cancer; MCF-7 SKBR-3; colon cancer; LDV0, DLD-1, prostate cancer: androgen-dependent LNCAP and androgen-independent PC-3, DU 145; neuroblasts Knob: N2A Cells were purchased from American Type Culture Collection. Cells were placed in a 37 ° C, 5% CO ₂ incubator with RPMI1640 medium containing 10% fetal bovine serum and penicillin-streptomycin. -

MTT and SRB: MTT and SRB experimental methods are briefly described as follows: Take cancer cells in logarithmic growth phase and inoculate them into 96-well plates with a cell density of 5000 cells / 200μ1 per well. After 24 hours, add serial dilutions of the drug. The cells were cultured for 72 hours under the influence of drugs. After removing 100 μΙ of the culture medium from each well, 50 μ1 of MTT [3- (4, 5-dimethylthiazol- 2-yl)-2., 5-diphenyltetrazolium bromide] solution was added and culture was continued for 4 hours. A 200 μl solution of hydrochloric acid-isopropanol was used to dissolve the purple black latent nail deposits, and the light absorption value was measured at a wavelength of 540 nm. If the SRB method is used, after 72 hours, all the culture medium is removed, the cells are fixed with 10% trichloroacetic acid for 1 hour, and the cells are naturally dried in the air for 24 hours, and 5 (^ l SRB (sulforhodamine B) is added to stain 20-30 After 5 minutes of washing with 1% glacial acetic acid, and air-drying in air, 200 μl of a 10 mM Tris-HCl (pH 10. 0) solution was added to dissolve the purple-red protein-SRB complex, and the light absorption value was measured at a wavelength of 600 nm. Calculate the average value of the test data of each group, and calculate the cytogenesis inhibition rate by the experimental group versus the control group. Draw the cell growth rate-drug logarithmic curve with the Sigma Plot plotting software, and find the half effective concentration of the drug (IC50).

(2) Results:

Using two analytical methods, MTT and SRB, we studied the anticancer activity of JN-2518. Table 1-3 lists the half effective inhibitory concentrations of JN-2518 and other chemotherapy drugs on various cancer cells. Figure 3 is the pharmacodynamic-drug concentration curve of the effect of J-2518 and other drugs on hormone-dependent prostate cancer cell LNCAP. As can be seen from Table 1-3 and Figure 3, whether it is breast cancer (MCF-7, SKBR-3, Table 1), colon cancer (L0V0, DLD-1, Table 2), hormone-dependent (LNCAP) and hormones Non-dependent prostate cancer (PC-3, DU-145, Table 3), or neuroblastoma (N2A, IC50 1.2 μM), 'is more sensitive to JN-2518, its half effective inhibitory concentration IC50 is 1- 9 μM (see Table 1-3), similar to common chemotherapeutic drugs. This result indicates that J-2518 has strong anticancer activity.

Table 1: Effective inhibitory concentration of JN-2518 on breast cancer cells (IC50, μΜ)

And comparison with other chemotherapy drugs (MTT method)

Fine moon package

Drug

MCF-7 SKBR-3

JN-2518 4. 72 + 0.54 3. 29 soil 0.47

Daunorubicin 0. 054 + 0. 011 0. 061 + 0. 005

Retinoic acid 21. 45 + 3. 78> 50

NS389>200> 200 Table 2: Effective concentration of JN-2518 for half inhibition of colon cancer cells (IC50, μM)

And comparison with other chemotherapy drugs (MTT method)

Table 3: Effective concentration of half inhibition of prostate cancer cells by JN-2518 (IC50, μ)

And comparison with other chemotherapy drugs (MTT method)

(III) Discussion

Through MTT and SRB experiments, it is not difficult to see that JN-2518 has strong anti-cancer activity, and its inhibitory effect on cancer cells is better than the cell-inducing differentiation agent Retinoid acid (IC50: 21. 5-> 50 μM) and a novel ring The oxidase II inhibitor NS389 (IC50> 200 μM) is much stronger. The inhibitory effect of JN-2518 on hormone-dependent prostate cancer cells LNCAP is also significantly stronger than Casodex (IC50: 57.4 μM) and Proscar (IC50: 40.6 μM) commonly used in clinical practice. More importantly, JN-2518 also has a good inhibitory effect on hormone-independent prostate cancer cells (PC-3, DU-145), which are clinically helpless. In contrast, PC-3 and DU-145 cells were nearly 10 times less sensitive to paclitaxel.

Neuroblastoma N2A cells are more sensitive to JN-2518, with IC50 of 1.2 μM. Since the compound has a fat-soluble structure, it may easily cross the blood-brain barrier, which is beneficial to the treatment of brain tumors.

Although compared with daunorubicin or paclitaxel, the anticancer effect of JN-2518 is weaker, but the former has already produced strong toxic and side effects at effective drug concentrations. For example, paclitaxel produces severe when the plasma drug concentration is 0.05 μM. Heavy bone marrow suppression. 9G / kg。 And JN-2518 toxicity is very low, the mouse LD50 of its precursor compound is 3.9 g / kg.

It is worth noting that it is not difficult to see from Figure 3 that although LNCAP cells are very sensitive to paclitaxel, it is difficult to measure the IC90 in the experiment, because no matter the concentration, about 15-20% of the cells are not sensitive. The reason may be that these cells enter the GO phase under the action of paclitaxel and evade the further action of the drug. In contrast, JN-2518 has a good curative effect-concentration curve. This result also shows that if JN-2518 is administered in a different order than paclitaxel, it may have a significant synergistic effect.

In addition, JN-2518 has no significant difference in the IC50 of the tumor cells tested, which is different from other chemotherapy drugs, such as colon cancer and breast cancer cells are more sensitive to daunorubicin than prostate cancer cells, which indicates that JN-2518 acts on cell formation Public target. Example 3: Effect of JN-2518 on prostate cancer ascites metastasis cancer cells:

(I) Materials and methods

Reagent: JN-2518 See Example 1. Daunorubicin, paclitaxel, arsenic trioxide (As ₂ 0 ₃ ), Triochstatin A (TSA) were purchased from Sigma Chemical Reagent Company, USA. Other chemical reagents used in the experiment were purchased from American Sigma Chemical Reagent Company unless otherwise specified.

Cell culture: The primary cultured prostate cancer ascites metastasized human cancer cell B1011 was collected from the patient's ascites by centrifugation. The cells were collected at 37 ° C, 5% (0: ₂ in an incubator, containing 10% fetal bovine serum). And penicillin-streptomycin in RPMI1640 medium. After one week of culture, cell growth was fast and stable. B1011 cancer cells in logarithmic growth phase were taken and the anti-cancer activity of JTN-2518 was studied by MTT method.

MTT: The MTT experimental method is as described in Example 2.

(II) Results and discussion

B1011 cancer cells come from ascites in a patient with advanced prostatic cancer. The patient has been resistant to hormones and other chemotherapy. To help patients choose a sensitive chemotherapeutic drug, we inoculated primary cultured cancer cells (B1011) into a 96-well plate and performed different chemotherapeutic drugs as described above. MTT experiment. The results are shown in Figure 4. Experiments show that B1011 cancer cells are more sensitive to JN-2518, and their sensitivity is slightly stronger than arsenic trioxide (As ₂ 0 ₃ ) (2.8 μM vs. 4.2 μM). However, arsenic trioxide is highly toxic, and its effective blood concentration does not reach 4.2 μM at clinically acceptable doses.

_ B1011 cells have been resistant to other commonly used anticancer drugs such as paclitaxel, daunorubicin, isovincin and so on. For example, the IC50 of LNCAP prostate cancer cells to paclitaxel is 0:35 ηM, but the IC50 of B1011 cells to this drug has increased to 5 nM. gp, B1011 cells were nearly 15 times less sensitive to paclitaxel! The resistance of B1011 cells to daunorubicin and isovincristine is equivalent to that of paclitaxel. In contrast, LNCAP and B1011 cells are equally sensitive to JN2518 (LNCAP IC50 2. 59 μM, for B1011, 2. 8 μM), indicating that JN2518 is effective not only for early hormone-dependent prostate cancer, but also for advanced metastatic prostate cancer The same works. Example 4: Anticancer effect of JN-2518 on rat W256 breast cancer:

(I) Materials and methods

Material: JN-2518, see Example 1. Wistar rats were provided by the Experimental Animal Center of the National Cancer Institute.

Method:

Preparation of drug suspension: Take a certain amount of JN-2518 in a mortar, add a few drops of Tween-100 and grind with the drug, and then add sufficient water to grind until fully suspended before use.

Rat W256 breast cancer model and dosing plan: Many years of research have shown that rat W256 breast cancer has a good correlation with human tumors, and has a wide range of applications. This patent then uses this model to study the anticancer effect of JN-2518. Forty Wistar rats with 60-80 grams were randomly divided into 4 groups according to body weight, ten rats in each group, and 1-2 x ^{10 6} W256 tumor cells were inoculated under the armpit. Twenty-four hours after the inoculation, JN-2518 was administered orally at 0, 50, and 100 mg / kg, once a day for ten days. Cyclophosphamide was used as a positive control (60 mg / kg administered intraperitoneally on the first and fifth days). Twenty-four hours after the last dose, the animals were sacrificed and all tumor masses were removed and weighed. Take the average tumor weight of the test group as compared to the average tumor weight of the control group, and obtain the tumor suppression rate according to (1-T / C). T test was used to determine the significant difference.

(II) Results and discussion

As shown in Table 4, JN-2518 has significant anticancer activity on W256, and its anticancer activity is directly proportional to the dose. At a dose of 100 mg / kg, the inhibition rate of JN-2518 on W256 reached more than 70%. Under the same test conditions, the cyclophosphamide positive control group also showed strong anti-cancer activity, but from the survival and weight changes of the animals, it can be seen that JN-2518 did not die at the dose of 100 mg / kg, and Animals gained weight without any signs of toxicity. In the cyclophosphamide group, not only the average body weight of the animals decreased, but also one rat died on the eighth day after administration, showing significant toxicity.

Table 4. Inhibition of JN-2518 on rat W256 breast cancer

*; Oral administration. (P. O.); **: Intraperitoneal administration (匸 P.). Example 5: Inhibition of cell cycle kinase by JN-2518:

(1) Materials and Methods-Materials: Polyacrylamide gel reagents for protein electrophoresis, SDS, electrophoresis buffer, protein electrotransfer buffer, purchased from Bio-Rad Life Sciences, USA. Other chemical reagents used in the experiment were purchased from American Sigma Chemical Reagent Company unless otherwise specified. Cell cycle kinase antibodies for testing, cdk2, cdk4, Cyclin Dl Antibodies were purchased from Santa Cruz Biotechnology Company Sepharose A + G from Oncogene Inc.

[ ³² P]-ATP was purchased from PerkinElmef Life Sciences.

Cell culture: See Example 2.

Inhibition of cell cycle kinases: We used HUVEC and LNCaP cells as experimental models to study the effects of JN-2518 on cell cycle kinases and other related kinases. HUVEC and LNCaP cells were cultured in EMB (endothelial cell basal medium) and RPMI 1640 medium containing 10% fetal bovine serum, respectively. Cells in logarithmic growth phase were treated with a given concentration of JN-2518 for 24 hours. Cells were collected, washed, and total protein was extracted by conventional methods. 100 μ _§ protein was immunoprecipitated with cdk2, cdk4, Cyclin Dl, ERK2 and other specific antibodies in the presence of various protease inhibitors at 4 ° C overnight. The immunoprecipitation complex was washed four times with a buffer and then once with a buffer for measuring cell cycle kinase activity. The immunopurified cell cycle kinase was suspended in its buffer, and reacted with histone HI for 10 minutes in the presence of [ ³² Ρ] -ΑΤΡ (0.5 μ0ΐ / 100 μM). Phosphorylated histones, which respectively represent the corresponding cell cycle kinase activities, were isolated and purified and recorded in X-ray diffraction pictures and quantified with a density scanner. Compared with the control group, the inhibition rate was determined as (1-T / C)%. In the positive control group, cells were treated with different concentrations of PD98058 (ERKl / 2, a podocyte-kinase-specific inhibitor), and the enzyme activity was measured in the same way.

(II) Results and discussion

Figure 5 is a typical example of the effect of JN-2518 on cell cycle kinases and other related kinases. Experiments show that using the above-mentioned cell system, JN-2518 has a significant inhibitory effect on the cell cycle kinases cdk2, cdk4 / 6, and its IC50 is between 1.5-6.2 μM. We speculate that this inhibition may be more pronounced if a pure enzyme system is used. However, under the same conditions, this compound has no effect on protein kinase C (PKC), MAP kinase, etc., indicating that JN-2518 has good selectivity for cell cycle kinase inhibition. Although, according to the median effective concentration, Flavopiridol JN-2518 reported in the literature as good as that of cyclin kinase pure enzyme systems 1, 2 and 4 EC50 in the ^10-9 to ^10-8 molar concentration range. It should be noted that, when some compounds use pure enzymes, although 'has very good activity, some of these compounds have no biological activity because they cannot pass through the cell membrane, let alone effective on the whole animal. Example 6: Induction of -2518 on apoptosis:

The degradation of PARP [Poly (ADP-ribose) -Polymerase] protein and the formation of DM trapezoidal bands are important features of apoptosis and are widely used in the study of apoptosis. We induced apoptosis by JN-2518 The role was studied.

(I) Materials and methods

Materials: DNA agarose gel, reagents used for protein polyacrylamide electrophoresis, purchased from Bio-Rad Life Sciences, USA. Other chemical reagents used in the experiment were purchased from American Sigma Chemical Reagent Company unless otherwise specified. PARP monoclonal antibodies were purchased from Pharmingen, A Becton Dickinson, USA. Western blot detection reagents and other supplies were purchased from Amershan Life Sciences, USA. Cell culture: See Example 2.

Apoptosis analysis:

1) Degradation of PARP protein by Western blot:

Human neuroblastoma cells N2A in logarithmic growth phase were treated with different concentrations of JN-2518 or 20 nM paclitaxel (positive control) for 48 hours. Cells were collected, washed, and proteins were extracted by conventional methods and quantified. Take 100 proteins for SDS-polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to a nitrocellulose membrane for detection with a specific PARP antibody, and β-Actin antibody was used as an internal standard. The obtained X-ray diffraction pictures were quantified with a density scanner.

2) JN-2518 induced DNA ladder band formation: Human prostate cancer cells LNCAP and human neuroblastoma cells N2A in logarithmic growth phase were treated with different concentrations of JN-2518 or paclitaxel (positive control) for 48 hours. The cells were collected, washed, and the DNA was extracted by conventional methods. The DNA fragments were separated by 2% agar agarose gel electrophoresis, and photographed and recorded.

(II) Results and discussion

As mentioned earlier, inhibition of the cell cycle kinase cdk4 / 6 and other cell cycle kinases, such as cdk2, leads to impeded cell cycle operation, which in turn promotes cell differentiation or induces apoptosis. To confirm this inference, we observed JN-2518-induced apoptosis.

As shown in Figure 6, under the action of JN-2518, the PARP protein band of llOKda was significantly reduced, but under the same experimental conditions, there was no change in β-Actin protein as an endogenous control. This result indicates that JN-2518 activates the apoptotic information pathway and induces apoptosis. This conclusion is fully confirmed by the DNA ladder formation experiment.

As can be seen from Figures 7A and B, whether it is LNCAP prostate cancer cells or neuroblast N2A, JN-2518 can obviously cause the formation of DNA ladder bands. Its intensity of action is consistent with the MTT experiment. N2A cells are more sensitive to DNA ladder formation than LNCAP cells. When the drug concentration is less than 5.0 μM, it can significantly induce DNA ladder formation.

Conclusion: JN-2518 and the compounds listed in this patent are an attractive class of anticancer compounds. They are a specific class of cell cycle kinase inhibitors that block the normal operation of tumor cell cycles and induce apoptosis. It has a strong anti-cancer effect on a variety of human tumor cells, and its IC50 is between 1 and 9 μM. It also has a strong inhibitory effect on experimental animal tumors. So JN-2518 represents a new class of anticancer drugs. Example 7: Both isomers have anticancer activity

The conventional isomer resolution method was used to resolve the JN-2518 isomer mixture to obtain two isomers (I-A and I-B) with a resolution ratio of 3: 7 ~ 7: Between 3. The anticancer activity was measured in the same manner as in Example 2-6.

The results show that both isomers have anticancer activity, and both are comparable in activity.

Claims

1. An indole compound of formula I or a pharmaceutically acceptable salt thereof,

Right

among them-

! ^ Is 11, CI-C6 alkyl, CI-C6 alkoxy, a C ₂ H ₄ 0H, or —C¾0H;

Is 11, CI-C6 alkyl, -C6 alkoxy, a C ₂ H ₄ 0H, or —CH ₂ 0H;

Is 11, or a halogen atom, or a S0 ₃ H;

Is 11, or a halogen atom, or a S0 ₃ H. begging

2. The compound according to claim 1, wherein and are 11.

3. The compound according to claim 1, wherein: At least one of ^ and R ₂ is H or a C¾0H.

4. The compound according to claim 1, wherein the compound is a mixture of isomers, and its structural formula is:

5. A pharmaceutical composition, comprising a safe and effective amount of an indole compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier.

6. The composition according to claim 5, wherein the dosage form of the composition is a tablet or a pill Agents, capsules, creams, creams, patches, powders, injections, sprays, implants, suppositories, drops or fat emulsions.

7. The composition according to claim 5, wherein the pharmaceutically acceptable salt is sulfate, hydrochloride, phosphate, formate, acetate, butyrate, succinate Salt, tartrate, amino acid salt, or a combination thereof.

The use of an indole compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that it is used for preparing a medicament for treating tumors and inhibiting cell cycle kinase activity.

9. A method for preparing a compound of formula I according to claim 1, characterized by comprising the steps:

(b) the compound of formula IV is reacted with 1, wherein is: (1- 1-C6 alkyl), thereby forming a compound of formula V

Among them, 11, CI-C6 alkyl, C1-C6 alkoxy, -C ₂ H,

Wherein, the definitions of R ₃ , R ₃ and R ₄ in each structural formula are as described in claim 1.

10. The method according to claim 9, wherein it is 11, R ₂ is a CH ₂ 0H, R _; and 11 is.