TW201444864A - Hexadepsipeptide analogues as anticancer compounds - Google Patents

Abstract

This invention relates to an isolated compound of Formula (1) or derivatives or pharmaceutically acceptable salts thereof. The invention also includes all isomeric and tautomeric forms of the compound of Formula (1) or the derivatives thereof. The present invention further relates to processes for the production of the compound of Formula (1) by fermentation of the fungal strain of Actinomycetes (PM0895172/MTCC5684), pharmaceutical compositions comprising the compound of Formula (1) as the active ingredient; and use of the said compounds or composition containing them in the treatment of cancer.

Description

Hexapeptide analog as an anticancer compound

The invention is related to a compound of formula (1) (as described herein), a pharmaceutically acceptable salt or a derivative thereof. The present invention is further related to a compound production procedure of structural formula (1) from an isolated microorganism belonging to a radiation strain (PM0895172/MTCC 5684). The present invention is also related to a pharmaceutical composition comprising the compound of the formula (1), a pharmaceutically acceptable salt or derivative thereof as an active ingredient; and the use thereof for the treatment of cancer.

Cancer is a general term for a large group of diseases caused by uncontrolled cell growth and spread, which can affect any part of the body. Several treatment options are available for cancer today, including chemotherapy, surgery, and radiation to localized lesions or a combination of such treatments. The choice of therapy depends on the location of the cancer and also on the extent to which the cancer spreads at the time of diagnosis. Chemotherapy is one of the most common forms of cancer treatment involving the use of anticancer drugs that can destroy cancer cells. Although the treatment of cancer continues to progress, the disease remains one of the leading causes of death in the world, and the most likely reason is that the available treatment options are associated with poor side effects and limited effects. Therefore, there is a need for new anticancer agents/drugs that exhibit the clinical benefit of treating cancer.
There are reports of anticancer compounds from natural sources, such as taxol, vincristine, torreyanic acid, and camptothecin (Natural Product Communications, 2009, 4 (11), 1513).
The marine environment, covering 70% of the Earth's surface and 95% of tropical life, represents thirty-four of the thirty-six lives and offers a variety of fascinating biodiversity. Biodiversity beyond terrestrial environmental performance (Life Sciences, 2005, 78, 442-453). A number of drug candidates have been produced for the exploration, refinement and screening of biodiversity and intrinsic knowledge for the bio-exploration of marine natural products with commercially valuable genetic and biochemical resources (Drug Discovery Today, 2003, 8 (12) ), 536-544).
There have been reports of drug discovery involving screening of natural products from marine organisms and microorganisms. Anticancer agents derived from marine sources include cytarabine, bryostatin-1, aplidine, dolastatin, and ET-743 (Current) Opinion in Pharmacology, 2001, 1, 364-369).
Some hexaester peptides are reported in the art. For example, JP09040559 discloses hexaester peptide compounds having anti-inflammatory activity which are effective for allergic and non-allergic inflammation.
Considering that many therapeutically active compounds have been obtained from natural sources, it is prudent to explore this field of research to obtain new compounds that are effective for the treatment of diseases such as cancer. The inventors of this patent specification have provided their efforts to provide novel compounds found for use in cancer therapy. Also provided is a method of producing this compound which, via fermentation and, further, chemical modification of a compound of formula (1) to obtain a derivative thereof.

The invention relates to compounds which are designated herein as compounds of formula (1) (as described herein).
The present invention relates to the isomeric form, tautomeric form of the compound of the formula (1) or a pharmaceutically acceptable salt thereof or a derivative thereof.
According to the invention, the compound of formula (1) is a compound of formula (1a) or a compound of formula (1b).
According to the invention, the derivative of the compound of the formula (1) is contained in the compound of the formula (1c).
The present invention is also related to a purified compound of the formula (1) which is isolated from a culture solution obtained by fermentation of one of marine growth strains (PM0895172/MTCC 5684) or a variant or mutant thereof. Thus, the compound of the present invention is an isolated compound of the formula (1).
The invention also relates to a process for the production of a compound of formula (1) and/or its isomers or tautomers thereof from a marine radiation strain (PM0895172/MTCC 5684).
The invention further relates to a process for the isolation of microorganisms belonging to a radiation strain (PM0895172/MTCC 5684) which produces a compound of formula (1), or an isomer or tautomer thereof.
The compound of formula (1) or an isomer or tautomer thereof or a pharmaceutically acceptable salt or derivative thereof is useful for the treatment of cancer.
The present invention relates to a method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula (1) or an isomer or tautomer or pharmaceutically acceptable salt thereof. Or a derivative.
The invention also relates to a compound comprising structural formula (1) or an isomer or tautomer thereof or a pharmaceutically acceptable salt or derivative thereof as an active ingredient, and at least one pharmaceutically acceptable A pharmaceutical composition of a form, carrier or vehicle.

no

Figure 1 illustrates the ¹ H NMR (CDCl ₃ ; 500 MHz; instrument: Bruker) spectrum of the compound of formula (1a).
Figure 2 illustrates the ¹³ C NMR (CDCl ₃ ; 75 MHz; instrument: Bruker) spectrum of the compound of formula (1a).
Figure 3 illustrates the ¹ H NMR (CDCl ₃ ; 500 MHz; instrument: Bruker) spectrum of the compound of formula (1b).
Figure 4 illustrates the ¹³ C NMR (CDCl ₃ ; 75 MHz; instrument: Bruker) spectrum of the compound of formula (1b).
Figure 5 illustrates the effect of a compound of formula (1a) on apoptosis of HeLa cancer cells.

The present invention provides a compound of the formula (1) as shown below:



Structural formula (1)
Wherein in the compound of formula (1), when the bondRepresents a single button and R¹Is H; a compound is referred to as a compound of formula (1a);
When the keyIs a double bond and R¹When not present, the compound is referred to as a compound of formula (1b). Thus, the compound of formula (1) is a compound of formula (1a) wherein the bondRepresents a single button and R¹a compound of H) or structural formula (1b) (wherein the bondIs a double bond and R¹Not present) or a mixture thereof.
The compound of the formula (1) belongs to a hexaester peptide compound.
The compound of formula (1a) and the compound of formula (1b) can be characterized by any one or more of physico-chemical and spectral properties, such as high performance liquid chromatography (HPLC), high resolution as discussed herein below. Mass spectrometry (HR MS), far infrared (IR), and nuclear magnetic resonance (NMR) spectral data.
According to one aspect of the invention, the compound of formula (1) is a compound of formula (1a) having the formula C₃₇H₆₂N₈O₁₃And the molecular weight of 826.9.
The compound of the formula (1a) is expressed as follows:



Structural formula (1a)
The chemical name of the compound of formula (1a) is N-(6,18-dihydroxy-22-isopropyl-7,19-dimethyl-5,8,11,17,20,24-hexa-oxa Tetrahydrodioxazino[6,1-f:6',1'-o][1,4,7,10,13,16]pentazaoxacyclohexadecandin-23-yl)-2 - Hydroxy-2-(2-hydroxy-5-isobutyl-6-methyltetrahydro-2H-pyran-2-yl)propanamine.
According to one aspect of the invention, the compound of formula (1) is a compound of formula (1b) having the formula C₃₇H₆₀N₈O₁₃, and the molecular weight of 824.4.
The compound of the formula (1b) is represented as follows:



Structural formula (1b)
The chemical name of the compound of formula (1b) is N-(6,18-dihydroxy-22-isopropyl-7,19-dimethyl-5,8,11,17,20,24-hexaoxy- 3,4,4a,5,6,7,8,9,10,11,13,14,15,16,16a, 17,18,19,20,22,23,24-tetrahydrofuran Pyrazino[6,1-f:6',1'-o][1,4,7,10,13,16]pentazaoxacyclohexenyne-23-yl)-2-hydroxy-2- (2-Hydroxy-5-isobutyl-6-methyltetrahydro-2H-pyran-2-yl)propanamine.
The microorganism which can be used for the production of the compound of the formula (1) is a radioactive strain (PM0895172/MTCC 5684), hereinafter referred to as the number PM0895172, which is collected from the deep sea of the offshore area of Mumbai, Maharashtra, India. Separation in sediments.
An aspect of the invention provides a production procedure for a compound of formula (1) from culture number PM0895172, comprising the steps of:
a) cultivating one of the culture numbers PM0895172 or one of its variants or mutants under water aerobic conditions, comprising one or more sources of carbon and one or more sources of nitrogen and nutritive inorganic salts and/or traces Growing in a nutrient medium of the element to obtain a culture solution containing the compound of the formula (1);
b) separating the compound of formula (1) from the culture broth;
c) Purification of the compound of formula (1).
The compound of the formula (1) produced according to the above procedure may be a compound of the formula (1a) or a compound of the formula (1b).
In a specific embodiment, the culture solution in step (a) comprises a mixture of a compound of formula (1a) and a compound of formula (1b).

In a specific embodiment, in step (b) of the procedure, the compound of formula (1) is a compound of formula (1a).
In a specific embodiment, in step (b) of the procedure, the compound of formula (1) is a compound of formula (1b).
In a specific embodiment, in step (c) of the procedure, the compound of formula (1) is a compound of formula (1a).
In a specific embodiment, in step (c) of the procedure, the compound of formula (1) is a compound of formula (1b).
In a specific embodiment, the invention provides a production procedure for a compound of formula (1a) from culture number PM0895172, comprising the steps of:
a) cultivating one of the culture numbers PM0895172 or one of its variants or mutants under water aerobic conditions, comprising one or more sources of carbon and one or more sources of nitrogen and nutritive inorganic salts and/or traces Growing in a nutrient medium of the element to obtain a mixture culture solution comprising the compound of the formula (1a) and the compound of the formula (1b);
b) separating the compound of formula (1a) from the culture broth;
c) Purification of the compound of formula (1a).
In another embodiment, the invention provides a production procedure for a compound of formula (1b) from culture number PM0895172, comprising the steps of:
a) cultivating one of the culture numbers PM0895172 or one of its variants or mutants under water aerobic conditions, comprising one or more sources of carbon and one or more sources of nitrogen and nutritive inorganic salts and/or traces Growing in a nutrient medium of the element to obtain a mixture culture solution comprising the compound of the formula (1a) and the compound of the formula (1b);
b) separating the compound of formula (1b) from the culture broth;
c) Purification of the compound of formula (1b).
Step (c) of any of the above procedures relates to the purification of the compound of the formula (1), and is carried out by using a purification procedure generally used in the related art.
The compound of the formula (1), in particular the compound of the formula (1a) and the compound of the formula (1b) which are produced according to the procedure of the present invention, are substantially pure compounds. Thus, the compound of formula (1), in particular the compound of formula (1a) or the compound of formula (1b), is an isolated pure compound.
Typically, the compound of formula (1a) and the compound of formula (1b) are purified via column chromatography techniques.
As used herein, the term "mutant" means an organism or a cell in which one or more genes in the genome have been altered by one or more genes responsible for the organism's production. The ability of the compounds of the invention. Such mutant strains can be produced in a manner known per se using physical means such as ultraviolet or X-ray irradiation or chemical mutagen.
As used herein, the term "variant" means an individual organism that recognizes any standard type different from that species.
The term "whole broth" can be used interchangeably with the terms "nutrient broth", "culture broth" or "fermented broth".
As used herein, the term "mammal" means both human and non-human mammals including, but not limited to, cows, horses, pigs, dogs, and cats. The term "mammal" can be used interchangeably with the term "patient" or "subject".
The phrase "active ingredient" or "active compound" may be used interchangeably, and as used herein, the term means a compound of formula (1) or a compound of formula (1a). Or a compound of formula (1b) or an isomer or tautomer thereof, or a pharmaceutically acceptable salt or derivative thereof. In the context of the present invention, the term "active ingredient" or "active compound" also means a compound of formula (1c) which comprises a derivative of a compound of formula (1) as described herein.
The phrase "compound of the formula (1)" includes a compound of the formula (1a) or a compound of the formula (1b) or a mixture thereof; and an isomer, tautomer or pharmaceutically acceptable salt thereof. class.
The term "compound of the formula (1c)" includes a derivative of the compound of the formula (1), which comprises a compound of the formula (1a) or a compound of the formula (1b) or an isomer thereof, tautomer A derivative of a construct or a pharmaceutically acceptable salt.
The term "substantially pure" as used herein, means a compound of formula (1), particularly a compound of formula (1a) or a compound of formula (1b) or an isomer thereof. The isomer is pure enough that further purification does not detectably alter its physical and chemical properties as well as the enzyme and biological activity of the compound. The compound of formula (1) can be substantially purified according to methods known to those skilled in the art.
As used herein, the term "therapeutically effective amount" refers to the use of a compound of formula (1), especially a compound of formula (1a) or a compound of formula (1b) or a pharmaceutically acceptable compound thereof. The treatment of a cancer (as exemplified herein) of a salt or derivative (a compound of formula (1c) (as described herein) means that one or more of the following effects can be caused in a patient receiving the compound of the invention Amount: (i) to some degree of tumor growth inhibition, including slowing and complete growth arrest; (ii) reduction in the number of tumor cells; (iii) tumor size reduction; (iv) tumor cells infiltrating into surrounding organs Inhibition (ie, reduction, slowing or complete cessation); (v) inhibition of metastasis (ie, reduction, slowing or complete cessation); (vi) enhancement of anti-tumor immune response, which may but does not necessarily result Reconstruction of the tumor; and/or (vii) some degree of relief of one or more symptoms associated with the cancer being treated.
The term "pharmaceutically acceptable salts" as used herein, is intended to include a compound of formula (1), especially a compound of formula (1a) or a compound of formula (1b), and derivatives thereof. Those salts of the compounds of the invention (compounds of formula (1c)) which are safe and effective in mammals and possess the desired biological activity. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, phosphate, acetate, lactate, salicylate, citrate , tartrate, ascorbate, succinate, maleate, fumarate, formic acid salt, benzoate, glutamate, methanesulfonate, besylate, Tosylate. Suitable base addition salts include, but are not limited to, calcium, lithium, magnesium, potassium, sodium or zinc salts.
Preliminary identification of culture number PM0895172 was carried out via evaluation of its colony characteristics, and the compound of formula (1) was generated from the culture number PM0895172. A microscopic study of the isolated culture number PM0895172 strain was carried out on a modified radiobacteria separation agar medium. Observation was carried out after 20-22 days of incubation at 20 ° C to 30 ° C until colonies were observed. The culture number PM0895172 has been confirmed as a marine radiation strain.
The culture number PM0895172 has been deposited in the Microbial Type Culture Collection (MTCC, School of Microbiology and Technology (Sector 39-A, Chandigarh-160 036, India)) (World Intellectual Property Organization (WIPO)) Approved International Depository Authority (IDA) and has been provided with accession number MTCC 5684.
It should be understood that in addition to the specific microorganisms described herein, mutant strains of PM0895172, such as those produced by chemical or physical mutagenic agents (including X-rays, UV light), and organisms that have been modified by molecular biotechnology, may also be cultured, To produce a compound of formula (1).
The biological activity of the active compound can be determined by HPLC, NMR, IR, MS and/or the accumulation of the active compound, for example, via the test compound anticancer activity or via its binding, it is confirmed that a suitable mutation can be produced for the compound according to the invention. Screening of variants and variants.
The medium and/or nutrient medium isolated and cultured using the culture number PM0895172 for the production of the compound of the structural formula (1) preferably contains carbon, nitrogen and nutrient inorganic salt sources. The carbon source is, for example, one or more of starch, glucose, sucrose, dextrin, fructose, molasses, glycerin, lactose or galactose. A preferred carbon source is glucose. The nitrogen source is, for example, one or more of soy flour, peanut meal, yeast extract, beef extract, peptone, malt extract, corn steep liquor, gelatin or casein amino acid. Preferred nitrogen sources are peptone and yeast extracts. The nutritive inorganic salt is, for example, sodium chloride, potassium chloride, calcium chloride, manganese chloride, magnesium chloride, barium chloride, cobalt chloride, potassium bromide, sodium fluoride, sodium hydrogen phosphate, potassium hydrogen phosphate. , dipotassium hydrogen phosphate, disodium hydrogen phosphate, calcium carbonate, sodium hydrogencarbonate, sodium citrate, sodium nitrate, ammonium nitrate, potassium nitrate, sodium sulfate, ammonium sulfate, ammonium heptamolybdate, iron citrate, copper sulfate, sulfuric acid One or more of magnesium, ferrous sulfate, zinc sulfate or boric acid are preferred to sodium chloride and calcium chloride.
The maintenance of the culture number PM0895172 can be carried out at a temperature ranging from 24 ° C to 32 ° C. Typically, culture number PM0895172 is maintained at 27 °C - 29 °C. The well-grown culture plants can be stored in a refrigerator at 4 ° C - 8 ° C.
The inoculum culture of the culture number PM0895172 can be carried out at a temperature ranging from 27 ° C to 33 ° C and at a pH of about 5.5 to 8.5 at 210 to 260 rpm (revolutions per minute) for 60 to 80 hours. Typically, the number PM0895172 is grown at 29 ° C - 31 ° C and at a pH of about 6.5-7.5 and incubated at 230-250 rpm for 70-74 hours.
The production of the compound of the formula (1) can be carried out by fermenting the culture number PM0895172 in a shaken flask at a temperature ranging from 27 ° C to 33 ° C and at a pH of about 5.5 to 8.5 at 200-250 rpm. It took 110 hours. Typically, culture number PM0895172 is cultured at 210-230 rpm for 90-100 hours at 29 °C - 31 °C and pH of about 6.5-7.5.
The production of the compound of the formula (1) can be carried out by fermenting the culture number PM0895172 in the fermentation tank at a temperature ranging from 27 ° C to 33 ° C and from about 5.5 to 8.5 pH at 40-70 rpm and 200-300 lpm ( The liters per minute is incubated for 20-40 hours under aeration. Typically, culture number PM0895172 is incubated at 28 ° C - 31 ° C and pH between about 6.5 and 7.5, and aerated at 50-60 rpm and 240-260 lpm for 20-30 hours.
The production of the compound of formula (1) can be carried out by culturing the culture number PM0895172 in a suitable nutrient medium under the conditions described herein, preferably under water-containing conditions, for example, shaking. In the flask. The progress of the fermentation and the production of the compound of the formula (1) can be detected by high performance liquid chromatography (HPLC) and detected by measuring the biological activity of the culture solution by testing different cancer cell lines.
Fermentation is the process of growing microorganisms that produce various chemical or pharmaceutical compounds. The microorganisms are normally cultured under specific conditions in which nutrients are present. The culture solution is obtained after the completion of the fermentation process. Several cultures are centrifuged, which results in the formation of cell pellets and culture filtrate, which can be further processed in the procedures described herein.
The compound of the formula (1) present in the culture solution may exist in the form of a mixture of the compound of the formula (1a) and the compound of the formula (1b), and the compound or structure of the formula (1a) may be The compound of formula (1b) is separated from the chromatographic technique using different extraction methods.
The compound of the formula (1), in particular the compound of the formula (1a) or the compound of the formula (1b), can be used as a water-immiscible solvent such as petroleum ether, dichloromethane, chloroform or acetic acid from the culture filtrate. Ester, diethyl ether or butanol extraction or via the use of polymeric resins such as Diaion HP-20^R(Mitsubishi Chemical Industries Limited, Japan), Amberlite XAD^R(Rohm and Haas Industries, USA) or hydrophobic interaction chromatography for activated carbon adsorption. These techniques can be used repeatedly, individually or in combination.
The compound of the formula (1), in particular the compound of the formula (1a) or the compound of the formula (1b), may be derived from a cell mass in a water-miscible solvent such as methanol, acetone, acetonitrile, n-propanol or isopropyl. The alcohol is recovered by extraction with a water-immiscible solvent such as petroleum ether, dichloromethane, chloroform, ethyl acetate or butanol.
Alternatively, the culture solution is extracted with a solvent selected from petroleum ether, dichloromethane, chloroform, ethyl acetate, methanol, acetone, acetonitrile, n-propanol or isopropanol or butanol.
Typically, the compound of formula (1) is extracted from the entire culture using ethyl acetate as a solvent. The concentration of the extract provides an active crude material having a mixture of a compound of the formula (1a) and a compound of the formula (1b).
The compound of the formula (1) according to the invention, in particular the compound of the formula (1a) or the compound of the formula (1b), can be recovered from the crude material by fractional distillation of any of the following techniques: normal phase layer Analytical method (using alumina or tannin as stationary phase; extract such as petroleum ether, ethyl acetate, dichloromethane, acetone, chloroform, methanol, isopropanol or a combination thereof); reversed phase chromatography (using reverse A phase gum such as dimethyl octadecyl decane phthalate (RP-18) or dimethyl octyl decyl phthalate (RP-8) as a stationary phase; and an extract such as water, a buffer (for example , phosphate, acetate, citrate (pH 2-8)), and organic solvents (for example, methanol, acetonitrile, acetone, tetrahydrofuran or a combination of these solvents); gel permeation chromatography (using resin Sephadex LH) -20^R(Pharmacia Chemical Industries, Sweden), TSKgel^RToyopearl HW (TosoHaas, Tosoh Corporation, Japan) in a solvent such as methanol, chloroform, acetone, ethyl acetate or a combination thereof; or via countercurrent chromatography (using a two-phase extract system, the composition of which is two or more Solvents such as water, methanol, ethanol, isopropanol, n-propanol, tetrahydrofuran, acetone, acetonitrile, dichloromethane, chloroform, ethyl acetate, petroleum ether, benzene, and toluene). These techniques can be used repeatedly, individually or in combination. A typical method is chromatography via a normal phase gel and a reverse phase gel such as RP-18.
As used herein, the term "isomer" is a generic term for all isomers of a compound of formula (1) that differ only in their atomic orientation. The term isomers include enantiomers, mirror image isomers (racemates, racemic mixtures) and more than one palm center, which are not mutually Is a mirrored compound isomer (non-an image isomer). The compounds of the invention may have asymmetric centers and may occur as racemates, racemic mixtures, individual non-image or isomers, or may exist as geometric isomers, all isomeric of the compounds The form of matter is included in the present invention.
As used herein, the term "tautomer" means the coexistence of only one (or more) mobile atoms and the distribution of two (or more) compounds different from each other, for example, a ketone. - keto-enol tautomer.
The compound of formula (1), its isomer or tautomer can be converted into a pharmaceutically acceptable salt or derivative thereof in the present invention.
The pharmaceutically acceptable salts of the compounds of formula (1) are prepared by standard procedures known to those skilled in the art, for example, salts such as sodium and potassium salts can be obtained by formulating compounds of formula (1) or The isomers or tautomers or derivatives thereof are prepared by treatment with a suitable sodium or potassium base such as sodium hydroxide, potassium hydroxide and the like. Similarly, salts such as hydrochlorides and sulfates can be treated with a suitable acid by, for example, hydrochloric acid, a compound of formula (1) or an isomer or tautomer or derivative thereof. Prepared by sulfuric acid and the like.
All derivatives of the compound of the formula (1), particularly a compound of the formula (1a) or a derivative of the compound of the formula (1b), are included in the scope of the present invention. Derivatives of the compound of the formula (1), particularly the compound of the formula (1a) or the compound of the formula (1b), which are derived from those in which the amine group and the hydroxyl group are derived One or more of the compounds, particularly the N-hydroxy group of the compound of formula (1), especially the compound of formula (1a) or the N-hydroxy group of the compound of formula (1b).
Accordingly, in one aspect of the invention, there is provided a derivative of a compound of formula (1) which is represented by the following structural formula (1c),



Structural formula (1c)
among them,
R¹With R⁴Representing H independently or not;
R²With R³Is independently selected from H, hydroxyl, -O (C₁-C₆)alkyl and -OC(O)(C₁-C₆)alkyl;
Represents a single or double bond;
among them,
(C₁-C₆An alkyl group is unsubstituted or substituted with one or more groups independently selected from halogen, hydroxy, -O (C)₁-C₆)alkyl, nitro, cyano, -COOH, -NH₂,-NH(C₁-C₆)alkyl, -N[(C₁-C₆)alkyl]₂,-NHC(O)O(C₁-C₆)alkyl, -NHC(O)O(C₁-C₆)alkyl (C₆-C₁₀) aryl, -NH-PEG or (C₆-C₁₀)Aryl;
(C₆-C₁₀An aryl group is unsubstituted or substituted with one or more groups independently selected from halogen, halo (C)₁-C₆) alkyl, hydroxy, cyano, nitro, (C₁-C₆)alkyl, -O(C₁-C₆)alkyl, -COOH and -NH₂;
And PEG is polyethylene glycol, which is selected from O, O'-bis[2-(N-succinimide-succinyl)ethyl]polyethylene glycol (α,ω-bis-NHS- PEG), methyl-PEG-NHS ester (MS (PEG)_n, wherein n is 24), and a branched trimethyl and amber succinimide derivative of polyethylene glycol (TMS(PEG)n, where n is 12);
And stipulates if R²With R³Both are hydroxyl, then R¹With R⁴For non-existent andRepresents a double bond;
And all isomeric and tautomeric forms or pharmaceutically acceptable salts thereof.
It will be understood that "substitution", "substituted" or "substituted with" means that one or more hydrogen atoms of a given motif are substituted with a suitable substituent and include implied conditions. Substituting for this type is based on the allowable valence of the substituted atom and the substituent, and produces a stable compound.
The term "(C₁-C₆"Alkyl" or "alkyl", as used herein alone or as part of another group, means an unsubstituted or substituted straight or branched chain hydrocarbon comprising from 1 to 6 carbons, for example Methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, isobutyl, pentyl and hexyl. Substituted alkyl means substituted by one or more groups (C₁-C₆An alkyl group, the group being selected from, but not limited to, halogen, hydroxyl, -O (C)₁-C₆)alkyl, nitro, cyano, -COOH, -NH₂,, -N[(C₁-C₆)alkyl]₂,-NHC(O)O(C₁-C₆)alkyl, -NHC(O)O(C₁-C₆)alkyl (C₆-C₁₀) aryl, -NH-PEG or (C₆-C₁₀)Aryl.
As used herein, the term "(C₆-C₁₀"Aryl" or "aryl" means a monocyclic or bicyclic hydrocarbon ring system having up to ten ring carbon atoms, wherein at least one carbocyclic ring has a p-electron system. (C₆-C₁₀Examples of aryl ring systems include, but are not limited to, phenyl or naphthyl. Unless otherwise indicated, an aryl group can be unsubstituted or substituted with one or more groups independently selected from, but not limited to, halogen, halo (C).₁-C₆) alkyl, hydroxy, cyano, nitro,, -O(C₁-C₆)alkyl, -COOH and -NH₂.
As used herein, the term "-O(C₁-C₆"Alkyl" or "alkoxy" means unsubstituted or substituted (C₁-C₆An alkyl radical having an oxygen atom bonded thereto. Representative alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy, n-butoxy, 1-methylpropoxy, 2-methyl Propyloxy or 1,1-dimethylethoxy. Substituted alkoxy group means -O(C₁-C₆An alkyl group wherein the alkyl group is substituted with one or more groups selected from, but not limited to, halogen, hydroxy, -O (C)₁-C₆)alkyl, nitro, cyano, -COOH, -NH₂,-NH(C₁-C₆)alkyl, -N[(C₁-C₆)alkyl]₂,-NHC(O)O(C₁-C₆)alkyl, -NHC(O)O(C₁-C₆)alkyl (C₆-C₁₀) aryl, -NH-PEG or (C₆-C₁₀)Aryl.
As used herein, the term "halogen (C₁-C₆"Alkyl" or "haloalkyl" means a radical wherein one or more of the hydrogen atoms of the alkyl group are substituted with one or more halogens. The monohaloalkyl radical, for example, may have a chlorine, bromine, iodine or fluorine atom. The dihalo and polyhaloalkyl radicals may have two or more identical or different halogen atoms. "halogen (C₁-C₆Examples of "alkyl" or "haloalkyl" include, but are not limited to, chloromethyl, dichloromethyl, trichloromethyl, dichloroethyl, dichloropropyl, fluoromethyl, difluoromethyl, tri Fluoromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl or difluoropropyl.
As used herein, the term "halogen" means fluoro, chloro, bromo and iodo.
As used herein, the term "PEG" means a polyethylene glycol polymer. PEG is non-toxic, non-immunogenic, non-antigenic and highly soluble in water. A variety of different molecular weights of PEG are commercially available, based on intramolecular ethylene oxide (ie, -OCH₂CH₂-) The number of repeating units. For example, PEG can be obtained from commercial suppliers such as Sigma-Aldrich Co. LLC or NOF Corporation. Some examples of PEG are but are not limited to: (a) O, O'-bis[2-(N-succinimide-succinyl)ethyl]polyethylene glycol (α,ω-bis-NHS- PEG), with a molecular weight in the range of 2000 Da to 10,000 Da, which is structurally represented below:
;

(b) Methyl-PEG-NHS ester (MS (PEG)_n, where n is 24) and has a molecular weight of 1214.39 Da, which is structurally represented below;
;
as well as;
(c) Branched trimethyl and amber quinone imide derivatives of polyethylene glycol (TMS (PEG)_n, where n is 12 and the molecular weight is 2420.80 Da). Typically, PEG has a molecular weight in the range of 1000 to 20,000 Da, which is preferably used in the preparation of PEG conjugates of compounds as described herein.
In the context of the present invention, the term "compound of formula (1c)", "derivative of compound of formula (1)" or "detail of formula (1c) is used interchangeably, and the terms Each means a compound of the formula (1) represented by the formula (1c); and a derivative thereof, an isomer, a tautomer or a pharmaceutically acceptable salt.
According to a specific embodiment, the present invention provides a compound of formula (1c),
among them,
R¹And R⁴Is H;
R²For -O(C₁-C₆)alkyl or -OC(O)(C₁-C₆)alkyl;
R³Hydroxyl or -O(C₁-C₆)alkyl;
Among them, (C₁-C₆An alkyl group is unsubstituted or is -NH₂,-NHC(O)O(C₁-C₆)alkyl (C₆-C₁₀Aryl or -NH-PEG substituted;
And all isomeric or tautomeric forms or pharmaceutically acceptable salts thereof.
According to another specific embodiment, the present invention provides a compound of formula (1c),
among them,
R¹With R⁴Representing H independently or not;
R²With R³Is independently selected from H and a hydroxyl group;
Represents a single or double bond;
And stipulates if R²With R³Both are hydroxyl, then R¹And R⁴For non-existent andRepresents a double bond;
And all isomeric or tautomeric forms or pharmaceutically acceptable salts thereof.
Representative compounds of formula (1c) according to the invention include:









And all isomeric or tautomeric forms or pharmaceutically acceptable salts thereof.
A derivative of the compound of the formula (1), which is represented by the structural formula (1c), can be produced by a method known in the art. The reagents employed in the preparation of the compounds of formula (1c) may be commercially available or may be prepared by procedures known in the art.
R¹And R⁴H; and R²With R³Is independently selected from hydroxyl and -O (C₁-C₆)alkyl, where R²And R³At least one of them is -O(C₁-C₆A compound of the formula (1c) of the alkyl group can be prepared by reacting a compound of the formula (1), which is a compound of the formula (1a) in an alcohol selected from the group consisting of methanol, ethanol, propanol or butanol. In combination with toluene or benzene, an alkylating agent such as trimethylformane diazomethane in an organic solvent such as hexane or toluene is reacted at a temperature ranging from -5 ° C to 5 ° C for 1 to 5 hours. Compounds 1 and 2 were prepared in this manner.
R¹And R⁴H; and R²With R³Is independently selected from hydroxyl and -OC(O) (C₁-C₆) alkyl to make R²With R³At least one of them is -OC(O)(C₁-C₆Alkyl; where (C₁-C₆)alkyl group is -NHC(O)O(C₁-C₆)alkyl (C₆-C₁₀The compound of the formula (1c) substituted with an aryl group can be produced by reacting a compound of the formula (1), which is a compound of the formula (1a) in a solvent such as tetrahydrofuran (THF), dichloromethane ( DCM) or dimethylformamide (DMF) with concomitant reagents such as N-benzyloxycarbonyl-L-alanine (Cbz alanine) or N-(9-fluorenylmethoxycarbonyl)-L-propylamine Acid (Fmoc alanine), in the presence of a coupling agent such as benzotriazol-1-yl-oxytripyrrolidinium hexafluorophosphate (PyPOP), (benzotriazol-1-yloxy) three (two Methylamino) hexafluorophosphate (BOP), N, N'-dicyclohexylcarbodiimide (DCC) or propanephosphonic anhydride (T3P), and bases such as N,N-diisopropyl B The amine (DIPEA), triethylamine or 4-dimethylaminopyridine (DMAP) is reacted at a temperature ranging from -5 ° C to 5 ° C for 1 to 20 hours. Compound 3 was prepared in this way. The compound thus obtained can be deprotected via catalytic reduction in an acidic environment created using an acid such as diluted HCl using a catalyst such as Pd/C or Pt/C in a solvent such as methanol, ethanol, ethyl acetate or THF. To obtain a compound of the formula (1c), wherein With R³Is independently selected from hydroxyl and -OC(O) (C₁-C₆) alkyl to make R²With R³At least one of them is -OC(O)(C₁-C₆Alkyne; where the (C₁-C₆)alkyl group -NH₂Replaced. Compound 4 was prepared in this way.
R¹And R⁴H; and R²With R³Is independently selected from hydroxyl and -OC(O) (C₁-C₆) alkyl to make R²With R³At least one of them is -OC(O)(C₁-C₆Alkyne; where the (C₁-C₆)alkyl group -NH₂The substituted compound of formula (1c) can be used for the action of polyethylene glycol by dissolving the compound in an organic solvent such as DCM, methanol or DMF in the presence of a base such as triethylamine or DIPEA. And adding a polyethylene glycol (PEG) polymer such as O, O'-bis[2-(N-succinimide-succinyl)ethyl]polyethylene glycol (α,ω-bis-NHS- PEG) or methyl-PEG-NHS ester, and stirred at a temperature ranging from 25 ° C to 35 ° C for 10-20 hours to obtain a compound of formula (1c) wherein R²And R³Is independently selected from hydroxyl and -OC(O) (C₁-C₆) alkyl to make R²With R³At least one of them is -OC(O)(C₁-C₆Alkyne; where the (C₁-C₆The alkyl group is substituted by -NH-PEG. Compound 8 was prepared in this manner.
The compound of formula (1) can be oxidized to remove at R¹With R⁴Position of hydrogen via a reagent such as m-chloroperoxybenzoic acid in an organic solvent selected from dichloromethane or dichloroethane at a temperature ranging from 25 ° C to 35 ° C for 10-20 hours to Obtaining a compound of formula (1c) wherein R¹And R⁴For non-existence, and R²And R³It is a hydroxyl group. Compound 5 was prepared in this way.
The N-hydroxy group of the compound of the formula (1) can be converted to NH (deoxygenation) by adding a compound of the formula (1) in an organic solvent selected from methanol, ethanol or DMF to a solvent For example, a solution reagent in THF, DCM or DMF such as bis(cyclopentadienyl)titanium(IV) dichloride and activated zinc, and stirred at a temperature ranging from -5 ° C to -50 ° C for 0.1 hr to 1 hr, To obtain a compound of formula (1c) wherein R¹And R⁴H or non-existent, and R²And R³H. Compound 6 was prepared in this way.
The mercapto derivative of the N-hydroxy group of the compound of formula (1) can be passed via a compound of formula (1) in a solvent such as dichloromethane, pyrimidine or dichloroethane with an accompanying reagent such as ethyl chloroform. Or acetic anhydride, prepared in the presence of a base such as pyrimidine, TEA or DMAP at a temperature ranging from -5 ° C to 30 ° C for from 1 hr to 5 hr to obtain a compound of formula (1c), wherein R²And R³Is independently selected from hydroxyl and -OC(O) (C₁-C₆) alkyl to make R²With R³At least one of them is -OC(O)(C₁-C₆)alkyl. Compound 7 was prepared in this way.
The compound of formula (1c) which is a derivative of the compound of formula (1) can be optionally converted into a pharmaceutically acceptable salt thereof.
The compound of the formula (1) or a pharmaceutically acceptable salt thereof or a derivative of the formula (1c) has an anticancer activity. This has been demonstrated by testing a representative compound of the invention for a wide range of cancer cells, including compounds of structural formula (1), structural formula (1a), structural formula (1b) and structural formula (1c).
The compound of the formula (1) or an isomer or tautomer thereof or a pharmaceutically acceptable salt or a derivative of the formula (1c) can be administered as a medicament and administered as a pharmaceutical composition. Patients who have a need for it. The compound of the formula (1) or an isomer or tautomer thereof or a pharmaceutically acceptable salt or a derivative of the formula (1c) can be administered to a patient diagnosed with cancer.
Accordingly, the invention is also related to a compound of formula (1), in particular a compound of formula (1a) or a compound of formula (1b) or an isomer or tautomer or pharmaceutically acceptable salt thereof Use of a derivative of the class or formula (1c) for the manufacture of a therapeutic agent for cancer.
The invention further relates to a therapeutically effective amount of a compound of formula (1) or an isomer or tautomer thereof or a pharmaceutically acceptable salt thereof or a derivative of formula (1c), and a pharmaceutical A pharmaceutical composition of an acceptable excipient or carrier. The pharmaceutical composition is provided for the treatment of cancer. A therapeutically effective amount of a compound of the formula (1) or a stereoisomer thereof or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a derivative thereof of the structural formula (1c), which is an active ingredient of a pharmaceutical preparation, may be It may range from about 0.01 mg to 1000 mg; or may range from about 0.1 mg to 750 mg; or may range from about 0.5 mg to 500 mg; or may range from about 1 mg to 250 mg.
The use of the compound of the present invention, comprising a compound of the formula (1) or an isomer or tautomer thereof or a pharmaceutically acceptable salt or a derivative of the formula (1c), for the treatment of cancer . The compounds of the invention are used to reduce, inhibit or reduce the proliferation of tumor cells and provide for a reduction in tumor size. Representative cancers that can be treated with the compounds of the invention include, but are not limited to, leukemia, lung cancer, brain cancer, non-Hodgkin's lymphoma, Hodgkin's disease, liver cancer, kidney cancer, bladder cancer, urinary tract cancer, breast cancer, head and neck Cancer, endometrial cancer, lymphoma, melanoma, cervical cancer, thyroid cancer, gastric cancer, germ cell tumor, cholangiocarcinoma, extracranial cancer, sarcoma, mesothelioma, malignant fibrous histiocytoma, retinoblast Tumor, esophageal cancer, multiple myeloma, pancreatic cancer, ependymoma, neuroblastoma, skin cancer, ovarian cancer, recurrent ovarian cancer, prostate cancer, testicular cancer, colorectal cancer, lymphoproliferative disease, refractory Multiple myeloma, anti-multiple myeloma or myeloproliferative disease.
According to a specific embodiment of the present invention, the cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, adult acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, non- Small cell lung cancer, small cell lung cancer, brain stem glioma, glioblastoma, cardiac cell tumor including cerebellar cardiac cell tumor and cerebral heart cell tumor, visual pathway glioma, thalamic glioma, cerebellar primordial Neuroectodermal, pineal tumor, medulloblastoma, primary central nervous system lymphoma, mantle cell lymphoma, Hodgkin's disease, hepatocellular carcinoma, renal cell carcinoma, embryonal carcinosarcoma (Wilms 'tumor', bladder cancer, urinary tract cancer, Ewing's sarcoma tumor family, osteosarcoma, rhabdomyosarcoma, soft tissue sarcoma, mesothelioma, breast cancer, endometrial cancer, oral cancer, melanoma, cervical cancer, thyroid cancer, stomach cancer , germ cell tumor, cholangiocarcinoma, extracranial cancer, bone malignant fibrous histiocytoma, retinoblastoma, esophageal cancer, multiple Myeloma, pancreatic cancer, ependymoma, neuroblastoma, skin cancer, ovarian cancer, recurrent ovarian cancer, prostate cancer, testicular cancer, colorectal cancer, lymphoproliferative disease, refractory multiple myeloma, anti- Multiple myeloma or myeloproliferative diseases, etc.
According to another embodiment of the present invention, the cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, adult acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, Non-small cell lung cancer, small cell lung cancer, brain stem neuronoma, glioblastoma, cardiac cell tumor including cerebellar cardiac cell tumor and cerebral heart cell tumor, visual pathway glioma, pineal tumor, medullary cell Tumor, primary central nervous system lymphoma, mantle cell lymphoma, Hodgkin's disease, hepatocellular carcinoma, renal cell carcinoma, bladder cancer, urinary tract cancer, osteosarcoma, breast cancer, endometrial cancer, oral cancer , melanoma, cervical cancer, thyroid cancer, gastric cancer, malignant fibrous histiocytoma, retinoblastoma, esophageal cancer, multiple myeloma, pancreatic cancer, neuroblastoma, skin cancer, ovarian cancer, prostate cancer, Testicular cancer, colorectal cancer, lymphoproliferative disease or myeloproliferative disease, etc.
According to a further embodiment of the invention, the cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, adult acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, non- Small cell lung cancer, small cell lung cancer, brain stem neuronoma, glioblastoma, cardiac cell tumor including cerebellar cardiac cell tumor and cerebral heart cell tumor, medulloblastoma, renal cell carcinoma, bladder cancer, urinary tract cancer , breast cancer, oral cancer, melanoma, cervical cancer, thyroid cancer, stomach cancer, pancreatic cancer, prostate cancer or colorectal cancer.
According to a specific embodiment, the present invention provides a method of treating cancer in a patient by administering to a patient a therapeutically effective amount of a compound of the formula (1) or a derivative or isomer thereof of the formula (1c) Or tautomer or pharmaceutically acceptable salt.
According to another embodiment, the invention provides a method of treating cancer in a patient by administering to the patient a therapeutically effective amount of a compound of formula (1a) or a compound of formula (1b) or an isomer thereof Or tautomer or pharmaceutically acceptable salt.
According to yet another embodiment, the invention provides a method of treating cancer in a patient by administering to the patient a therapeutically effective amount of a derivative of formula (1c) or an isomer or tautomer thereof or A pharmaceutically acceptable salt.
a compound of formula (1) or a pharmaceutically acceptable salt thereof or a derivative of formula (1c); or an isomer or tautomer or pharmaceutically acceptable salt thereof, can be administered orally, It is administered nasally, topically, subcutaneously, intramuscularly, intravenously or in other modes of administration.
The method of administration suitable for a particular situation depends on the type of cancer to be treated and the stage of the cancer. Further, the method of administration can be optimized by a physician using methods known in the art.
By convention, the range of dosages that are appropriate for a particular situation depends on the type of cancer being treated and the condition of the individual condition or disease. The selected dose level can be determined by the skilled physician according to the relevant circumstances, including the symptoms to be treated (cancer), factors such as the age, weight and physical health and response of the individual patient, pharmacokinetics, severity of the disease, etc. The factors known to the medical field are determined immediately by the chosen route of administration. The actual dosage of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient effective to achieve a desired therapeutic response to a particular patient (patient in need of treatment), a composition, and a non-toxic to the patient. Medicine mode. On average, the daily dose of the active compound of the patient (one or more of the compounds of the invention) may range from about 0.05 mg to about 500 mg per kg or from about 0.5 mg to about 200 mg per kg or about A range of from 1 mg to about 100 mg per kg, or any dosage range falling within the broader range of dosage ranges as indicated herein. In the case of higher body weight patients in need of treatment, the dosage of the active compound may range from about 1 mg to about 1000 mg per kg or from about 5 mg to about 500 mg per kg. The desired dose of the active compound, i.e., the compound of the present invention, can be selected within a wide range. The daily dose administered is selected to achieve the desired therapeutic effect in patients receiving cancer treatment. Higher or lower daily doses may also be administered if desired.
A pharmaceutical composition comprising a compound of formula (1) or an isomer or tautomer thereof or a pharmaceutically acceptable salt or a derivative of formula (1c), which is accompanied by other pharmaceutically acceptable Excipients such as wetting agents, solubilizing agents such as surfactants, vehicle liquids, isotonic agents, fillers, colorants, masking perfumes, lubricants, disintegrating agents, diluents, binders, plasticizers, emulsifiers, Ointment bases, emollients, thickeners, polymers, lipids, oils, solubilizers, complexing agents or buffer substances for tablets, coated tablets, capsules, granules, powders, creams, ointments, gels, A syrup, emulsion, suspension or form of a solution suitable for parenteral administration.

In a specific embodiment of the invention, a compound comprising a compound of formula (1) or a derivative of formula (1c); or an isomer or tautomer thereof, a pharmaceutically acceptable salt The composition can be used with one or more anticancer agents such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil , cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin, doxorubicin, soft Equivalent, etoposide, fluorouracil, hexamethyl melamine, hydroxyurea, ifosfamide, leucovorin, lomustine, double Mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, hydrogen splash Nisson (prednisolone), prednisone, procarba Procarbazine, streptozocin, tamoxifen, thioguanine, vinblastine, vincristine, vindesine, Aminoglutethimide, 5-azacytidine, cladribine, busulfan, diethylstilbestrol, 2',2'-difluorodeoxygenate Glycosides, docetaxel, red-9-(2-hydroxy-3-indolyl) adenine, ethinyl estradiol, 5-fluorodeoxyuridine, 5- Fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin , interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotan, pacific violet Alcohol (paclitaxel), pentostatin, N-phosphinium carboxyacetamido-L-aspartate (PALA), lucamycin, teniposide, propionic acid Testosterone, thiotepa, trimethyl melamine, uridine, vinorelbine, alsterpaullone, butyrolactone I, 2-(2-hydroxyethylamino)-6- (3-Chloroanilino)-9-isopropyl hydrazine, indirubin-3'-monohydrazine, kenpaullone, olomoucine, iso-oromoxin, N⁹-Isopropyl-Oromoxin, purvalanol A, roscovitine,S- the isomer Rosvitine and WHI-P180 [4-(3'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline] binding; for cancer as described herein treatment.
It is to be understood that modifications that do not materially affect the activity of the various embodiments of the invention are included within the scope of the invention disclosed herein. Accordingly, the following examples are intended to illustrate but not to limit the scope of the invention.

example
The following terms/symbols/abbreviations/chemical formulas are used in the examples:


Example 1
Separation of culture number PM0895172 from marine sources
a) Separation medium composition:
1% peptone, 2% glucose, 0.2% calcium carbonate, 0.001% cobalt chloride hexahydrate, 1.5% agar, pH (at 25 ° C) 7.5.
Artificial seawater (ASW) composition: 2.46% sodium chloride, 0.067% potassium chloride, 0.136% calcium chloride, 0.629% magnesium sulfate, 0.466% magnesium chloride, 0.018% sodium hydrogencarbonate; water.
b) procedure
Deep sea sediments were collected from the offshore area of Mumbai, Maharashtra, India, and sent to the Piramal LifeSciences division of Piramal Enterprises Limited (Formerly Piramal Healthcare Limited) in Mumbai, India. Store at -20 °C during the trip.
A molecular biology method is performed on the separation of uncultured marine radiobacteria from the collected sediment samples. For this, genomic DNA was isolated from sediment samples and amplified for the presence of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS). The culture of interest is then separated from the sediment sample with the NRPS pathway.
Samples were stored at -20 ° C to 22 ° C and later thawed to room temperature (25 ± 2 ° C) for microbial isolation. Soil samples (~2 g) were suspended in 25 ml of sterile artificial seawater (ASW) under hot pressed mash and underarm and thoroughly comminuted. 1 ml of this suspension was transferred to a test tube and vortexed for 30 seconds. Vortex the tube for 30 seconds. Prepare up to 10 in sterile ASW^-5Serial dilution. Put 100 μl of 10^-3The dilution is surface coated in a dish containing the separation medium. The plates were incubated at room temperature (25 ± 2 ° C) until colonies were observed. After 20-22 days of culture, the colonies present in this medium were streaked on a Petri dish containing the separation medium (as mentioned above). The isolate was purified and provided with culture ID number PM0895172. Thus, the culture number PM0895172 was separated from the grown microorganisms into a single isolate.

Example 2
Purification of culture number PM0895172
a) Purification medium composition:
1% peptone, 2% glucose, 0.2% calcium carbonate, 0.001% cobalt chloride hexahydrate, 1.5% agar; pH (at 25 ° C) 7.5.
b) Procedure: The culture number PM0895172 was streaked into a culture dish containing the purification medium. The dishes were incubated at 27 ° C for ten days. One isolated colony from the culture dish was transferred to fresh slant medium containing ISP agar (HiMedia, India). The slant medium was incubated at 27 ° C for ten days.

Example 3
Maintenance of producer strain - culture number PM0895172
This particular strain was maintained in a slant medium containing ISP4 medium (HiMedia, India). After dissolving the medium by heating, the resulting solution was distributed in a test tube and sterilized at 121 ° C for 30 minutes. The tube was cooled and allowed to cure in an inclined position. The agar tilt medium was streaked with well-grown culture number PM0895172 and incubated at 27 ° C to 29 ° C until good growth was observed. The well-grown culture strain was stored in a refrigerator at 4 ° C to 8 ° C.

Example 4
Fermentation of culture number PM0895172 in a shaking flask
a) seed medium composition:
0.2% calcium carbonate, 0.5% sodium chloride, 0.5% corn syrup, 0.75% peptone, 1.5% glucose, 0.75% yeast extract; demineralized water; pH 7.
b) Procedure:
50 ml of the above medium was distributed in a 500 ml Erlenmeyer flask and sterilized at 121 ° C for 30 minutes. The conical flask was cooled to room temperature (25 ± 2 ° C) and each conical flask was inoculated on a bevel with a ring of well-growing production (culture number PM895172) and rotated at 30 ° C with a rotating shaker (± 1 ° C) was shaken at 230 rpm to 250 rpm for 72 hours to provide a seed culture.
c) Production medium composition:
1% peptone, 2% glucose, 0.2% calcium carbonate, 0.001% cobalt chloride hexahydrate, 1.5% agar, and pH (at 25 ° C) 7.5.
d) Procedure:
100 ml of production medium in a 500 ml Erlenmeyer flask was sterilized at 121 °C for 30 minutes, cooled to 29 °C to 30 °C and mentioned in Example 4b at 3% (v/v) Plants are grown.
e) Fermentation parameters:
The production flask was incubated on an shaker at 30 ° C for 96 hours at 220 rpm. The production flasks were harvested (harvest pH: 7.0 to 8.0) and the entire culture was extracted from each of the medium flasks with the same volume of solvent mixture (methanol: ethyl acetate (1:9)). These harvested flasks were maintained at room temperature for 4-6 hours for extraction of the supernatant after separation. The supernatant was used for testing anticancer activity.

Example 5 Preparation of a culture strain in a shake flask of a fermentation tank batch
a) Seed medium 274 (1) composition:

Glucose 15 g, corn syrup 5 g, peptone 7.5 g, yeast extract 7.5 g, calcium carbonate 2 g, sodium chloride 5 g, demineralized water 1.0 L, pH 6.5-7.5 (before sterilization).
b) Procedure:
200 ml of the above medium was distributed in a 1000 ml Erlenmeyer flask and sterilized at 121 ° C for 30 minutes. The conical flasks were cooled to room temperature, and each conical flask was inoculated on a bevel with a ring of well-growing production line (PM0895172) and oscillated at 230-250 rpm at 29 ° C -30 ° C with a rotary shaker. 70-74 hours to obtain a seed culture.

Example 6
Culture number No. PM0895172 in the fermentation tank
a) Composition of the production medium:
Soy protein 腖 10 g, glucose 20 g, calcium carbonate 2 g, cobalt chloride 0.001 g, demineralized water 1.0 L, pH 7.0 (before sterilization).
b) Procedure:
500 L of production medium was sterilized in situ in a 1000 L fermentation tank with 200 ml desmophen as an antifoaming agent at 121 ° C for 20 minutes, cooled to 29 ° C to 30 ° C and in 8 to 10 L in Example 5b The obtained seed culture plants are planted.
c) Fermentation parameters:
Temperature 29 ° C - 30 ° C, tip speed 0.94 m / s, aeration 250 lpm and harvest time 24 hr. The harvested pH of the culture broth was 6.5-7.5. Production of a compound of formula (1a) or a compound of formula (1b) which is active in the fermentation broth is determined via HPLC and the biological activity is tested against anticancer activity.
Example 7
Separation and characterization of compounds of structural formula (1a)
The whole broth (500 L) harvested in step c) of Example 6 was extracted with ethyl acetate (500 L). The organic layer was separated using a stacked disc separator (Alfa Laval (USA), model LAPX404) and concentrated to give a crude extract (150 g).

The crude extract was treated by column chromatography (gel, solvent: isopropanol / chloroform). The active compound was extracted with 1-2% isopropanol in chloroform and concentrated to give a concentrated compound (30 g).
This material was purified by column chromatography (RP C-18 gum, solvent: water / acetonitrile). The obtained active compound is extracted with 40% acetonitrile in water and evaporated to obtain 2 g of the desired semi-pure compound of formula (1) (compounds of formula (1a) and formula (1b)) mixture).
Further purification was carried out via reversed phase preparative HPLC as follows:
Column: Water’s X-Bridge RP-18 (250 mm X 19 mm, 10 μ)
Effervescent solution: acetonitrile: water (50:50).
Flow rate: 25 ml/min
Detection (UV): 220 nm
The collected active peaks were evaporated to dryness to obtain 1 g of the active compound (compound of formula (1a)).
Final purification of the active compound via gelatin preparative HPLC as follows:
Column: Water’s sunfire silica, (150 mm X 19 mm, 5 μ)
Effervescent solution: chloroform: methanol (98.5: 1.5)
Flow rate: 15 ml/min
Detection (UV): 240 nm
Rt time: 8-9 min.
The extract containing the active compound from the repeated injection was concentrated to obtain 145 mg of the compound of the pure structural formula (1a). The physical and spectral properties of the compound of formula (1a) are provided in Table 1.
Analytical HPLC conditions are as follows:
Column: Kromasil RP-18 (150 mm X 4.6 mm, 3.5 μ)
Eluent: Gradient (10% acetonitrile to 100% acetonitrile to water at 20 minutes followed by an additional 5 minutes with 100% acetonitrile)
Flow rate: 1 ml/min
Detection (UV): 220 nm
Residence time: 17-18 min (purity > 99 %)

Table 1: Physical and spectral properties of compounds of structural formula (1a)

Example 8
Isolation and Characterization of the Compound of the Structural Formula (1b) The semi-pure compound comprising the compound of the formula (1a) and the compound of the formula (1b) obtained after the RP-18 preparative HPLC in Example 7 is subjected to RP- 18 Preparative HPLC to obtain 15 mg of the pure structural formula (1b). The physical and spectral properties of the compound of formula (1b) are provided in Table 2.
Analytical HPLC conditions are as follows:
Column: Kromasil RP-18 (150 mm X 4.6 mm, 3.5 m)
Eluate: Gradient (10% acetonitrile to 100% acetonitrile to water at 20 minutes followed by an additional 5 minutes with 100% acetonitrile)

Flow rate: 1 ml/min
Detection (UV): 220 nm
Residence time: 15-17 min (purity > 99%)

Table 2: Physical and spectral properties of compounds of structural formula (1b)

Example 9
2-hydroxy-N-(18-hydroxy-22-isopropyl-6-methoxy-7,19-dimethyl-5,8,11,17,20,24-hexaethoxytetrahydrogen Pyridazine[6,1-f:6',1'-o][1,4,7,10,13,16]pentazaoxacyclohexadecane-23-yl)-2-(2- Hydroxy-5-isobutyl-6-methyltetrahydro-2H-pyran-2-yl)propanamide (Compound 1)
2-hydroxy-2-(2-hydroxy-5-isobutyl-6-methyltetrahydro-2H-pyran-2-yl)-N-(22-isopropyl-6,18-dimethoxy Base-7,19-dimethyl-5,8,11,17,20,24-hexaoxatetrahydrodiazine-[6,1-f:6',1'-o][1, Preparation of 4,7,10,13,16]pentazaoxycyclohexadecano-23-yl)propanamide (Compound 2) TMSCHN ₂ (trimethylformamidinyldiazomethane, 48 μl, 0.097 Methanol (2M solution in hexane) was added dropwise to a compound of formula (1a) (described in Example 7, 20 mg, 0.024 mmol) in methanol (1 ml) and toluene (1 ml) The solution was stirred and the resulting reaction mixture was stirred at 0 ° C for 3 h then warmed to room temperature. After completion of the reaction, the solvent was removed to obtain a solid, which was purified by column chromatography (yield, 1% methanol in dichloromethane) to obtain 5 mg of compound 1 and 3 mg of compound 2.
Compound 1: ¹ H NMR (CDCl _3, 300 MHz): δ 7.98, 7.10, 6.92, 6.54, 6.20, 5.52, 5.50, 5.30, 5.16, 5.14, 5.07, 4.96, 4.54, 4.51, 4.13, 4.05, 3.80, 3.18, 3.12, 2.79, 2.14, 2.04, 1.98, 1.95, 1.77, 1.71, 1.68, 1.60, 1.52, 1.45, 1.15, 1.10, 1.05, 0.98, 0.95, 0.90, 0.88, 0.85; ESI-MS: m/e 839 (MH ) ^- .
Compound 2: ¹ H NMR (CDCl _3, 300 MHz): δ 7.85, 7.15, 7.10, 6.20, 5.82, 5.42, 5.38, 5.30, 5.15, 4.98, 4.80, 4.60, 4.15, 3.95, 3.85, 3.70, 3.25, 3.15 , 2.85, 2.23, 2.15, 1.98, 1.95, 1.77, 1.71, 1.68, 1.65, 1.60, 1.52, 1.44, 1.15, 1.10, 1.05, 0.98, 0.95, 0.90, 0.88, 0.85; ESI-MS: m/e 853 ( MH) ^- .

Example 10
18-Hydroxy-23-(2-hydroxy-2-(2-hydroxy-5-isobutyl-6-methyltetrahydro-2H-pyran-2-yl)propanylamino)-22-isopropyl Base-7,19-dimethyl-5,8,11,17,20,24-hexaoxydisuccinazine [6,1-f:6',1'-o][1,4 ,7,10,13,16]pentazaoxycyclohexadecane-6(7H,13H,22H)-yl 3-(((benzyloxy)carbonyl)amino)propionate (compound 3) preparation.
Method I:
Benzyloxycarbonylalanine (Cbz alanine, 27 mg, 0.12 mmol) and PyBOP (75 mg, 0.14 mmol) were added to a compound of formula (1a) in THF (5 ml) at 0 ° C (described in the example) In a solution of 7,40 mg, 0.048 mmol), DIPEA (51 μl, 0.29 mmol) was added dropwise. The resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (5 mL) andEtOAcEtOAc Saturated NaHCO _3, the organic layers were washed with brine, dried by anhydrous Na ₂ SO ₄ and concentrated to obtain crude compound which was purified by column chromatography (silica gel in 1% methanol in dichloromethane) To obtain pure compound 3.
Yield: 12 mg; ¹ H NMR (CDCl _3, 300 MHz): δ 7.90, 7.39, 7.2, 6.45, 6.34, 6.15, 5.65, 5.50, 5.38, 5.30, 5.15, 5.10, 4.90, 4.60, 4.50, 4.10, 3.85, 3.7, 3.65, 3.55, 3.4, 3.2, 3.15, 2.7, 2.6, 2.5, 2.3, 2.19, 1.98, 1.95, 1.77, 1.71, 1.68, 1.65, 1.60, 1.52, 1.44, 1.15, 1.10, 1.05, 0.98, 0.95, 0.90, 0.88, 0.85; ESI-MS: m/e 1030 (MH) ^- .

Method II:
Benzyloxycarbonylalanine (Cbz alanine, 67.5 mg, 0.302 mmol) in T3P (0.216 ml, 0.363 mmol, 50 wt.% in ethyl acetate) in acetonitrile (20 ml, 0.121 mmol) Cool the solution and stir for 10 min. The compound of formula (1a) (described in Example 7, 100 mg, 0.121 mmol) in acetonitrile (20 ml, 0.121 mmol) was added to the above solution, followed by dropwise addition of triethylamine (0.051 ml, 0.363 mmol) . The resulting reaction mixture was stirred for 4 hrs. After the reaction was completed, water (10 ml) was added to the reaction mixture and the mixture was extracted with ethyl acetate (3 x 20 ml). The organic layers were washed with saturated NaHCO _3, washed with water, brine, dried and concentrated to obtain crude compound via over anhydrous Na ₂ SO _4, which was purified by column chromatography (silica gel, in dichloromethane 1-2 % methanol) to obtain compound 3. Yield: 38 mg.

Example 11
18-Hydroxy-23-(2-hydroxy-2-(2-hydroxy-5-isobutyl-6-methyltetrahydro-2H-pyran-2-yl)propanamine)-22-isopropyl -7,19-Dimethyl-5,8,11,17,20,24-hexaoxydisuccinazine [6,1-f:6',1'-o][1,4, Preparation of 7,10,13,16]pentazaoxycyclohexadecane-6(7H,13H,22H)-yl 3-propionate (Compound 4).
1 equivalent of HCl and a catalytic amount of 10% Pd/C were added to a solution of compound 3 (30 mg, 0.029 mmol) obtained in Example 10 in methanol (3 ml) and the resulting reaction mixture Stir at room temperature for 2 hours. After the reaction was completed, the reaction mixture was filtered through celite and concentrated to give Compound 4.
Yield: 15 mg; ¹ H NMR (DMSO-d _6; 300 MHz): δ 7.92, 7.30, 7.10, 6.75, 5.85, 5.45, 5.35, 5.15, 5.00, 4.85, 4.16, 3.86, 3.46, 3.10, 2.85, 2.73, 2.56, 2.15, 1.85, 1.80, 1.70, 1.68, 1.65, 1.60, 1.52, 1.44, 1.15, 1.10, 1.05, 0.98, 0.95, 0.88, 0.82, 0.80; ESI-MS: m/e 896 (MH) ^- .

Example 12
N-(6,18-dihydroxy-22-isopropyl-7,19-dimethyl-5,8,11,17,20,24-hexaoxy-3,4,4a,5,6,7 ,8,9,10,11,15,16,16a,17,18,19,20,22,23,24-tetrahydrodiazine-[6,1-f:6',1'-o ][1,4,7,10,13,16]pentazaoxycyclohexadecano-23-yl)-2-hydroxy-2-(2-hydroxy-5-isobutyl-6-methyltetra Preparation of hydrogen-2H-pyran-2-yl)propanamide (Compound 5).
A solution of the compound of formula (1a) (30 mg, 0.036 mmol) obtained in Example 7 obtained in Example 7 with m-chloroperoxybenzoic acid (18 mg, 0.104 mmol) at 0 °C The resulting reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (20 ml) and extracted with dichloromethane. The organic layers were washed with brine, washed with water, and dried through anhydrous Na ₂ SO _4, and concentrated to obtain crude compound, which was purified by column chromatography (silica gel, 5% methanol in dichloromethane) to obtain to Compound 5.
Yield: 20 mg; ¹ H NMR (DMSO-d _6, 300 MHz): δ 10.34, 9.62, 7.84, 7.52, 7.45, 7.43, 7.07, 6.68, 6.19, 5.77, 5.64, 5.51, 5.41, 5.04, 5.02, 4.94, 4.17, 4.12, 2.20, 1.98, 1.90, 1.60, 1.51, 1.33, 1.23, 1.04, 0.90, 0.87, 0.82; ESI-MS: 846 (M+Na).

Example 13
2-hydroxy-2-(2-hydroxy-5-isobutyl-6-methyltetrahydro-2H-pyran-2-yl)-N-(22-isopropyl-7,19-dimethyl -5,8,11,17,20,24-hexaoxytetrahydrotetrapyrazine[6,1-f:6',1'-o][1,4,7,10,13,16 Preparation of pentanitroxacyclohexadecano-23-yl)propanamide (Compound 6).
A clean, flame dried round bottom flask equipped with a stir bar was evacuated and rinsed with Ar. Cp ₂ TiCl ₂ (193 mg, 0.774 mmol) and a solution of activated zinc (411 mg, 6.29 mmol) in degassed THF (3 ml) were stirred for 45 min. The color of the reaction mixture changed from dark red to olive green. The reaction mixture was cooled to -30 ° C and was added dropwise with a solution of the compound of formula (1a) (40 mg, 0.048 mmol) in methanol (3 ml) obtained in Example 7 over 3 min. The reaction mixture was stirred for 45 min maintaining the bath temperature between -30 °C and -10 °C. The reaction mixture was warmed to room temperature and saturated at _{5% K 2 CO 3 (5} ml) and partition between ethyl acetate (20 ml). The organic layer was removed by pipette and filtered through a Whatman glass microfiber filter (GF/F type) to remove the insoluble titanium salt. The aqueous layer was extracted with ethyl acetate (4×20 ml) and the organic layer was filtered through watman glass microfiber filter (GF/F type) after each extraction. The combined organic _layers were dried over anhydrous Na ₂ SO via and concentrated to obtain a solid, which was purified by column chromatography (silica gel, 70% ethyl acetate in hexanes) via, to obtain Compound 6.
Yield: 8 mg; ¹ H NMR (DMSO-d _6, 300 MHz): δ 8.32, 7.78, 6.87, 6.65, 6.25, 5.75, 5.59, 5.32, 5.28, 5.21, 5.15, 4.95, 4.77, 4.73, 4.45, 4.23, 4.12, 3.94, 3.34, 2.73, 2.27, 2.18, 2.08, 1.99, 1.88, 1.63, 1.35, 1.30, 1.23, 1.04, 0.90, 0.87; ESI-MS: 817 (M+Na).

Example 14
18-Hydroxy-23-(2-hydroxy-2-(2-hydroxy-5-isobutyl-6-methyltetrahydro-2H-pyran-2-yl)propanamine)-22-isopropyl -7,19-Dimethyl-5,8,11,17,20,24-hexaoxydisuccinazine [6,1-f:6',1'-o][1,4, Preparation of 7,10,13,16]pentazaoxycyclohexadecane-6(7H,13H,22H)-ylacetic acid (Compound 7).
Pyridine (0.0255 ml, 0.30 mmol) was added to a solution of the compound of formula (1a) (0.050 g, 0.060 mmol) obtained in Example 7 in dichloromethane (10 ml) and stirred for 15 min. Then, acetamidine chloride (0.043 ml, 0.60 mmol) was added. The resulting reaction mixture was stirred at room temperature for 2 hr. The reaction mixture was extracted with ethyl acetate (3 x 30 ml). The combined organic layers were diluted with 1% HCl (5 ml), water (10 ml) wash, dried by anhydrous Na ₂ SO ₄ and concentrated. The crude product was purified by column chromatography (2% methanol in dichloromethane) to afford compound 7.
Yield: 15 mg; ¹ H NMR (CDCl _3, 300 MHz): δ 7.92, 7.28, 6.85, 6.45, 6.15, 5.45, 5.36, 5.20, 5.15, 4.90, 4.85, 4.65, 4.21, 3.80, 3.15, 2.35, 2.25, 2.12 , 1.98, 1.95, 1.77, 1.71, 1.68, 1.60, 1.52, 1.40, 1.15, 1.05, 0.98, 0.95, 0.90, 0.88, 0.86; ESI-MS: 891 (M+Na).

Example 15
Preparation of a PEGylated conjugate (Compound 8).
N-Ethyl-N-isopropylpropan-2-amine (135 ml, 0.779 mmol) was added at 0 ° C to compound 4 (200 mg, obtained in Example 11 in DMF (10 ml, 0.223 mmol) a solution of 0.223 mmol), and a portion of O,O'-bis[2-(N-succinimido]-succinylamino)ethyl]polyethylene glycol (722 mg, 1.448 mmol) was added and The resulting reaction mixture was stirred at room temperature overnight. DMF was removed under high speed vacuum to obtain a solid which was purified via a sephadex G-15 size exclusion chromatography column.
The standard curve was constructed using a series of free structural formula (1a) concentrations in DMF and water for its absorbance at 269 nm. The concentration of the PEGylated conjugate is determined according to the content of the compound of the formula (1a) using the following formula:
Concentration of PEG conjugate = PEGylated conjugate absorbance - (intercept / slope)
The content of the compound of the formula (1a) in the PEGylated conjugate form (Compound 8) was found to be 10%.

Example 16
Biological evaluation of compounds of formula (1a):
The following terms/symbols/abbreviations/chemistry are used in pharmacological tests:


In vitro assays are designed as described in the cited documents BMC Cancer, 2010, 10, 610, 1-11; the disclosures of which are incorporated by reference for the purposes of reference.
Monolayer cell test step 1
Maintenance of cell lines will be human tumor cell lines: Panc-1 (pancreatic cancer), HCT 116 (colorectal cancer), ACHN (renal cell carcinoma), Calu-1 (lung cancer), MiaPaca2 (pancreatic cancer), FADU (head and neck) Carcinoma), PC3 (prostate cancer), G361 (melanoma), MDA-MB435S (melanoma), HeLa (cervical cancer) in minimal essential medium with Eagle's basic salt obtained by AMIMED (BioConcept - Switzerland) Growing in (MEM – EBS). Tumor cell lines: MDA-MB231 (breast cancer), JURKAT (T cell leukemia), H460 (small cell lung cancer) were cultured in RPMI 1640 (AMIMED, BioConcept, Switzerland) medium. All tumor cell lines were supplemented with 10% fetal bovine serum (FBS) (GIBCO), 1% penicillin/streptomycin (Sigma), and 1% anti-anti (GIBCO) and grown in T-175 tissue. Culture flask (Nunc). The non-tumor cell line MCF-10A was cultured in Mammary Epithelial Basal Medium Mammary Epithelial Basal Medium (MEBM) with all standard supplements (Lonza, Catalog. No. CC-3150). All cells were grown in a 5% CO ₂ incubator at 37 °C. Cells were subcultured at 80-90% confluence. The attached cells were trypsinized and maintained using trypsin-EDTA (Sigma). All cell lines were purchased from ATCC (Rockville, MD, USA).

Step 2
Sample Preparation Compounds of formula (1a) (described in Example 7) were dissolved in DMSO to provide the desired 20 mM stock solution. Eight different concentrations of the compound of formula (1a) were prepared via serial dilution of the stock solution, resulting in a 200-fold higher concentration (compared to the test concentration). The compound of formula (1a) was tested at concentrations of 0.0001 μM, 0.001 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM and 3 μM, respectively. Each concentration was evaluated in triplicate.

Step 3
Method for determining the IC ₅₀ of a compound of the formula (1a):
a) Plant different cancer/normal cells in tissue culture grade 96-well plates at a density of 3000 cells/200 μL well and restore them in a 5 ± 0.2% CO ₂ incubator humidified at 37 ± 0.5 °C. hour.
b) After 24 hr, add 1 μL of 200 X (200 times higher than the desired concentration to 200 X) of the compound of formula (1a) to the above tissue culture plate grown in cancer/normal cells. . The final concentration of DMSO was 0.5% in the wells. DMSO was used as the vehicle control group.
c) After 24 hr, the plates were removed from the CO ₂ incubator and 5 μL of CCK-8 (Dojindo Molecular Technologies, Inc. USA, catalog. no. CK04-20) was added to each well.
d) Place the plate at 37 °C for 2 hr.
e) Record the absorbance at 450 nm.
f) Calculate the percentage of cytotoxicity using the following formula.

Check visually evaluate the effect IC ₅₀ value of the curve - via the concentration. Select the geometric mean value of ₅₀ for testing IC fourteen cell lines of average calculations.
The results are presented in Table 3, as provided below.

Table 3: IC cancer cell structure different from the compound of formula (1a) of the normal cells ₅₀ value

Conclusion In vitro studies have shown that compounds of structural formula (1a) exhibit an IC _{50 in the} range of 0.0013 μM to 0.025 μM in different cancer cells; whereas in normal cells (MCF-10A), they exhibit an IC _{50 of} 0.019 μM. This data shows that compounds of structural formula (1a) are active against highly proliferating cancer cell lines.

Example 17
test
Detection of FITC Annexin V cell apoptosis:
The classic technique for detecting apoptosis, the annexin V assay, is the most commonly used method for detecting apoptosis by flow cytometry. One of the earliest features of apoptosis is the translocation of phospholipidylserine from the inner layer to the outer layer of the cell membrane, thereby exposing the phospholipid lysine to the environment.
Annexin V (cat. no. 556420, BD Biosciences, USA) binds to phospholipid lysine exposed to the cell surface and recognizes cells at an earlier stage of apoptosis.
a) HeLa cancer cells (described in step 1 of Example 16) were planted in tissue culture grade 96-well plates at a density of 0.5 × 10 ⁶ cells/2000 μL well and allowed to humidify at 37 ± 0.5 °C. 5 ± 0.2% CO ₂ incubator for 12 hours to recover.
b) After 12 hr, add 5 μL of 200 X (200 times higher than the desired concentration to 200 X) of the compound of formula (1a) (Example 16 Step 2) to HeLa cancer cells The above tissue culture plate. The final concentrations of the compounds of formula (1a) are 0.0003 μM, 0.003 μM and 0.01 μM. The final concentration of DMSO in the wells was 0.5%.
c) After 48 hr, the plates were removed from the CO ₂ incubator and stained for FITC annexin using the Apoptosis Detection Kit (catalog number: 556547, BD biosciences, USA) according to the manufacturer's instructions.
d) The treated cells were analyzed for Annexin V detection in a BD flow cytometer. The population present in M1 and M2 was determined using FACS analysis.

RESULTS: The vehicle control cells showed 2.01% Annexin V positive cells (M2), while the cells exposed to the compound of formula (1a) at 0.0003 μM, 0.003 μM and 0.01 μM for 12 hr showed 33.80 %, 40.27 %, respectively. It was positive (M2) for Annexin V staining with 59.05% of cells (as shown in Figure 5).
Conclusion: This data indicates that the compound of formula (1a) induces cell death via apoptosis in HeLa cancer cells.

Example 18
High-content screening of protein expression studies in HeLa cells with high levels of Cellomics images of cyclin D, nuclear factor-Κb (p65) [NFkB (p65)], ribosomal protein S6 (pS6), Protein expression of tumor protein 53 (p53), protein c-jun N-terminal kinase (pJNK), cleaving caspase 9, cleaving caspase 3 and cleaved poly(ADP-ribose) polymerase (PARP) (described in step 1 of Example 16). These primary resistance systems were purchased from Santacruz Biotechnology, USA. Briefly, 1 X 10 ⁴ cells were seeded in clear-bottom 96-well tissue culture grade black plates (Nunc, USA) and allowed to attach for 24 h, then replaced with fresh medium at 0.01 μM, 0.003 μM and 0.0003 The compound of formula (1) at a concentration of μM (described in step 2 of Example 16) was treated and cultured for 1 h and 3 h, respectively. At each time point, cells were fixed with 3.7% formaldehyde (Sigma St. Louis, MO) for 10 minutes at RT, followed by permeabilization with 0.15% Triton X-100 (Sigma St. Louis, MO) for 10 minutes. The cells were blocked with 5% bovine serum albumin for 2 hours. Following the blocking step, a specific primary antibody was added for 1 h, and primary antibodies of different proteins were localized with Dylight548 (red) labeled secondary antibody (Thermofisher, USA). After secondary antibody incubation, the nuclei were stained with Hoechst 3342 (blue) (Sigma, USA). Immunofluorescence was determined by scanning the disk on a Cellomics Array Scan ^R VTI HCS reader (Thermo-Firis Scientific Inc., Waltham, MA). All data points were analyzed using Cellomics' Analysis Analysis bio-algorithm and the quantitative data was expressed as % activation compared to untreated cells. 1000 cells were counted for each replicate well and the results were expressed as mean ± SE.
The results are summarized in Table 4 and Table 5.
Table 4: Effect of compounds of structural formula (1a) on the expression of p53, pJNK, cleaving caspase 9, cleaving caspase 3 and cleaving PARP proteins in HeLa cells after 3 hours


Table 5: Effect of Compounds of Structural Formula (1a) on Cyclin D, NFkB (p65) and pS6 Protein Expression in HeLa Cells After 3 Hours


Conclusion Compounds of structural formula (1a) significantly downregulate the protein expression of NFkB (p65), pS6 and cyclin D in cancer cells. It also upregulates p53, Pjnk, and cleaved caspase-9 expression, followed by a significant upward regulation of cleavage of caspase 3 and cleavage of PARP that drive cancer cells toward apoptosis.

Example 19
The biological evaluation of the compound of the formula (1b) and the compounds 1 to 8 is as described in the above-mentioned Example 16 design test. Determination of the IC ₅₀ values of the compounds prepared in vitro as described in Example 16 and the compounds of formula (1b) (described in Example 8) and the compounds obtained in Examples 9 to 15 (corresponding to compounds 1 to 8). .
Check visually evaluate the effect IC ₅₀ value of the curve - via the concentration. Calculating an average value for the geometric mean of six selected IC cell lines tested (PC3, MDA MB, HCT116, MCF7, MIAPaCa2, MCF10A) of _50.
The results are presented in Table 6, which is provided below.
IC ₅₀ value of the compound with a compound of different cancer cell lines and normal cells in the structural formula (1b) of 2-9: Table 6


- not tested Conclusion vitro test shows that the compound (i) of the formula (1b) of the display range of 0.018 μM to 0.071 μM of IC ₅₀ in various cancer cells; in normal cells (MCF-10A) which shows the IC ₅₀ of 0.030 μM (ii) Compounds 1 to 8 showed an IC _{50 in the} range of 0.009 μM to 1.045 μM in different cancer cells; whereas in normal cells (MCF-10A) it showed an IC _{50 in the} range of < 0.010 μM to 0.830 μM.
These data show that the compound of formula (1b) and compounds 1 to 8 are active against highly proliferating cancer cell lines.

no

Claims (22)

a compound of formula (1);





Structural formula (1)
Wherein in the compound of formula (1), when the bond Representing a single bond and R ¹ is H; the compound is referred to as the compound of formula (1a); and when the bond When a double bond and R ¹ is absent; This compound is referred to the structure of the compound of formula (1b) of the;
Or a homoisomer or a tautomer or a mixture thereof, or a derivative; or a pharmaceutically acceptable salt thereof. The compound of claim 1, wherein the compound is the compound of the formula (1a), which is characterized by:
a) the molecular weight is 826.9,
b) the molecular formula C ₃₇ H ₆₂ N ₈ O ₁₃ ,
c) IR (KBr) spectra 3325, 2960, 2872, 1755, 1641, 1524, 1445, 1331, 1306, 1288 and 1254 cm ^-1 ,
d) ¹ H NMR spectrum (500 MHz, CDCl ₃ ): δ 9.20, 8.00, 7.10, 6.90, 6.40, 6.10, 5.40, 5.38, 5.29, 5.10, 4.90, 4.80, 4.70, 4.60, 3.90, 3.70, 3.15, 2.70 , 2.30, 2.10, 1.98, 1.95, 1.77, 1.71, 1.68, 1.60, 1.52, 1.40, 1.15, 1.10, 1.05, 0.98, 0.95, 0.90, 0.88 and 0.85 (as provided in Figure 1),
e) ¹³ C NMR spectrum (75 MHz, CDCl ₃ ): δ 176.0, 172.0 (3), 171.0, 170.0, 169.0, 99.9, 78.9, 77.2, 71.7, 53.5, 52.9, 49.3, 49.0, 47.1, 46.7, 46.2, 42.2, 41.0, 39.8, 29.9, 27.4, 24.8, 24.6, 24.2, 24.1, 23.2, 21.7, 21.5 (2), 20.6, 19.9, 19.4, 18.1, 12.9 and 11.7 (as provided in Figure 2). The compound of claim 1, wherein the compound is a compound of the formula (1b), which is characterized by:
a) molecular weight 824.4,
b) the molecular formula C ₃₇ H ₆₀ N ₈ O ₁₃ ,
c) IR (KBr): 3342, 3030, 2953, 1752, 1650, 1524, 1421, 1293, 1255 and 1239 cm ^-1 ,
d) ¹ H NMR spectrum (500 MHz, CDCl ₃ ): δ 9.20, 8.00, 7.19, 7.0, 6.18, 6.14, 5.80, 5.41, 5.39, 5.30, 5.10, 4.80 (2), 4.20, 3.70, 3.19, 2.62 , 2.27, 2.10, 2.01, 1.98, 1.77, 1.71, 1.62, 1.58, 1.44, 1.40, 1.39, 1.37, 1.20, 1.10, 1.05, 0.96, 0.91, 0.90, 0.86 and 0.85 (as provided in Figure 3),
e) ¹³ C NMR spectrum (75 MHz, CDCl ₃ ): δ 176.0, 171.8, 171.7, 170.8, 170.6, 170.3, 168.9, 144.6, 98.8, 79.0, 77.2, 71.7, 53.6, 53.5, 50.6, 49.1, 47.1, 46.4, 42.6, 41.0, 39.8, 29.9, 27.4, 24.7, 24.6, 24.2, 24.0, 22.1, 21.5, 21.4, 19.8, 19.5, 19.4, 18.1, 17.2, 12.9 and 11.8 (as provided in Figure 4). The compound of claim 1, wherein the compound is a derivative of the compound of the formula (1), which is represented by the formula (1c),



Structural formula (1c)
among them,
R ¹ and R ⁴ independently represent H or are absent;
R ² and R ³ are independently selected H, hydroxy, -O (C ₁ -C ₆₎ alkyl and _{-OC (O) (C 1 -C} 6) alkyl;
Represents a single or double bond;
among them,
(C ₁ -C ₆ )alkyl is unsubstituted or substituted with one or more groups independently selected from halogen, hydroxy, -O(C ₁ -C ₆ ) Alkyl, nitro, cyano, -COOH, -NH ₂ , -NH(C ₁ -C ₆ )alkyl, -N[(C ₁ -C ₆ )alkyl] ₂ , -NHC(O)O ( C ₁ -C ₆ )alkyl, -NHC(O)O(C ₁ -C ₆ )alkyl(C ₆ -C ₁₀ )aryl, -NH-PEG or (C ₆ -C ₁₀ )aryl;
(C ₆ -C ₁₀ ) aryl is unsubstituted or substituted with one or more groups independently selected from halogen, halo(C ₁ -C ₆ )alkyl, hydroxy, cyano , nitro, (C ₁ -C ₆ )alkyl, , -COOH and -NH ₂ ;
And PEG is polyethylene glycol, which is selected from O, O'-bis[2-(N-succinimide-succinyl)ethyl]polyethylene glycol (α,ω-bis-NHS- PEG), methyl-PEG-NHS ester (MS(PEG) _n , where n is 24), and a polyethylene glycol-branched trimethyl and amber quinone imide derivative (TMS(PEG) _n , Where n is 12);
With the proviso that if both R ² and R ³ are hydroxyl groups, then R ¹ and R ⁴ are absent and Represents a double bond;
Or a homoisomer or a tautomer or a mixture thereof; or a pharmaceutically acceptable salt thereof. A compound according to claim 4, which is a compound of the formula (1c);
R ¹ and R ⁴ are H;
R ² is -O(C ₁ -C ₆ )alkyl or -OC(O)(C ₁ -C ₆ )alkyl;
R ³ is hydroxy or -O(C ₁ -C ₆ )alkyl;
Wherein (C ₁ -C ₆ )alkyl is unsubstituted or is taken as -NH ₂ , Or -NH-PEG substituted;
Or a homoisomer or a tautomer or a mixture thereof; or a pharmaceutically acceptable salt thereof. For example, the compound described in claim 4,
among them,
R ¹ and R ⁴ independently represent H or absent;
R ² and R ³ are independently selected from H and hydroxy;
Represents a single or double bond;
With the proviso that if both R ² and R ³ are hydroxyl groups, then R ¹ and R ⁴ are absent and Represents a double bond;
Or a homoisomer or a tautomer or a mixture thereof; or a pharmaceutically acceptable salt thereof. The compound of any one of clauses 4 to 6, wherein the compound is:



Or a homoisomer or a tautomer or a mixture thereof; or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 7 or a homoisomer or a tautomer thereof or a pharmaceutically acceptable Accepted salts; and a pharmaceutically acceptable excipient, diluent and/or carrier. A compound according to any one of claims 1 to 7, or a homoisomer or a tautomer or a pharmaceutically acceptable salt thereof; for use in cancer therapy . A compound for use according to the invention of claim 9, wherein the cancer is selected from the group consisting of leukemia, lung cancer, brain cancer, non-Hodgkin's lymphoma, Hodgkin's disease, liver cancer, kidney cancer, bladder cancer , urinary tract cancer, breast cancer, head and neck cancer, endometrial cancer, lymphoma, melanoma, cervical cancer, thyroid cancer, gastric cancer, germ cell tumor, cholangiocarcinoma, extracranial cancer, sarcoma, mesothelioma, bone malignancy Fibrosial cell tumor, retinoblastoma, esophageal cancer, multiple myeloma, head and neck cancer, pancreatic cancer, ependymoma, neuroblastoma, skin cancer, ovarian cancer, recurrent ovarian cancer, prostate cancer, testicular Cancer, colorectal cancer, lymphoproliferative disease, refractory multiple myeloma, anti-multiple myeloma or myeloproliferative disease. A compound for use according to the invention of claim 9 or claim 10, wherein the cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia in adults, acute lymphoblastic leukemia, chronic Lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, non-small cell lung cancer, small cell lung cancer, brain stem glioma, glioblastoma, cardiac cell tumor including cerebellar cardiac cell tumor and cerebral heart cell tumor, vision Accessory neurofibroma, pineal tumor, medulloblastoma, primary central nervous system lymphoma, mantle cell lymphoma, Hodgkin's disease, hepatocellular carcinoma, renal cell carcinoma, bladder cancer, urinary tract cancer , osteosarcoma, breast cancer, endometrial cancer, oral cancer, melanoma, cervical cancer, thyroid cancer, gastric cancer, bone malignant fibrous histiocytoma, retinoblastoma, esophageal cancer, multiple myeloma, pancreatic cancer, nerve Blastoma, skin cancer, ovarian cancer, prostate cancer, testicular cancer, colorectal cancer, lymphoproliferative disease or myeloproliferative disease. A compound for use according to any one of claims 9 to 11, wherein the cancer is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, adult acute myeloid leukemia, acute lymphatic Embryonic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, non-small cell lung cancer, small cell lung cancer, brain stem neurotroph, glioblastoma, including cerebellar heart cell tumor and cerebral heart cell tumor Cardiomyoma, medulloblastoma, renal cell carcinoma, bladder cancer, urinary tract cancer, breast cancer, oral cancer, melanoma, cervical cancer, thyroid cancer, gastric cancer, pancreatic cancer, prostate cancer or colorectal cancer. The pharmaceutical composition according to item 8 of the patent application, for use in the treatment of cancer. A compound according to any one of claims 1 to 7; or an isomer or a tautomer or a pharmaceutically acceptable salt thereof for use in cancer therapy The use of a pharmaceutical agent. A method of treating cancer in a patient; comprising administering to the patient a therapeutically effective amount of the compound or any of the same according to any one of claims 1 to 7 Isomer or a tautomer or a pharmaceutically acceptable salt. A preparation procedure for a compound as described in claim 1, which comprises the following steps;
a) growing one of the culture number PM0895172 or one of its variants or mutants under aerobic conditions under a nutrient medium comprising carbon, nitrogen and nutritive inorganic salts and/or trace elements to obtain a culture solution of a compound of formula (1);
b) separating the compound of formula (1) from the culture solution;
c) Purification of the compound of formula (1). The procedure of claim 16, further comprising converting a compound of formula (1) to a pharmaceutically acceptable salt thereof. The process of claim 16, wherein the culture solution in the step (a) comprises a mixture of the compound of the formula (1a) and the compound of the formula (1b). The process of claim 16, wherein the compound of the formula (1) in the step (b) is a compound of the formula (1a). The process of claim 16, wherein the compound of the formula (1) in the step (b) is a compound of the formula (1b). The process of claim 16, wherein the compound of the formula (1) in the step (c) is a compound of the formula (1a). The process of claim 16, wherein the compound of the formula (1) in the step (c) is a compound of the formula (1b).