TWI228417B - A composition for inhibiting neoplastic cells - Google Patents

Abstract

A composition for inhibiting neoplastic cells comprising an effective amount of a compound of formula I to inhibit the neoplastic cells. The composition of this invention further comprises a chemotherapy agent.

Description

Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1228417 A7 ^ -------- B7 _ V. Description of the invention () Manj field: _ The present invention relates to a method that can inhibit cancer cells and can be applied to chemotherapy The invention relates to a composition for reversing the multiple drug resistance of cancer cells. Ming background: _ Camptothecin ((20S) -camptothecin, "CPT") is a natural plant base with anti-cancer effect isolated from Chinese plant-camptothecin extract in 1966 (see wa 丨 j, et al. Wang, J. Am. Chem. Soc, (199.6) 8 8: 3 8 8 8). The results show that camptothecins, including their derivatives or analogs, have attracted attention because they interfere with the activity of DNA topoisomerase I in cells. A new generation of anticancer drugs. The functional system of DNA topoisomerase I can unravel or relax the helical structure of DNA. In cancer cells that are continuously replicating, the DNA topoisomerase I is overexpressed and its content is higher than that of normal cells. Many, inhibiting DNA topoisomerase I can trigger a series of cascade reactions in cancer cells that can induce cell death programs, and then cause cancer cell death (see Slichenmeyer α /., J. iVd /. Cancer Inst. (1993) 85: 271; Giovanella eta l.,

Science (1989) 246: 1046). Since camptothecin compounds are mostly pentacyclic, highly hydrophobic aromatic acetic acid compounds, the solubility in physiological conditions is extremely low, which limits the clinical effects of these compounds. In the early days, in order to improve the solubility, even sodium hydroxide was used. Page 4 This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) (Please read the precautions on the back before filling this page)

1228417 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention () The lactone ring on the camptothecin compounds is opened to form the sodium camptothecin, but its anticancer effect is greatly reduced. Subsequent research reports have found that the closed lactone ring is an indispensable structure for the anticancer activity of camptothecin compounds (see Kingsburyeta l., J. Med. C hem., (1 9 9 1) 3 4 : 98, Sawada etal ·, C hem · P harm · Bull., (1991) 3 9: 1 4 4 6; Luzzio et al., J. Med. Chem., (1 9 9 5) 3 8: 3 9 5; Abigerges et al, J. Clin. Oncol., (19 9 5) 1 3: 2 1 0). After the structure and activity test, the two camptothecin analogues, t octopotecan (TPT) and irin otecan (CPT-11), are soluble due to their solubility. It has excellent anti-cancer effect and was first approved by the US Food and Drug Administration for cancer treatment. TPT was first approved as a second-line anticancer drug for epithelial ovarian cancer in 196, and then approved for small cell lung cancer in 1988; c PT-1 1 was in 198 He was approved as the first-line anticancer drug for colorectal cancer in 2000. In addition, it has been found that these compounds can be used in combination with radiation therapy and have an additive effect (Goldwasser M a / ·, c / zi Cwcer 7? ~., (1 9 6) 2: 6 8 7) 〇 Although there are many The research department is working towards preparing camptothecin compounds with good water solubility, but the current trend is still to develop towards fat-soluble formulations. Sugaram et al. Proposed that free-state camptothecin can accumulate in cancer cells to achieve anti-cancer effects (see sugarmane ί α /., Cancer Chemother. Pharmacol ,, (1 9 9 6) 3 7: 5 3 1). In addition, 'camptothecin is known to be rapidly metabolized in the body and loses its anti-cancer activity (Hertzberg w α /., 5 magic cAem Qin " y (1 9 8 9)) Page 5 (Please read the back Note to fill out this page again}

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This paper size applies to China National Standard (CNS) A4 specification (210X297 public love) 1228417 A7 B7 V. Description of the invention (28: 4629; Hsiang and Liu, Cancer Res, (1988) 48: 1 722; Jaxel et al. 9 Cancer Res (1 9 8 9) 4 9: 5077). Therefore, there are a lot of researches now, not for the research direction of preparing water-soluble camptothecin compounds, but to improve the physiological properties of camptothecin compounds. Stability under conditions to promote its bioavailability (see Lavergne w β /, human cAem. (1 99 8) 4 1:54 1 0; B〇mer ./., Y. CAem (1 9 9 9) 4 2: 3 0 1 8). Curran et al., U.S. Patent No. 6,136,978, which was granted on January 24, 2000, proposed a novel, silicon-containing Camptothecin compounds and a method for preparing the same increase bioavailability by adding a silicon substituent to the B ring of camptothecin. In addition, cwun et al., US Patent No. No. 6,207,832 B1, is to expand the E-ring of camptothecin from a six-membered ring to a seven-membered ring, making it more stable under physiological conditions. Within these patents The structure of Camptotheca acuminata and some of its common derivatives, such as decancer and anti-cancer, are as follows: (Please read the notes on the back before writing (This page)

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Page 6 This paper size applies to China National Standard (CNS) A4 (210X297 mm) 1228417 A7 B7 V. Description of the invention (

Topotecan, TPT

P (Please read the precautions on the reverse side before writing this page}, although a bit of cell death; ',: 纟' The literature reveals camptothecin compounds and their anticancer effects inducing death, but it does not disclose Or it is suggested that the effect of such compounds when cancer cells have multiple drug resistance. One of the main factors limiting the clinical use of anticancer drugs is the multiple drug resistance developed by cancer. Platinum ketamine (c 丨 s _ p 丨 at 丨 n), 5-fluorofluorouracil (5-fU〇ur〇Uracil, referred to as "5-FU"), vinca alkaloids (also known as vinblastine) ) And ancycline page 7 This paper size applies to China National Standard (CNS) A4 specifications (2) 0X297 mm

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1228417 V. Description of Invention () A7 B7 (anthracyclines (also known as doxorubicin)) These anti-cancer drugs for cancer treatment will not only cause cancer cells Increased tolerance for these drugs will also develop cross-resistance with other anticancer drugs. In some cases, the patient did not even respond to the initial chemotherapy, and it is generally believed that this situation is due to the endogenous (i n t r i n s 丨 c) resistance of the cancer cells. The endogenous resistance of the cancer cells themselves and the resistance developed by them against various cancer treatment drugs are collectively referred to as multiple drug resistance. Molecular biologists After years of research on multidrug resistance, several genes for proteins associated with multidrug resistance have been identified. These multi-drug resistant proteins include P-glycoprotein (P-gp) (Weinstein d α /, PW / 2 (9 / · (1 9 9) 2 1: 3 4), multi-drug resistance Multidrug resistance associated protein (MRPl) (Cole α / ·, Sc zewce (1992) 258: 1650), the lung resistant protein (LRP) (Scheper et al., Cancer Res. (1993) 53 : 1475) and the breast cancer resistance protein BCRP (Miyake etal., Cancer Res. (1999) 59: 8.

Ross e t a l., J. Natl. Cancer Inst. (1999) 91: 429; Doyle et a L, Proc. Natl. Acad. Sci. USA (1999) 95: 15665). These multiple drug-resistant proteins are basically transmenbrane proteins that can cross cell membranes. The mechanism by which cancer cells become resistant is that they can effectively transport anti-cancer drugs out of the cell membrane or keep them away from resistance. Location of organelles intended for cancer drugs (G 〇11 esma η eta l., J. CI ini c al 〇 nc ο lo gy (1 9 8 9) 7: 4 0 9; page 8 of this paper applies to China National Standard (CNS) A4 specification (210x297 mm) (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1228417 A7 B7 Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs and printed by the Consumer Cooperative (Invention Note) (1) tain et al. 9 J. Nat./ Cancer Inst. δ i · ^ Fojo et al., Cane er Res. (1985) 45; / 0 2). Therefore, the concentration of anticancer drugs in cancer cells can be effectively reduced, so that the cancer cells can survive and grow. A number of compounds are currently known to inhibit the activity of multiple proteins and cause cancer cells to accumulate in their target cells. One believes that these compounds can be used as receptors for multidrug-resistant proteins in the body. By occupying a large number of multidrug-resistant proteins, anticancer drugs will not be sent out of cells, further causing the death of cancer cells. Such first-generation inhibitors that inhibit multiple drug resistance proteins include verapamil, q nidine, and cyclosporine A. Among them, Wu Mai'an is the most clearly studied curse. Wu Mai'an itself is a calcium ion channel inhibitor and is widely used in various clinical applications. The most important one is to treat high blood pupa in clinical research curse ( Gottesman " a / ·, j · (1 9 8 9) 7: 4 0 9), Wu Mai An has a good inhibitory effect on multi-drug resistant proteins and can increase the mortality of cancer cells. But unfortunately, the dose of promethasan required to inhibit multiple drug resistance proteins is too high, and its side effects can cause toxicity to the patient's cardiovascular system, making this drug unable to be further developed and used in cancer patients. Body. Second- and third-generation multidrug-resistant protein inhibitors include PSC 8 3 3 (Krishna μ α / ,, Anticancer Res. (1997) 17: 3329), Gf 1 2 0 9 1 8 (Hyafil et al ·, Cancer Res. (1993) 5 3: 4 5 9 5) ^ VX-710 (Germann er a /.? Anti.Cancer Dap (1 9 9 7) 8: 1 4 1) and LY 3 3 5 9 7 9 (Dantzig β /., Cwcer (1996) 56: 4171). But these inhibitors and page — page 9 This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling and writing this page)

Printed by% 1228417 A7 B7 Printed by the Intellectual Property Office of the Ministry of Economic Affairs and Consumer Affairs Co., Ltd. V. Description of the invention () Substituting inhibitors also have cytotoxicity and are not easy to remove. Therefore, a non-toxic agent that can inhibit multi-drug resistant proteins will make an important contribution to the treatment of cancer patients. Therefore, there is an urgent need to develop a safe and effective multidrug resistant protein inhibitor. Regarding the treatment of multi-drug resistant cancer cells, US Patent No. 5,371,081 discloses a N-substituted phenoxazines compound having a substituent on the nitrogen atom. These compounds are not chemically similar to the camptothecin compounds discussed above. In addition, the patent does not disclose any benefits when using such compounds alone, and the toxicity of this substance may also make it difficult to use it clinically. The most serious problem in traditional chemotherapy is the toxicity of the chemotherapeutic agent itself. Typical chemotherapeutic agents' such as cis-chloramine platinum 丨 "丨. Has some unavoidable side effects, such as hair loss, weight loss, damage to the liver and kidneys, etc. Because of the high toxicity, it is not possible to administer this kind of medicine for a long time every day. Generally, the administration of this kind of medicine is several days a week , Continuous administration for two to three months, followed by a few weeks of non-administration, and then repeat the drug administration process. Therefore, no traditional anticancer drug or a drug that can enhance the anticancer effect is non-toxic. In short, None of the previous techniques have disclosed or suggested a compound that can kill cancer cells without causing side effects and non-toxicity in cancer cells with multiple drug resistance. The present invention provides a method for inhibiting cancer cells. And you ^ ^ ^ ^ Jiu Congjin treated with chemotherapy The page size of this paper applies Chinese National Standard (CNS) A4 (210X297 mm) (Please read the note on the back first (Fill in this page again)

1228417 A7 B7 V. Description of the invention (the effect of cancer cells on the human body θ θ '1 " is particularly a composition that can reverse the multiple drug resistance of cancer cells. Purpose and sincerity of the invention: Derivatives are used to treat cancer, especially in cancer cells with multiple drug resistance, using camptothecin derivatives as the main chemotherapeutic agent or as an adjuvant to other chemotherapeutic agents to treat cancer and kill cancer cells. One object of the present invention is to provide a composition capable of inhibiting cancer cells, which uses camptothecin derivatives as the main chemotherapeutic agent to achieve the purpose of reversing the multiple resistance of cancer cells and inhibiting cancer cells. Another object is to provide a composition that can promote the therapeutic effect of multi-drug resistant carcinogen therapy, which is used to reverse the multi-drug resistance of cancer cells by using a camptothecin derivative as an adjuvant to its chemotherapeutic agent. And the purpose of suppressing cancer cells. One of the main aspects of the present invention is to provide a composition capable of suppressing cancer cells, which comprises an effective amount of a compound of formula (j) (Please read the notes on the back before filling this page)

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1228417 A7 B7 V. Description of the invention ()

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and a pharmaceutically acceptable carrier. In the above composition, the compound of formula (I) is (20S) -10-hydroxy-7- (tert-butyldimethylsilyl) camptothecin (abbreviated DB-67). Even if the cancer cells have multiple drug resistance, the above compounds can effectively inhibit cancer cells. The above compounds include the camptothecin derivatives disclosed in U.S. Patent No. 6,136,978 to Curran et al., The entire contents of which are incorporated herein by reference. However, in Patent No. 978, only the general anticancer activity of these insoluble camptothecin derivatives (anti a c t i v i t y) 'is not suggested or disclosed for the therapeutic effect of these compounds on cancer cells with multiple drug resistance. The applicant of the present invention has found that 'the camptothecin derivative described above unexpectedly exhibits a higher anticancer activity against cancer cells having multiple drug resistance compared to similar derivatives currently used. It can effectively treat cancer cells including human oral epithelial cancer cells, human uterine cancer cells, human breast cancer cells, human colorectal cancer cells, human lung cancer cells, human liver cancer cells, human gastric cancer cells, and human nasopharyngeal cancer cells. Page 12

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1228417 A7 B7 V. Description of the invention () Furthermore, the camptothecin derivative of the present invention can be used as an adjuvant to other chemotherapeutics in addition to being a main chemotherapeutic agent. By administering camptothecin derivatives together with other chemotherapeutic agents to patients with cancer, especially for cancer cells with activated multidrug resistance genes, the camptothecin derivatives described above and other such as When used in combination with chemotherapeutic agents such as cis-aminoplatin, 5-fluorouracil, vinblastine, and androidmycin, synergistic effects (syneTgism) that enhance anti-cancer effects are shown. Therefore, another important object of the present invention is to provide a composition capable of enhancing the therapeutic effect of cancer cells with multiple drug resistance. The composition is an effective amount of a compound of the formula Acceptable salts. The aforementioned more suitable compounds and cancers can be included in this composition. The camptothecin derivatives of the present invention cause cancer cells to die by accumulating a large amount of anticancer drugs in these cancer cells, and are therefore very effective in reversing human cancer cells and drug-resistant cancers. Detailed description of the invention: The inventors of the present case found that the camptothecin derivative of the present invention can be applied to the body to suppress cancer cells. Unless otherwise stated or defined, the term "inhibition (1 n h 1 b i t i ο η)" as used herein refers to inhibiting the growth of cancer cells and / or killing cancer cells. Our research shows that when camptothecin derivatives are applied to naked gas with human cancers, they can 'inhibit cancer development by about 70-75% (under the experimental conditions of the present invention)' i.e. 'in animals Without significant change in body weight, it effectively reduces the volume of cancer. Moreover, this medicine is used alone or with other treatments. Page 13 This paper scales _ ⑽) Α4 ^ (21 () χ 297 mm) (Please read the precautions on the back before filling this page) Pack.

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. 1228417 A7 _ B7 V. Description of the invention () method is applied together, even for cancer cells with multiple drug resistance, the drug is still effective. As discussed in the foregoing background to the invention, one of the main factors limiting the clinical application of anticancer drugs is the resistance that cancer cells develop to them. Recent studies have pointed out that two mechanisms that make cancer cells develop resistance include (i) replication and amplification of multiple drug resistance gene families; and (π) inhibition of the activation of the cell death program (apoto sis). In cells with large multiplicity of resistance genes, the mechanism of drug resistance mainly lies in the ability of anticancer drugs to be continuously pumped out of cells through multiple drug resistance proteins to reduce the concentration of anticancer drugs in cells. The second major drug-resistance mechanism in cancer cells has only recently been discovered. This is achieved by inhibiting the initiation of the cell death program. This not only enables cancer cells to fight chemotherapy but also cancer cells to resist radiation therapy ( Desoize w β / ·, Xin Xin [, (1 994) 14.221). It is known that the protein that can inhibit the cell death program is the product of the B c 1-2 gene. This protein is expressed in large quantities in cancer cells that are resistant to drugs and radiation. Many rapid and unique molecular events occur in cells undergoing a cell death program, such as the aggregation of nuclear chromatin, changes in cell size, and activation of endogenous nuclear enzymes, all of which can lead to the generation of DNA fragments. Multi-drug resistant cancer cells have the activity to inhibit the cell death program. This result greatly increased the proportion of 4 cancer cells that were killed in the cancer cell death analysis test. Our research results show that camptothecin derivatives can effectively increase the sensitivity of anti-cancer drugs to anti-cancer cancer cells and produce antisense effect. Therefore, this drug has great potential for cancer treatment in the future. . Page 14 This paper ruler National Standard (CNS) A4 size (210x 297 public love) (Please read the precautions on the back before copying this page)

1228417 A7 B7 V. Description of the invention (The following will be discussed in more detail. It is generally believed that the composition of the present invention can effectively inhibit cancer cells because of its advantages in overcoming the resistance mechanisms of the above two types of cancer patients. Camptothecin Derivatives The camptothecin derivative of the present invention is a compound tBu having the structure of the following formula I

(I) (Please read the notes on the back before transcribing this page)

The name of the camptothecin derivative of the formula (I) printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is (20s) _ 丨 〇_ hydroxy-7- (tert-butyldimethylsilyl) ) Camptothecin (abbreviated db_67). A compound having a structure similar to the present invention, i.e., a compound having an aromatic pentacyclic structure similar to camptothecin alkaloid, was not found to have any significant effect on inhibiting drug-resistant cancer cells. Take the structure # db_67 that is almost the same, except that an extra carbon repulsion on the E ring has been reversed and changed from ό member ring to 7 member% D Β-9 1 as an example. Its beneficial voice side I # and t _ total … Both in the tank and / or in vitro cytotoxicity tests' are not capable of reversing multiple drug-producing drugs, which can produce proteins and inhibit cancer cells. Therefore, such a property of inhibiting drug-resistant cancer cells in pharmacy appears only in the compounds defined by the present invention. The structure of DB_91 is shown below: Page 15% 1228417 A7 B7 V. Description of the invention (

0 Preparation of Camptothecin Derivatives The compound of formula I according to the present invention can be disclosed in the approved and granted US Patent No. 6,136,978, with the title of "Shi, Bismuth is an amygrine analog and its preparation method (Camptothecin AnalQ2S Λ δ And Methods of Preparation Thereof) ", the entire contents of which are incorporated herein by reference.

Curran et al. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Pharmaceutical application In accordance with the present invention, a step-by-step aspect of the present invention 'The drug capable of inhibiting cancer cells is a drug containing a compound of formula I as its active ingredient. The compound of formula I of the present invention has been recognized as a medicine for suppressing cancer cells because of its extraordinary effect on reversing the multiple drug resistance of cancer cells. The compound of Formula I and its pharmaceutically acceptable dosage will vary depending on the course of cancer development, the type of cancer, the degree of its multiple drug resistance, and the chemotherapy used in combination. Generally speaking, it can be about 〇 · 〇 i m g / K g to page 16 First please read the notes on the back #Fill this page)

1228417 A7 B7 V. Description of the invention () 100 mg / K g is applied to the patient, preferably in an amount of about 0.05 mg / K g to 75 mg / Kg, in order to effectively inhibit cancer cells and Reverse its multiple resistance. Applicable cancer cells are human oral epithelial cell lines (KB cell lines) and drug-resistant subtype cell lines (including KB · 100, KB-VinlO, KB-Taxol, and KB-7D), and human uterine cancer cells. (MES-SA and MES-SA / DX5), human liver cancer cells (HepG2), human breast cancer cells (MCF7), human lung cancer cells (A-549), human nasopharyngeal carcinoma cells (HONE-1 / CPT30), human large intestine Rectal cancer cells (SW480) and human gastric cancer cells (MKN-45). As for the formulation of various medicines, a compound of formula I is generally mixed with a pharmaceutical carrier to prepare a pharmaceutical composition. Examples of suitable pharmaceutical carriers include diluents or carriers (such as fillers, adhesives, disintegrating agents, and lubricants). The dosage of this medicine can exist in various injection, powder, capsule, granule, tablet or injection vial types. In the case of tablets, the carrier used can be selected from the following, such as lactose, sucrose, sodium chloride, glucose solution, starch, calcium carbonate, cellulose crystals or silicic acid; adhesives such as water, ethanol, Propanol, glucose, starch solution, gelatin solution, methylcellulose carbonate, methylcellulose or potassium phosphate; decomposing agents such as dried starch, sodium bath, gel powder, sodium bicarbonate, calcium carbonate, hard Glycerol fatty acid, starch or lactose; or lubricants such as stearates, boric acid powders or well-known polyethylene glycol solids. The tablets can be coated with sugar or gelatin or film if necessary. In the case of injection, the diluent used can be selected from, for example, page 17 The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling (This page)

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V. Description of the invention (1228417, such as 7, ethanol m-oxy sorbate, ethyl sorbate, or liposomes. In this case, 'a sufficient amount of chlorination, glucose or glycerin can be added to form an isotonic solution. Commonly added substances that can help to dissolve, buffer solutions, pain-relieving drugs or preservatives are added. The camptothecin derivatives of the present invention can also be used alone or combined with chemotherapeutic agents, or their chemotherapeutics such as radiation therapy. Use. When the Camptothecin derivative of the present invention is used in combination with a chemotherapeutic agent such as cis-chloramine, 5-fluorouramine, changchun, and androidmycin, it exhibits a synergistic anti-cancer effect. Derivatives of cucumine can be administered at the same time as the chemotherapeutic agent, and the camptothecin can also be administered in a uniform pharmaceutical form with any chemotherapeutic agent. The present invention will be described by the following specific examples and text descriptions, but this The scope of the invention is not limited thereby. The test compounds are synthesized by the methods disclosed in the literature. The purpose of the examples is to show compounds of formula And reverse the extraordinary effects of its multiple drug resistance. Yi_ 1: DB-6 7 withered when alone. Method DB-67 (20S) -10 -hydroxy-7- (tert-butyldi) Methylsilyl) camptothecin ((2〇S) -10-hydroxy- (ieri-butyldimethylsilyl) camptothecin)) Page 18 This paper applies Chinese National Standard (CNS) A4 (210X297 mm) (Read the notes on the back and fill out this page)

Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1228417 A7 B7 V. Description of Invention () tBu

〇 Printed by the Consumer Property Cooperative, Member of the Intellectual Property Bureau of the Ministry of Economics. In this experiment, two methods of in vitro and in vivo toxicity analysis were used to measure the anticancer activity of the compounds of the present invention. ! · Vitrorn ^ C ^ LLotoxicUy measurement): The cultured drug-resistant cancer cells were used for the cytotoxicity test. Cultivate in a culture dish with 24 culture wells at a density of 5,000 cells per ml per well until the cells exhibit logarithmic growth, and the drug-resistant cells are tested as if 'systematic. Transgenics were grown in drug-free growth medium for 3 days. It was then treated with various test drugs for 72 hours. After drug treatment, the effect of test compounds on cell growth was evaluated by methyl blue staining (Finiey G. J. er α /., U984) (139: 272) and the number of cells remaining in each culture well was confirmed Then, draw the residual cell curve from the experimental data and calculate its IC50 (the concentration of the drug when it reaches 50% lethality). 2. Colony analysis: Human cancer cells are directly implanted into nude mice. Page 19 This paper is based on paper towels_ 绪 ^^ ϋΠ_Κ) × 297 mm) (Please read the precautions on the back before filling in this page}

Order · 1228417 A7 B7 V. Description of the invention () Subcutaneous test for inhibiting the growth of cancer. When the cancer begins to grow under the skin of nude mice, it is treated with the compound of the present invention. The advantage of this analytical method is the ability to test the effects of chemotherapeutic agents in vivo. Sixty-week-old male nude mice weighing approximately 20 to 24 grams were inoculated with 1 x 106 human gastric cancer cells MKN45 or human colorectal cancer cells SW480 expressing p-coprotein. After allowing the cancer cells to grow subcutaneously in nude mice for about 2 weeks, when the cancer volume reaches 50_1 (0) cubic millimeters, the doses of 3- 10 mg / K: g per animal per day are subcutaneously > Administer db-67 by main shot (SC) or intravenous (IV). One administration period is 21 days. 'Each cycle is administered for 5 consecutive days in the first 5 days only. After two administration cycles, the size of the colonized cancer and the animal weight are measured and compared with the data of the control group without treatment. Cell lines used All cell lines used in the present invention are from the American Type Tissue Culture Laboratory (Japanese Type Research Culture Collection Center) and their own research. Developed human cancer cell lines' were cultured according to their own methods. The following are the cell lines and tissue sources used in this experiment: (Please read the precautions on the back before filling this page)

Printed by the Employees 'Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Page 20 This paper size applies to the Chinese National Standard (CNS) A4 specification (210X 297 mm) A7 B7 Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1228417 V. Invention Description ()

Editing cell line tissue notes -----— 1 KB oral cavity 2 KB-100 oral cavity can resist CPT 3 KB-VinlO oral cavity can resist vincristine, showing excessive p-glycoprotein 4 KB-Taxol oral 5 KB-7D oral cavity Anti-VP-16, MRP showing overdose 1 6 MES-SA Uterus 7 MES-SA / DX5 Uterus 8 A-549 Lung 9 HepG2 Liver 10 MCF7 Breast 11 MKN45 Stomach 12 SW-480 Colorectal manifestation p -Cool protein 13 Hone- 1 / CPT30 Nasopharyngeal cancer cells can resist CPT results 1) DB-6 7 and cancer cell in vitro toxicity test analysis: Cancer cell toxicity test analysis is very important for the in vitro analysis of the anticancer activity of compounds. These cancer cells have undergone a long period of time. (72 hours) treatment with anticancer drugs. Table 1 shows the effects of various anticancer drugs (including camptothecin, TPT, CPT-11, and D B-67 of the present invention) on cancer cell lines. When DB-6 7 is used alone, it can effectively inhibit humans at lower concentrations than Camptothecin and two other FDA-approved anticancer drugs (TPT and CPT-1 1). Page 21 This paper size applies to China National Standard (CNS) A4 (210x297 public love) '' (Please read the precautions on the back before filling this page}

1228417 A7 B7 V. Description of the invention () Cancer cells (oral epithelial cell lines KB, KB-100, KB-VinlO, KB-Taxol and KB-7D, uterine cancer cell lines mes sa and mes_ SA / DX5 'Hepatoma cell line HepG2 The breast cancer cell line MCF7, the lung cancer cell line A-549, the colorectal cancer cell line SW480 and the gastric cancer cell line MKN-4 5) have more significant anticancer activity. In addition, 'Compared to the effect of the compound DB_67 of the present invention on drug-resistant cancer cells, the compound DB-9 which has the same aromatic pentacyclic structure as DB-67' has been tested in vitro for cytotoxicity. Does not have the ability to inhibit cancer cells. DB-91 against KB, KB-100, KB-7D, KB-VinlO, KB-Tax () 1, MES_SA / DX5 1 (: 5 of each cancer cell line. 78, 1, 93, 917, 917, 4,158, 450, 134, and 1.065 nM, which are much higher than DB · 67, and are almost the same as those in the control group. Therefore, the property of suppressing drug-resistant cancer cells pharmacologically appears only in (Please read the notes on the back before filling out this page} Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, page 22 This paper is sized to the Chinese National Standard (CNS) A4 (210X297 mm) ) 1228417 A7 B7 V. Description of the invention (Table 1) Table 1 Analysis results of in vitro toxicity test of camptothecin and its derivatives against cancer cells Cancer cell line IC50 (nM) camptothecin TPT CPT-11 DB-67 KB 55 45 9300 35 KB-100 1,286 2,100 ND 750 KB-VinlO 380 1,134 66,000 2,271 KB-Taxol ND ND ND 50 KB-7D ND ND ND 95 MES-SA 2 81 6,431 3 MES-SA / DX5 2 182 > 20,000 35 A- 549 56 520 13,437 87 HepG2 17 45 2,280 3 MCF7 80 369 11,586 105 MKN45 2 6 6,431 2 SW-480 68 395 > 20,000 65 ND: Unknown (Please read the notes on the back before filling in this page)

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Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 2) In vivo tests, the effect of DB-67 on the growth of human colorectal and gastric cancer cells: Human cancer cells were implanted subcutaneously in nude mice. This analysis method provides a A convenient system to study the effects of drugs on the growth of cancer cells in the body. Colorectal and gastric cancer cells were used to test the effect of DB-6 on the growth of the cancer system. In two doses of DB-67 (3 mg / K g and 10 mg / K g) by intravenous or subcutaneous injection respectively, the Chinese National Standard (CNS) A4 specification (page 4) applies to this paper standard ( 210X297 mm) 1228417 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention () 2 groups of animals (9 animals per dose, implanted into colorectal and gastric cancer cells) for testing. As shown in Figure 丨 and Figure 2, the degree of tumor volume reduction in mice was related to the dose of DB-67. Referring to Figure 1, subcutaneous injection of 3 mg / Kg of DB-67 does not have much effect on reducing the volume of cancer. However, when the DB-6 67 dose is increased to 10 mg / κg, it has obvious antitumor effect. . However, if DB-67 was administered by intravenous injection, the effect was even more significant. On the 75th day after the administration, it inhibited the growth of human colorectal rectal tumors (ie, tumor volume reduction) exhibiting pgp by as much as about 70%. Similarly, the antitumor effect of DB-6 was also seen in nude mice implanted with gastric cancer cells. DB-67 at 10 mg / Kg inhibited tumor growth by about 75% 45 days after administration (see Figure 2). This test is the result of a separate test of d B-6 7 without any other related anticancer drugs. These results confirm the potential of the compounds of the present invention as a new generation of anticancer drugs. Conclusion In Example 1, when D B-67 was used alone, it could show excellent anti-cancer activity against selected multi-drug resistant cancer cells. Yiyi blanket example 2: DB-6 6 synergistic anti-cancer methods and results to promote the effects of other chemotherapeutics. The isobologram analysis revealed by Berenbaum was used to determine the synergistic resistance of DB_67 when used with other chemotherapeutics. Cancer activity (See Berenbaum, page 24. This paper size applies Chinese National Standard (CNS) A4 specification (210X297 public love) " " " (Please read the precautions on the back before filling this page))

1228417 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 5. Description of the invention () (198 1) Mv. And 3 · 5: 2 6 9 -29〇). This analytical method has been widely used in various fields to evaluate the synergistic effect or resistance effect of biologically active substances or agents. During the operation, first determine the effectiveness of an active substance (in this experiment, DB-6 7, cis-chloramine platinum, or 5-fluorouracil) to inhibit the growth of cancer cells alone, and then combine them under the same biological effect. Different doses of different active substances, observe the combined dose used to inhibit the growth of cancer cells. If the dose curve of the combined use shows a concave-up phenomenon, it means that the combined agents have a synergistic effect with each other. The contrary ' The dose curve when combined use shows a concave downward (c ο nca V e-d 0 w η) phenomenon, which means that the combined agents are resistant to each other. If the combined agents are used together, 'Don't interact', the resulting dose curve will be a straight line, indicating that they only have an additive effect, not a synergistic effect. The cell line used The cell line used in the previous analysis method is for camptothecin The drug resistance of human nasopharyngeal carcinoma cell line (Hone-1 / CPT30) is caused by the large number of multi-drug resistant proteins. The results are shown in Figure 3. It shows that whether it is cis-chloramine platinum (Figure 3a) or 5-fluorouracil (Figure 3B), when it is used in combination with DB-6 7, the dose curve shows a concave upward phenomenon, which represents the combined use of DB- 6 7 and cis-chloramine platinum or combined use of DB-67 and 5-fluorouracil, the combination of these two drugs page 25 (Please read the precautions on the back before filling in this page j

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This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1228417 A7 B7 V. Description of the invention () There is a synergistic effect between each other, which can further inhibit the growth of cancer cells with multiple drug resistance. Conclusion Camptotheca derivatives can effectively kill cancer cells when used alone, but when used with standard chemotherapy agents such as cis-chloramine platinum and 5-fluorouracil in resistant cancers, they can show more significant The anti-cancer activity is the best example of this phenomenon. Therefore, in clinical trials, first, a compound of one of the camptothecin derivatives of the present invention is applied to cancer patients as a main chemotherapeutic agent (regardless of the presence of multiple drug-resistant proteins). When this compound is used alone When no effect can be achieved, the b chemotherapy procedure can be introduced as an adjuvant treatment method, such as adding other chemotherapeutic agents and administering the camptothecin derivative according to the present invention. (Please read the notes on the back before filling this page}

Those who print the knowledge of temple effects in the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs should understand that without exceeding the scope of the present invention, they can make more changes or improvements to the present invention, and such equivalents should be regarded as Still within the scope of the invention. The embodiments herein are only used to illustrate the present invention, and the scope of the present invention is not limited thereto. The scope of the invention is defined by the scope of the accompanying patent applications. Brief description of the figure: Figure 1 shows the relationship between the change in cancer volume and the dose of DB-6 in nude mice with or without DB-67 treatment. Page 26 This paper size applies to China National Standard (CNS) A4 specifications (210X297 public f

1228417 A7 B7 V. Description of the invention (2) Figure 2 shows the relationship between the change in cancer volume and the dose of DB-6 in nude mice with or without DB-67 treatment. Figures 3A-3B show the combination of DB-67 and cis-chloramine platinum (Figure 3A) or the combination of DB-67 and 5-fluorouracil (Figure 3B) for the treatment of human nasopharyngeal carcinoma cells (Hone-I) , Its synergistic anti-cancer effect. (Please read the notes on the back before filling this page}

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Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, page 27. This paper size applies to China National Standard (CNS) A4 (210X297 mm)

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1228417 | D8 6. The scope of patent application is between 100mg / Kg. 5. The composition according to item 1 of the scope of patent application, wherein the content of the compound of formula I is more preferably between 0.5 mg / Kg per day and 70 mg / Kg per day. 6. The composition according to item 1 of the scope of patent application, which may further comprise a chemotherapeutic agent, the chemotherapeutic agent is selected from the group consisting of cis-gas amine platinum, 5-fluorouracil bite, periwinkle test, and Anhuan In the ethnic group. (Please read the notes on the back of IH before Θ this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Page 29 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)