TWI228045B - Vascular damaging agents - Google Patents

Abstract

Colchinol derivatives of formula wherein R1, R2, R3 and R6 are each independently H, optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, alkanoyl, PO3H2; X is carbonyl (CO), thiocarbonyl (CS), methylene (CH2) or a group CHR4 R4 is OH, O-alkyl or NR8R9; R5 and R7 are each independently H, alkyl, halogen, hydroxy, alkoxy, nitro or amino; R8 is H, optionally substituted alkyl, cycloalkyl, alkanoyl, thioalkanoyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, arylaminocarbonyl, alkylsulphonyl, arylsulphonyl, aminosulphonyl, alkylaminosulphonyl, dialkylaminosulphonyl or arylaminosulphonyl; and R9 is H, alkyl or cycloalkyl and the pharmaceutically acceptable salts, solvates, and hydrates thereof have been found to be useful for treatment of diseases involving angiogenesis. Some of these compounds are novel. Particularly preferred are those compounds in which R6 is PO3H2.

Description

1228045 V. Description of the invention (1) The present invention relates to a vascular disrupting agent, specifically a group of colchicine derivatives formed by the therapist, some of which are novel compounds that are now formed by blood vessels to form new organs. Is a key feature of some diseases (J Folkman, The New England Journal of Medicine, Physics 3 3 3, 1 7 5 7 (1 9 9 5)). For example, for a solid tumor to grow, it must develop its own blood supply, which mainly relies on its supply of oxygen and nutrients; if this blood supply is mechanically interrupted, the tumor is necrotic. Neovascularization is also a clinical feature of pruritus skin lesions, invasive vascular crests and atherosclerotic plaques in the joints of patients with rheumatoid arthritis. In macular degeneration and diabetic retinopathy, retinal neovascularization is pathological. In all of these diseases, the damage can be reversed by destroying the newly formed endothelial endothelium — "with beneficial medical effects. Derivatives of blood s into a pre-colchicol alcohol, such as N-ethyl colchicol, have been noted, and tumor effects have been noted in animal models [see, for example, JN. Journal of the Institute of Anti-Swelling, pp. 379-3, 92, 1952, 13: 1. The effect of cancer in the mysterious country is on the general damage (hemorrhage, softening, and necrosis). The study of the destruction of new blood vessel formation to treat inappropriate blood vessel formation has no suggestion for Tongqiao according to the following substructure.

A search of "Chemical Abstracts Years Later" reveals 1228045 V. Description of the invention (2) Any one of the binding agent is expected: anti = cancer and anti-cancer activity, and stabbing, so it is related to swollen alcohol. These compounds are neutral because tubulin cells have a direct effect. In the course of the work of the present invention, issues related to the possible effectiveness of tubulin-binding disks as anti-vascular agents were investigated, but no predictability was found. So Doce 独 axe 1 ("The Lancet", 344 ,,,

1 2 6 7- 1 2 7 1, 1 9 9 4), a tubulin-binding agent, has no vascular disruption effect even when it is administered at a tolerable dose. Even when Z = Z is equivalent to some compounds related to the present invention, it is found that the medical window [ratio of the most effective dose (MTD) to the minimum effective dose (MED)] has too much potential clinical effect. According to the present invention, there is provided a composition for treating diseases including blood & formation using a colchicol derivative, wherein the colchicol derivative has

(Wherein each of the alkyl groups h and I is hydrogen, and optionally substituted alkyl, cycloalkenyl, sulfo, alkynyl, calcined and phosphono, 1228045 V. Description of the invention (3) X is carbonyl (C0), thiocarbonyl (CS), methyl (CH2) or CHR4 groups; R4 is via group, 0-alkyl or NR8R9; one of R5 and R7 is nitrogen, alkyl, halogen, via, An alkoxy group, a schottyl group or an amine group, with a nitrogen atom as the substituent, an optionally substituted alkynyl group, a bad alkynyl group, an alkynyl group, a thioalkanoate group, an aryl group, a heteroaryl group, an aryl group, a heteroaryl group, Oxyalkyl, aryloxyzoyl, amidyl, alkynyl, dialkyl, arylamino, arylamino, arylamino, arylamino, amine Amine continuous, dialkylaminesulfonyl or arylaminesulfonyl; R9 is hydrogen, alkyl or cycloalkyl) and pharmaceutically acceptable salts, solvates and hydrates thereof. It is believed that although this is not limited to the present invention, the use of the compounds of the present invention destroys newly formed blood vessels, such as those of tumors, and thus effectively reverses the angiogenesis process. Compared with known anti-angiogenic agents, once the blood vessels have been Formation, the latter tends to be less effective. ‘Some of these compounds are new. In a specific context, the novel compounds are those of formula I, wherein at least one of h, R2, R3 and R6 is phosphate. In a specific preferred embodiment, R6 is acid-filled. The specific preferred compound is defined by formula

1228045 5. Description of the invention (4) (wherein each of R, R2 and R3 is hydrogen, optionally substituted alkyl, cycloalkyl, fine group, bulk group, burned group or acid filling group, R6 is phosphate group; R4 is hydrogen or NR8R9; each of R5 and R7 is nitrogen, alkynyl, hydrogen, alkynyl, sulfonyl, or amine; respectively, alkynyl, lactoyl, ammonyl, sulfur Burning group, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, aminecarbonyl, alkylaminecarbonyl, dialkylaminecarbonyl, arylamine group, burnout Acid group, aryl group, amine group, amine group, diamine group or diaryl group and S &group;

Rg is nitrogen, basal or soil basal) and pharmaceutically acceptable salts, solvates and hydrates thereof.

In another aspect of the invention, the novel compounds have formula II A

(Wherein each of R, R2 and R3 is hydrogen, and optionally substituted alkyl, cycloalkyl, fine, fast-based, courtyard or acid-filled,

1228045 5. Description of the invention (5) R6 is hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, or phosphate; hydrogen or NI Rg; each of R5 and R7 is a rat, an alkyl, Halogen, nitro or amine, R8 is hydrogen, optionally substituted alkyl, cycloalkyl, alkylfluorenyl, sulfanyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl , Aryloxycarbonyl, amine carbonyl, alkylamine carbonyl, dialkylamine carbonyl, arylamine carbonyl, continuous acid group, aryl continuous group, amine group, burned amine group, dioxane R9 is hydrogen, alkyl, or cycloalkyl, and the restriction is that, when R2 and R are all methyl, and R4 is hydrogen, acetamino, Acetylamido, amine, amidino or diamidoamine, etc., so R6 is not hydrogen, amidino, hydroxyethyl or ethoxyethyl ') and its pharmaceutically acceptable salts, solvates And hydrate. The compounds used in the present invention and preferred compounds of the present invention are those in which h, R2 and R3 are alkyl and those in which R4 is amidino. As used herein, the term \\ alkyl〃 (including any aliphatic structural chain associated with an alkyl group) means a straight or branched chain group containing one to seven (most preferably four) carbon atoms Groups such as fluorenyl, ethyl, propyl, isopropyl, butyl, second-butyl, t-butyl, and pentyl. Alternative substituents that may be present on the alkyl group include one or more substituents selected from the group consisting of: halogen, amine, monoalkylamino, dialkylamino, hydroxyl, alkoxy, alkylthio Group, alkanesulfonyl, amido, alkoxycarbonylamino, alkyl

Page 10 1228045 V. Description of the invention (6) Group, methoxy, carboxy, sulfate or phosphate. Examples of alkoxy are fluorenyloxy, ethoxy, propoxy, isopropoxy, butoxy and t-butoxy. The special term% 'halogen' means that it is fluorine, chlorine, bromine or Angstrom. Alkenyl is an alkenyl group containing two to seven carbon atoms, such as fluorenyl, ethynyl, η- phenylene, i-phenylene, η-butylene, i-butylene, s-butylene Butyl and t-butylene. An alkynyl is a group of 2 to 7 carbon atoms, such as ethynyl, propynyl or butynyl. The term aryl, alone or in combination, means an unsubstituted alkyl group or an equivalent group having one or more (preferably one to three) substituents. Examples of the substituents are halogen, alkyl, haloalkane Group, hydroxy, nitro, cyano, amine, and alkoxy. A haloalkyl group may have one or more halogen atoms, and examples of such groups are trifluorofluorenyl and dichloromethyl. The term heteroaryl is defined herein as a mono- or bi-cyclic aromatic group containing one to four heteroatoms selected from nitrogen, sulfur or oxygen atoms in any combination and up to 9 carbon atoms. Examples of heteroaryl include pyridyl, pyrimidinyl, furyl, thienyl, pyrrolyl, pyrazolyl, indolyl, bisfuranyl, benzothienyl, benzothiazolyl, oxazolyl, isopropyl Oxazolyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, quinolyl, and isoquinolyl. The term aralkyl is defined herein as one of the hydrogen atoms g in the alkyl (as defined above) being replaced by an aryl or heteroaryl (defined herein). When one or more of the functional groups in the compounds of formula I, Π, Π A are sufficiently basic or acidic, it is possible to form salts. Suitable salts include pharmaceutically acceptable salts, such as acid addition salts (including hydrochloride, hydroxamate, hyaluronate, sulfate, bisulfate, sulfonate, aromatic salt, acetate ,

1228045 V. Description of the invention (7) Annual osmate, citrate, maleate, fumarate, succinate, lactate and oleate), salts derived from inorganic bases [including alkali metal salts (Such as sodium salt or _ salt) and soil test metal salts (such as town salt or # 5 salt)] and salts derived from organic amines (such as morpholine, hexahydropyridine or diamine). Those skilled in the art will recognize that compounds of Formula I, Π, Π A may exist as stereoisomers and / or geometric isomers, and thus the present invention includes all such isomers and mixtures thereof. A useful group of compounds includes those whose centers, R2 and R3 are each alkyl. Another group of useful compounds include those in which each of h, R2 and R3 is an alkyl group and each of R5 and R7 is hydrogen. A particularly useful subgroup of this group includes compounds in which each of R, R2, and R3 is fluorenyl and R6 is hydrogen, alkyl, or acid-filled. Particularly useful compounds according to the present invention include: N-acetamcolchiol-0-fill salt and its salts, solvates and hydrates. Compounds of formula I, Π or Π A can be prepared in a number of ways, and the general description is as follows, more specifically described in the examples below. In the detailed description of the following method, unless otherwise specified, when used in the formulae described, it can be understood as representing the above-mentioned groups of formula I, Π or ΠA on behalf of B. In the scheme described below, it is necessary to use a protecting group and remove it at the final stage of the synthesis. For skilled artisans, the proper use of such protecting groups and their removal is obvious. Therefore, according to another aspect of the present invention, wherein each of R5, R6 and R7

Page 12 1228045 V. Description of the invention (8) Compounds of formula II or formula ΠA which are all hydrogen can be prepared by treating compounds of formula (2) with basic hydrogen peroxide. In a temperature range of, for example, 0-100 ° C (preferably at or near 60 ° C), in the absence or presence of a co-solvent (such as an alcohol, such as ethanol), the reaction can be carried out in the presence of hydroxide This is conveniently done in aqueous solution.

(2) The intermediate of formula II or IA can be prepared by acidolyzing the compound of formula (3). At elevated temperatures (e.g., 100 ° C or close to 1000), the reaction can be conveniently performed in an aqueous acid such as hydrochloric acid.

OHe OH (3) (2) Compounds of formula (3) are known or can be prepared from colchicine in a conventional manner. Compounds of formula I, Π or π A may also be chemically modified from other formulas I, Π

Page 13 1228045

5. Description of the invention (9) or ΠA compound preparation. Examples of such chemical modifications that can be applied are # quasi-alkylation, arylation, heteroarylation, fluorenation "do not use, thiolization, sulfonation, sulfation, acidification Effects, aromatic functionalization and coupling reactions. These reactions can be made to add new substituents or modify existing substituents. 4. In compounds of formula j, n or π A, existing substituents can be, for example, oxidation, Modification by reduction, elimination, hydrolysis or other cleavage reactions to obtain other compounds of formula jn 戋 Π A. Therefore, for example, compounds of formula π or π A containing an amine group can be used at a temperature range of, for example, -300 to 120 ° C. (Usually at or near room temperature) in a solvent such as a hydrocarbon solvent (such as dichloromethane) in the presence of a base (such as a third amine test such as triethylamine) on an amine group such as amidino The amine group of the compound of the formula π or Π A may be, for example, in the temperature range of -3 0 ° C to 1 2 0. (: generally At or near room temperature) For example, a solvent such as a hydrocarbon solvent (e.g., dichloromethane) in a solvent (e.g., a third amine base such as triethylamine) is treated with, for example, an alkyl or aryl group, chloro, or an alkyl or aryl group anhydride, and is continued. In another example, a compound of formula Π or Π A containing a hydroxyl group can be converted to the corresponding bis-tertiary-butyldiethylphosphine by treatment in the presence of a suitable catalyst (such as tetrazole). Hydrogen phosphate. In its temperature range of -40 to 40 (usually at or near room temperature), in a bath such as an ethereal solvent, followed by a temperature range of -78 ° C to 40 Inside (at-40 to -10 ° c, Xiao Fang Jia) is treated with an oxidant (such as 3-chloroperoxyfengying acid). The resulting intermediate

Page 14 1228045 V. Description of the invention (ίο) Phosphate triesters in a temperature range of -30 to 40 ° C (usually at 0 t: or close to 0 ° C) in chlorinated solvents (such as dichloromethane) Treatment with an acid such as trifluoroacetic acid to obtain a compound of formula (2) containing a dihydrogen phosphate. In another general example, compounds of formula (2) containing acid amines can be hydrolyzed at elevated temperatures (typically at reflux temperature) in a solvent such as an alcohol (e.g. methanol) with, for example, an acid (e.g. hydrochloric acid). In another general example, the 0-alkyl group can be cracked to the corresponding reaction with boron trioxide in a solvent such as a chlorinated solvent (such as dichloromethane) at a low temperature (for example, about -7 ° C). Alcohol (OH). In another general example, a compound of formula Π or Π A may be reacted with a suitable alkylating agent [such as an alkyl halide, an arsenite oxalate, an arsenite sulfonyl ester, or a trifluorosulfonate Ester (tr if 1 ate) alkyl ester] reaction and alkylation. The alkylation reaction can be carried out at a temperature of about -10 to 80 ° C in a suitable solvent such as an aprotic solvent (for example, difluorenamide) or an ether solvent (such as tetrahydrofuran) in a base [ For example, an inorganic test (such as a carbonate, such as cesium or potassium carbonate), a hydride (such as sodium hydride), or an alkoxide (such as potassium t-butoxide) is performed in the presence of. Preparation as a single enantiomer or when appropriate The preparation of diastereomeric compounds of formula Π or Π A can be accomplished as follows: by synthesis from the enantiomerically pure starting materials or intermediates, or by analyzing the final product in a conventional manner. Compounds of formula Π or Π A The acid addition salts can be prepared in a conventional manner as follows: A solution or suspension of the free base II or Π A is treated with about 1 equivalent of a pharmaceutically acceptable acid. The compound of formula I, Π, or Π A derived from an inorganic or organic base The salt can be prepared in a conventional manner as follows: The solution or suspension of the free acid I, Π or Π A is treated with about 1 equivalent of a pharmaceutically acceptable organic or inorganic base.

Page 15 V. Description of the invention (11) Both the salt-forming and alkali-derived salts are prepared by changing the resin to process the parent compound and preparing a two-level method using appropriate ion-interaction and recrystallization techniques. When isolating salts, the compounds according to the present invention can be broken down using conventional concentrations. The normal mature blood vessels are retained: A tumor blood vessels and newly formed blood vessels. The ability of compounds according to the invention: Temple :: can be described in the examples below. Φ Cancer including red tumors, as well as prevention and treatment for the prevention and treatment of diseases, including diseases such as diabetic retinopathy: = inflammation with inappropriate blood vessel formation, atherosclerosis and macular degeneration. Psoriasis, rheumatoid joints, the compounds of this 5 Ming can be used as: monotherapy + and joy. In order to treat solid tumors, Benlox, or in combination with other therapies, 丨 compounds with other anti-tumor substances can be combined with those of radiation therapy… mitotic inhibitors, such as Le, for example, selected from the following (PacUUxe " And Ducita, Paclitaxel, Siroplatin, Cardan and F _, alkylating agents, such as diamine digastric. Dingtangtang ;; antimetabolites, such as 4 urine bite, cytarabine glycoside And hydroxyurea; intermediary agents, lycomycin; enzymes, such as n 夂 _ 欧 酒 · 'Aria M π and Bo: J aspartate amidase, topoisomerase inhibitors, examples • / /, Topotecan and Ilinotecan) 'Thymine nucleotide synthetase inhibitors, such as ra 1 t tre χ ed; biological response Modifiers, such as interfering with 'something' such as edrecolomab; and anti-hormones such as tamoxifen. Such combined treatments may include the individual components of the therapy being applied simultaneously or sequentially.

1228045 V. Description of the invention (12) In order to prevent and administer the composition, choose. 001 Such pharmaceutical or rectal or gastric preparations or capsules can be used in the form of powders or ointments. Rectal administration intravenously, subcutaneously, for example, as a disinfectant solution for prevention or treatment depends on whether the selected compound is a dose to be administered by 0.001. To 100 milligrams of biological activity use the following selectivity. For the treatment of the disease, the compound can be administered parenterally. For example,. For the convenience of the solution, it can be used as intramuscular, suspension, to treat specific conditions, and administered alone. The dosage is determined by the physician. It is administered in oral or oral form, and administered according to the invention. Partial. For internal or infusion, the external drug is generally said to be 50 milligrams. According to the administration of the drug, take the appropriate form of the oral nasal cavity to supply the route required for suppository, vascular or milk conditions, or with another, but A compound of 0 · 1 can be used as a standard medicine. Silica Gelikou can be used in Cui, cheeks, nasal cavity, round ~ can be used conventionally σ »¥ ϋ A & agent can be administered by ^ method, compound i, when Can be used as ointment;, parenteral injection (including 'master'), the composition can be taken. The dosage of the indicated compound will be in the form and severity, as well as in combination. Therefore, the precise daily dose range may be g / kg. Better. Trials to clarify the activity and activity of the compounds according to the invention

Determination of tumor blood by radioactive tracer

The following experiments demonstrate that the compound selectively destroys the calcium ions of the tumor vessels. Subcutaneous CaNT tumors were initiated as follows: 12 to 16 weeks of age mouse back dorsal epithelium was injected subcutaneously with 0.05 ml of a crude tumor cell suspension containing approximately 106 cells. After about 3 to 4 weeks, when their tumors reached a geometric mean diameter of 5.5 to 65 mm, they were selected as treated animals. Dissolve compounds in sterile saline to

Page 17 1228045 V. Description of the invention (13) Intraperitoneal injection with a volume of 0.1 ml in weight. Six hours after intraperitoneal injection, tumors, kidneys, and liver were measured using 86RbC I extraction technology (Sap ir ste i η, American Journal of Physiology, 1 193, 16 1-1 6 8, 19 5 8). , Skin, muscle, bowel and brain tumor perfusion. Tissue radioactivity measured 1 minute after intravenous injection of 86 cents CI was used to calculate relative blood flow as part of cardiac output (Hi 1 1 and Denekamp, British Journal of Radiology, 55, 905-91 3, 1 9 8 2). The control group and the treatment group used 5 animals each. Results are expressed as a percentage of blood flow in the corresponding tissue of the vehicle-treated animal. Anti-tumor hemorrhagic activity measured by fish fluorescent dyes The following experiments further clarify the ability of compounds to destroy tumor blood vessels. A ?, S. Mith ("Central Cancer Research" 5 7, 2 4 7-2 5 3, 1 9 88) method, using the fluorescent dye Hoechst 33 342 to measure the tumor functional blood vessel volume with CaNT tumor stingy. The control group and the treatment group used 5 animals each. The fluorescent dye was dissolved in saline at 6.25 mg / ml, and was injected intravenously at 10 mg / kg 6 hours after the intraperitoneal drug treatment. • One minute later, the animals were killed and the tumors were excised and frozen; sections were taken at three different levels, and sacrifice was performed under ultraviolet irradiation using a ympus microscope equipped with epi-fluorescence. Vessel volume was identified based on its fluorescent profile, and the blood vessel volume was quantified using a point counting system as described by cha 1 k 1 ey (Journal of the National Cancer Institute, 4, $ 7 5 3, 1 943. All estimates are based on Counts from a minimum of 100 fields of view at three different levels of sections. A dose of this compound at 0 mg / kg or less reduces tumor functionality ^ Official volume is more than 20%. The following non-limiting examples illustrate This invention. Unless stated otherwise, in

1228045 V. Description of the invention (14)

All 1H NMR in the examples were run at 300 MHz. Obstructive chromatography was performed on silica gel. All temperatures are 0 ° C. The following abbreviations were used: THF-tetrahydrofuran; DMSO-dioxanine; MCPBA-chloroperoxy ^ carboxylic acid 0 Example 1 ^ Sian jl—〇 一 Carbonium _ in the presence of anhydrous tetrahydrofuran (2 ml ) N_Ethyl Akiyama Sin = ι89ϋ76 millimoles) solution with di + butyldiethylchloroamine C189g, 0.75 millimoles) and 1 (H) -tetrazole (〇 · 14 grams, J 99 millimoles), the solution was stirred at 2 (rc for half an hour. The solution was cooled to -40 ° C, force. 85% 3-chloroperoxy in anhydrous dichloroarsine (2 ml) Phosphoric acid (202) solution in bis (1.J9, penmor) was added at such a rate that the temperature was maintained at room temperature, and the bath was allowed to rise to room temperature, and ethyl acetate was added. Ml), ⑽ml), 5% bacillus acid aqueous solution (30 ml), the residue was subjected to column color ί and salt ί successively under reduced pressure to concentrate the organic solution, two _ t _. To get, containing N-acetamidine Narcissus-0-phosphoric acid: but to 0 .; white bubble bed (170 mg) 'redissolved in dichloromethane' # ϊ sub-treated with triacetic acid. Developed,, = ΐ ί 温, and reducing Mix for 1 hour before concentrating , With samura (d6 ^^ subsolid title compound, mp · 233-23 5 t: · (5 Η J 2 7 Hz), 7 \ d38U, Η, Hertz), 7.27U, 1Η, (s, 1H ), 4, 1H, J = 8 Hz), 7.10 (s, 1H), 6.77 • (m, 1H), 3.81 (S, 3H), 3.76 (s, 3H),

1228045 V. Description of the invention (15) 3.49 (s, 3H), 2.5 (the signal is masked by the value of dimethylamine), 1. 9-2 · 2 (m, 2Η), 1. 86 (s, 3Η). Phosphate compound activity was measured by the above-mentioned radiotracer analysis method: The compound of this example reduced tumor blood flow by 65% at a dose of 125 mg / kg, and there was no significant blood flow in the skin, muscle, liver, kidney, intestine, or heart Squamate compounds with parent N ~ acetonitrile (MTD) (no death in three animals) dishes and 1 ^ compared with = = tolerated doses of the fluorescent dye technology and medical & a / Bali D) , By dose). … (The amount of swords received at the Nongda University / minimum medical window minimum induced dose N-acetic acid colchicol N-ethyl brewed colchicol — 0-filling salt Although the minimum effective phosphate maximum effective dose minimum effective dose mg / 仟 mg / Gram weight grams weight 1 2 5 3 0 1 and agent 7 denier 50, 50 15 This is unexpected #. Phosphate :: J ^ window "is much larger. In order to compare colchicine (its structure and It is also large. Ducitaxel (the closest compound to the microtubule-binding protein YiXing) and Taxotere 〃, without damaging the blood vessel, the product name is ~ Medium: /), 14 data are presented in the table below (Other light-binding dyes are used in medical window I.)

1228045 V. Description of the invention (16) Minimum effective dose (mg / kg of body weight) Ducitexel> 30 (no effect at 30) Colchicin 2.5 Example 2 N-ethylcocitol (N- ethylcolchinol) During 15 minutes, a solution of n_ethyl colchicine (500 mg, 1.4 mmol) in tetrahydrofuran (5 ml) was added dropwise to a solution of tetrahydropyrene cooled in an ice bath. Ran (10 ml) in lithium aluminum hydride (106 mg, 2.74 mmol) in W float. The mixture was heated in a reflux device for 15 hours, allowed to cool, and heated in a reflux device for another three hours, and then treated with lithium aluminum hydride (53 mg, 1.4 mol). The mixture was cooled (ice bath), and water (10 ml) was added dropwise in three portions of acetate extract II 4 '. The combined dry (= magnesium acid) extracts were concentrated under reduced pressure to a green gum and triturated with ether to give the title compound as a pale green solid. 11) .185. (Decomposition), 111/6 343 (1+). Analysis: Calculated from C2qH25N04H20: c, 66.46; H, 7.53; N, 3.88. Per measurement. C, 66.50 79. Example 3 * Colchicol (625 mg "=) in N-caroxychicocolcol in dried pharyng (10 ml) > Valley liquid with chloroacetic acid (0 5 6 6 9 7 mmol Each 9δ = Wu Li, and 糌 拄, 人, t η η, six tj drop-free mixing this dropwise. For 16 hours. Remove the solvent under reduced pressure, add water, and show a large tolerated dose (mg / 仟 g body weight) 30 minimum effective dose of Bida < 5 2

Page 1228045 V. Description of the invention (17) The mixture is extracted with three portions of chloroform. The combined dry (as magnesium sulfate) extracts were concentrated under reduced pressure to a dark brown gum, and subjected to column chromatography on silica gel, eluting with 50% ethyl acetate / petroleum ether. The obtained orange gum was crystallized from diethyl ether / petroleum ether to give the title compound (346 mg) as a pale yellow solid (m.p. 79-81 ° C, m / e 449 (M +)). Analysis: · Calculated from C26H27N0 6 0 · 33Η2〇: C, 68 · 57; Η, 6 · 0 7; Ν, 3 · 0 8. Found: C, 6 8. 71; Η, 6 · 1 8; N, 2.91. Example 4 Colchisol (400 mg, 1.27 mmol) of N-(; aminoaminomethyl) colchicine in dry pyridine (10 ml) with isocyanate (〇 · 151 ml, 1, 39 mmol), dropwise, and stir the mixture for 18 hours before heating in the reflux device for 2 hours. The solvent was removed under reduced pressure, water was added, and the resulting mixture was extracted with three portions of chloroform. The combined dry (magnesium sulfate) extracts were concentrated under reduced pressure to a dark brown gum, and subjected to column chromatography on silica gel, eluting with 35% ethyl acetate / petroleum ether. The resulting gum was crystallized from diethyl ether / petroleum ether to give the title compound as a pale orange color (261 mg). mpl45-146 ℃, m / e 434 (M +) 〇 Example 5 N-sulfosulfacolchiol in acetol (8 ml) N, 0-dimethylsulfacolchicol (2 34 mg, 0.5 millimolar) solution was treated with sodium hydroxide (40 mg, 1 millimolar), and the mixture was heated in a reflux device for 3 hours. The solvent was removed under reduced pressure, and water (5 ml) was added. 1M hydrochloric acid was added to make the solution neutral, and it was extracted with three portions of dichloromethane. The combined dried (magnesium sulfate) extracts were concentrated under reduced pressure to give a pink

Page 22 1228045 V. Description of the invention (18) _ — The title compound of color solid (123 (M +). Magic. M · P · 234 ~ 2 3 6 ° C, m / e 393 used as the starting material N, 〇 one Methanesulfonium colchicine in two denier dried pyridine (15 ml) was prepared as follows: in the solution, methanesulfonyl chloride (0. 丨 3 5 mmol colchicol (500 mg, 1.6 mmol) Ear) Melting temperature and stirring for 3 6 hours. Add ^ liters, 1.7 mol), add the mixture to the room mol), continue to stir 丨 6 small Japanese servings of mesylate chloride (0 · 1 3 5 ml, 1 7 ml). The solution was taken in three portions of chloroform-Γ. Remove the solvent under reduced pressure and add water (5 milligrams) extract to obtain a brown gum = take 'reduced concentration and combined drying (eluted with ethyl sulfate to get light " ^ 贝'), which was subjected to a column on a silica gel Chromatography to B mg). 'Human Ugly Solid N, 0-Dimethyl Sulfonium Colchiol (2 92 Example 6 N-Dimethylamine Sulfonium Colchiol in Dry Ethyl Acetate (3 ml of Colchiol (5 ◦ Diethylamine) (0.022 ml, 0.16 mmol), treat the solution in the hair of the reflux device with '0 · 16 mmol') with dimethylsulfasulfonium chloride. Remove the solvent under reduced pressure, and heat for 15 hours, The mixture was stirred for 30 minutes. The combined water was concentrated under reduced pressure, and the resulting mixture was subjected to column chromatography (expressed with sodium sulfate) to a dark brown gum on silica gel in three portions. The title compound (4 6 = 1. Eluted with ethyl acetate to give a pale orange gum 42 2 Ui +). Wool 'it solidified. Ι 82 · 85 ° C, m / e Example 7 Drying dimethyl formaldehyde' Methylcolchiol

Page 23 N-ethylammonium ~ 〇 曱 oxymetamine-amine (5¾: liters) N-ethyl brewed colchicine (5001228045 V. Description of the invention (19) ___ Mao Ke '1 · 4 milligrams Moore) solution was treated with 2.1 millimoles) and sodium hydride (84 mg in i, ethyl acetate (322 mg, Moore)), and the mixture was stirred for 30 minutes. 60% of the total Suspension, 2.1 milligrams were extracted with four parts of ethyl acetate. Mandarin Thai water (50 ml), mixed with a gasified sodium hydroxide aqueous solution were washed successively, and dried (with four parts of water and two parts of saturant, The title compound (magnesium phosphate) was obtained as a white solid, and the solvent m / e 42 9 2 (M +) was removed under reduced pressure. Analysis: from "〇80mg" ° ρ · 82-83 t, 23η, 7 Ι \ ΙΠ λα 63. 45; Η, 6.40; Ν, 3.22. Calculated values: 0, 3, 3, and 20: C, 6.29; Ν, 3.17., m: C, 63.53; H, Example 8 〇N- Ethylylmethyl methyl colchiol e 415.3 62.71; 溶液 N-ethylfluorenyl, 0 (1 40 mg, 0.33 mol) solution in acetonitrile (5 ml) with -methoxycarbonylformaldehyde Colchicol alcohol 5 ml), mixture at 80 ° c Force σ heat $ gasified potassium aqueous solution (1.0 M, after cooling the mixture of P to 3, in four, f \ 0 minutes. Add 2M hydrochloric acid, adjust the extract with four parts of saturated sodium chloride aqueous solution, first Extracted with ethyl acetate. The combined extracts were concentrated under pressure. Acetone (2 mL) and strips (dried with sulphuric acid) were added, and the title compound (58 mg) was added to give white. Solid (M +). Analysis: Calculated from C22H25N07 〇33η2〇 6.14; Ν, 3.32. Measured value · (:, 6263; hn, 3.26 ° Example 9 Ν-ethenyl 10-cyclopentyl autumn N-Ethyl colchicine (2 0 0

Page 24, 1228045, Description of the invention (20) mg, 0.56 mmol) was washed at 0 t with a 60% suspension of sodium hydride, 0.84 mmol, and then washed with g of cyclopentane in oil. (0.884 mmol), and the mixture was stirred for 1 hour. Second, odor (125 milligrams of sodium (17 mg of a 60% suspension in oil, 42) plus another portion of hydrogen bromide (63 grams, 0.42 millimoles), and the mixture at room temperature俨 and pentyl water (10 liters), the mixture was extracted with four parts of ethyl acetate. The device was washed. Two parts of saturated aqueous sodium chloride solution were added, washed, and dried (to protect the extract to shrink.) The title compound was white solid (600 mM, and reduced pressure m / e 425.3 (M +). Analysis: from c η Ν〇 bucket g) ° mP89-WC, knife 甶 L25H31N05 leaf calculated: c, 7〇w · H m3.29. Found: c, 7055 · 54 '3.25 〇, Ν, Example 1 0 Ν -ethymyl-1 0-continued colchicol in ice ethyl (20 ¾ liter ) Colchicine acetate (100 mg, 0.27¾ mol) in 20) liters of concentrated nitric acid (0.34 liters) in acetic acid (100 ml) was slowly maintained at a temperature of about 1¾ treatment 1 2. Stir and mix at room temperature

Compound 18h 'Add another 1ml nitric acid / acetic acid solution and continue stirring for 2 hours. The mixture was poured into ice and extracted with three portions of ethyl acetate. The combined extracts were washed with two portions of saturated aqueous sodium carbonate solution, dried (over magnesium sulfate), and concentrated under reduced pressure. Purification on silica gel with ethyl acetate gave the title compound (50 g) as a yellow solid, Qm.p.ii, 7-8 ° C, m / e 401.9 (M +). Analysis. Calculated from C20H22N20.7 0 · 3 3H2O: C, 5 8 · 8 2; H, 5 · 5 6; N, 6. 86. Measured value: C, 5 8 · 8 7; 6, 5. 6 6; Ν, 6 · 5 5. Example 11

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Claims (1)

12 ^ 804 ^ Case No. 88100179 MM a Modification τ, patent application scope 1. A pharmaceutical composition for treating angiogenesis-related diseases including a colchicol derivative of the formula I 丨 Amended and supplemented 74 ° {) ¾ β where h, R2, R3, and 1-6 are each hydrogen, P03Η2 or an alkyl group selected from (: 7 alkyl, cyclopentyl, and Ci_7 alkyl, where alkyl The group may bear one or more substituents selected from a hydroxyl group, a carboxyl group, and a Ci_7 alkoxycarbonyl group; X is a carbonyl group (C0), a fluorenyl group (CH2), or a CHR4 group; a meridian group or nr8r9; each of R5 and 1 Are hydrogen, cw alkoxy or nitro respectively; the group is selected from the group consisting of halides, or Ci_7 alkyl, Cp alkyl, Ci_7 alkoxycarbonyl, < amines, Ci7 alkyl and di ) In the alkyl group of the amine group, the alkyl group may carry one or more substituents selected from the group consisting of halogen, mesityl, weyl, cv, alkyl, and di (Ci_7 alkyl). Or a pharmaceutically acceptable salt or excipient thereof 2. The pharmaceutical composition according to item 1 of the scope of the applied patent, wherein h and R8 are hydrogen, selected from the group consisting of Toxycarbonyl, phenylcarbonyl and phenylaminecarbonyl Any of the benzene rings may have one or more substituents, and the substituents are O: \ 56 \ 56624-930915.ptc Page 28 1228045 Q Case No. 88100179 Amendment No. 6: Patent application scope 至少 · At least one of R 3 and R 6 is P0 Η 2 ° tn compound, of which R6 is 3. Medical P03Η2 according to item 2 of the scope of patent application. 4. The pharmaceutical composition according to any one of claims 1 to 3 of the scope of patent application, wherein Ri, R2 and R3 are Ci_7 courtyards. 5. The pharmaceutical composition according to any one of claims 1 to 3 of the scope of patent application, wherein R4 is amidino. 6. The pharmaceutical composition according to item 1 of the scope of patent application, wherein R6 is P03H2, Ri, R2, and 1 are (^ _7 alkyl groups, and R4 is amidino group. 7.-Compound of Formula I R. / J Ο (I) R5, R6, R7, and 乂 are as defined in item 1 of the scope of the patent application, and the restriction is that at least one of R 1, R 2, R 3, and R 6 is P 0 3 Η 2, or Pharmaceutically acceptable salt. 8. The compound according to item 7 of the scope of patent application, wherein R6 is? 03 [12, or a pharmaceutically acceptable salt thereof. 9. The compound according to item 8 of the application, wherein I, R2, and 1 are Cj_7 alkyl groups, and 1 is amidino group, or a pharmaceutically acceptable salt thereof. O: \ 56 \ 56624-930915.ptc Page 29 1228045 D _Case No. 88100179 丨 Month _ Amendment VI. Application scope of patent 1 0. Compound according to formula Π according to item 7 of scope of patent application Wherein Ri, R2 and 1 to 3 are each hydrogen, P03Η2 or an alkyl group selected from the group consisting of Ch alkyl, cyclopentyl, and C ^ 7 alkyl, wherein the alkyl group may have one or more members selected from a hydroxyl group and a carboxyl group. And (: 7 substituents of alkoxycarbonyl group; is 6 ?? 3112; is hydrogen or N Rs Rg; each of R 5 and R 7 is hydrogen, Ci_7 alkyl or alkoxy group; R8 is hydrogen An aryl group selected from the group consisting of Toxycarbonyl, phenylcarbonyl, and phenylamine carbonyl, wherein any benzene ring may have one or more substituents, and the substituents are selected from the group consisting of II-alkyl or Ci_7 alkyl '(ν7 Alkyl group, alkoxycarbonyl group, Ci_alkylamine group, Ci_7 alkyl group, and di (Ch alkyl group) group, wherein the alkyl group may have one or more selected from halogen, Substituents for hydroxyl, carboxyl, (^ _7 monoalkylamino and di (Ci_7 alkyl) amino groups; Rg is nitrogen, or a pharmaceutically acceptable salt thereof. 1 1. A compound of formula Π A O: \ 56 \ 56624-930915.ptc Page 30 1228045. _ Case No. 88100179 '' Month _ Amendment VI. ¥ Please patent scope Wherein Ri, R2, and 1 are each hydrogen, P03Η2, or an alkyl group selected from (V7 alkyl, cyclopentyl, and (^ _7 alkyl group), wherein the alkyl group may carry one or more members selected from hydroxyl, carboxyl, and Ci_7. Substituents of alkoxycarbonyl groups; R6 is hydrogen, P03H2 or an alkyl group selected from Ci_7 alkyl groups and cyclopentyl groups, wherein the alkyl group may have one or more substituents selected from hydroxyl groups, carboxyl groups and Ci_7 alkoxycarbonyl groups R 4 is hydrogen or NR 8 R 9; each of R 5 and 1 is hydrogen or nitro respectively; R 8 is hydrogen, an aryl group selected from the group consisting of oxocarbonyl, phenylcarbonyl, and phenylamine carbonyl, wherein any benzene ring It may have one or more substituents, and the substituent is selected from the group consisting of ^ 7 alkyl, CV7 alkyl sulfonyl, (ν7 alkoxycarbonyl, ^ _7 alkylamine carbonyl, Ci_7 alkylsulfonyl and di (Ci_7 alkyl) sulfamoyl alkyl, wherein the alkyl group may have one or more substituents selected from the group consisting of halogen, hydroxyl, carboxyl, Cp7 monoalkylamino and di (Ci_7 alkyl) amino; and R 9 is hydrogen, and the restriction is that when R 1, R 2 and R 3 are all fluorenyl and R 4 is O: \ 56 \ 56624-930915.ptc Page 31 1228045 Case No. 88100179 p \ year p month said amendment 6. When applying for a patent for hydrogen, acetamido, amine or amido, R6 is not hydrogen or methyl Or hydroxyethyl; or a pharmaceutically acceptable salt thereof. 1 2. The compound according to item 11 of the scope of patent application, wherein Ri, R2 and R3 are Ci_7 alkyl groups and R4 is an amino group. 1 3. A pharmaceutical composition for treating an angiogenesis-related disease, which includes a compound and an excipient such as any one of items 7 to 12 of the scope of patent application. 14. The pharmaceutical composition according to item 13 of the scope of patent application, wherein the compound is N-acetamcolchiol-0-phosphate. 15. A compound which is N-acetamcolchiol-0-phosphate or a pharmaceutically acceptable salt thereof. 16. A method for preparing a compound according to any one of items 7 to 12 of the scope of patent application, which comprises: treating the compound of formula (2) with alkali hydrogen peroxide O: \ 56 \ 56624-930915.ptc Page 32 1228045 _ Case No. 88100179 6. The scope of the patent application is amended in which Ri, R2, R3, and 1 are as defined in items 7 to 12 of the scope of patent application; and R 5. R 6 and R 7 are hydrogen. 17. A method for preparing a dihydrogen phosphate-containing compound of formula II or IIa as in any one of claims 10 to 12 of the scope of the patent application, the method comprising: (1) the presence of a suitable catalyst The compound of formula II or Ila containing hydroxyl group is treated with di-tertiary-butyldiethylphosphoramideneamine, and then treated with an oxidizing agent; and (i 1) the intermediate obtained in step (i) is filled with triacetic acid to Acid treatment. 18. The method according to item 17 of the scope of patent application, which is used to prepare N-acetamcolchiol-0-phosphate, wherein the compound of formula II containing hydroxyl group used in step (i) is N-acetamidine Colchiol. O: \ 56 \ 56624-930915.ptc Page 33