EP1569928A1 - Therapeutische verbindungen und verfahren - Google Patents

Therapeutische verbindungen und verfahren

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Publication number
EP1569928A1
EP1569928A1 EP03769069A EP03769069A EP1569928A1 EP 1569928 A1 EP1569928 A1 EP 1569928A1 EP 03769069 A EP03769069 A EP 03769069A EP 03769069 A EP03769069 A EP 03769069A EP 1569928 A1 EP1569928 A1 EP 1569928A1
Authority
EP
European Patent Office
Prior art keywords
optionally substituted
compound
compound according
group
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03769069A
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English (en)
French (fr)
Other versions
EP1569928A4 (de
Inventor
Barbara Martha Meurer-Grimes
Jin Yu
Gino Luigi Vairo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sarawak Government of State of Malaysia
Original Assignee
Sarawak Government of State of Malaysia
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Filing date
Publication date
Priority claimed from US10/291,863 external-priority patent/US6710075B2/en
Application filed by Sarawak Government of State of Malaysia filed Critical Sarawak Government of State of Malaysia
Publication of EP1569928A1 publication Critical patent/EP1569928A1/de
Publication of EP1569928A4 publication Critical patent/EP1569928A4/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • R 11 and R 12 are preferably each independently hydrogen or, alternatively, OR 4 and R 11 , and/or OR 5 and R 12 together form a methylenedioxy group;
  • Y is selected from the group consisting of optionally substituted phenyl, optionally substituted benzyl, optionally substituted benzoyl, optionally substituted C 3 -C 8 cycloalkyl, (preferably optionally substituted C 5 -C 6 cycloalkyl) optionally substituted CH 2 -(C 3 -C 8 cycloalkyl) (preferably optionally substituted CH 2 -(C 5 -C 6 cycloalkyl), optionally substituted 5-6 rnembered heterocyclyl, and optionally substituted CH 2 -(5-6 membered heterocyclyl).
  • the invention provides a composition
  • a composition comprising a compound of Formula (I), such as Formula (i), or a salt or prodrug thereof, together with a pharmaceutically acceptable carrier, excipient or diluent.
  • Disease states or conditions associated with cellular hyperproliferation which may be treated by compounds of the invention may include atherosclerosis, restinosis, rheumatoid arthritis, osteoarthritis, inflammatory arthritis, psoriasis, peridontal disease or virally induced cellular hyperproliferation.
  • Figure 2 Effects of Compound A on cell cycle progression and viability of THP-1 cells.
  • FIG. 3 Effects of Compound A on the proliferation of A549 cells.
  • A549 cells were cultured for 6 days with the indicated concentration of Compound A or 1 ⁇ M paclitaxel then collected and fixed in 70% ethanol prior to staining with propidium iodide and DNA content determined by flow cytometry.
  • the numbers indicate the % of cells in the various cell cycle phases relative to all cells with >2N DNA content and also the % dead cells (ie. subdiploid ⁇ 2N cells) to the left of the marker that arose during the culture period.
  • FIG. 5 Compounds A and B induce G2/M phase accumulation of K562 leukemic cells
  • A549 cells were seeded at ⁇ 10,000 cells/well and cultured in the presence of the indicted concentrations of Compound A or paclitaxel and the viable cell numbers determined by haemocytometer counting of trypan blue stained cells at the various times. On day 5 some of the cells were washed, resuspended in fresh medium lacking the various treatments and cultured for another 4 days prior to counting.
  • A549 cells were seeded at 10,000 cells/well in 6 well plates in the presence or absence of varying concentrations of Compound A (10 -50 nM) or 250 nM doxorubicin for 10 days prior to their processing and staining overnight for senescence-associated ⁇ -galactosidase activity as described previously (Dimri et al., 1995, Proc Natl Acad Sci USA 1995 92(20):9363-7). For Compound A only the 10 nM treatment is shown but there was no detectable SA- ⁇ gal activity at any other concentrations tested.
  • PC phase contrast microscopy.
  • BF bright field microscopy. Magnification x200.
  • Figure 10 Compound A inhibits growth of human tumour cells in a mouse xenograft model
  • Athymic Balb/c nude mice (Rygard and Povisen, 1969, Ada Pathol Microbiol Scand, 11: 758) were inoculated subcutaneously in the dorsal flank with 2x 10 6 PC3 cells.
  • Compound A was administered (3 mg/kg) after eight days when the tumours became palpable by intraperitoneal injection three times a week.
  • Compound A was first solubilized in ethanol then mixed 1:1 with cremaphore and diluted in saline for injection. Control animals were treated in an analogous manner with the same vehicle but lacking Compound A.
  • the data represents mean tumour volume ⁇ SEM (B) Effect of Compound A on mean tumour mass. At the end of the experiment (29 days post inoculation of PC3 cells) the mice were sacrificed, the tumours excised and then weighed. The data represents mean tumour weight ⁇ SEM.
  • the compounds of the present invention carry a sterically bulky group at the 6-oxy-position, in particular, a dioxanyl group.
  • the dioxanyl group of Formula (ii) (depicted below as sub-Formula (a)) has not previously been reported from a natural source.
  • the invention includes within its scope pharmaceutically acceptable salts, derivatives, or prodrugs of compounds of Formula (I), particularly of Formula (i), such as Compounds A and B.
  • salt, or prodrug includes any pharmaceutically acceptable salt, ester, glycoside, solvate, hydrate or any other compound which, upon administration to the recipient subject is capable of providing (directly or indirectly, for example, by chemical or in vivo enzymatic or hydrolytic degradation) a compound of the invention as described herein.
  • salts can be carried out by methods known in the art. It will also be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention, since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts.
  • Y groups of Formula (I) in particular the dioxanyl groups of compounds as depicted in Formula (i) and (ii), may possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form.
  • the invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric (chiral) centres eg., greater than about 90% ee, such as about 95% or 91% ee, preferably greater than 99% ee, as well as mixtures, including racemic mixtures, thereof.
  • Such isomers may be resolved by conventional methods, eg, chromatography, or use of a resolving agent.
  • the present invention thus provides Compounds A and B.
  • an alkyl group may be optionally substituted by one or more optional substituents as herein defined.
  • the straight, branched or cyclic hydrocarbon group (having at least 2 carbon atoms) may contain one, two or more degrees of unsaturation so as to form an alkenyl or alkynyl group, preferably a C 2 . 2 o alkenyl, more preferably a C 2 - 6 alkenyl, or a C 2 . 2 o alkynyl, more preferably a C . 6 alkynyl. Examples thereof include a hydrocarbon residue containing one or two or more double bonds, or one or two or more triple bonds.
  • alkyl is taken to include alkenyl and alkynyl.
  • aryl when used alone or in compound words such as “arylalkyl”, denotes single, polynuclear, conjugated or fused residues of aromatic hydrocarbons or aromatic heterocyclic (heteroaryl) ring systems, wherein one or more carbon atoms of a cyclic hydrocarbon residue is substituted with a heteroatom to provide an aromatic residue. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. Suitable heteroatoms include O, N, S and Se.
  • aryl examples include phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, idenyl, azulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl, thienyl, furyl, pyrrolyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl,
  • acyl is taken to refer to optionally substituted acyl.
  • Optional substituents for alkyl, aryl or acyl include halo (bromo, fluoro, chloro, iodo), hydroxy, C ⁇ - 6 alkyl (eg methyl, ethyl, propyl (n- and i- isomers)), C ⁇ - 6 alkoxy (eg methoxy, ethoxy, propoxy (n- and /- isomers), butoxy (n-, sec- and t-isomers), nitro, amino, C ⁇ - 6 alkylamino (eg methyl amino, ethyl amino, propyl (n- and i- isomers)amino), C ⁇ - 6 dialkylamino (eg dimethylamino, diethylamino, diisopropylamino), halomethyl (eg trifluoromethyl, tribromomethyl, trichloromethyl), halomethoxy (eg trifluoromethoxy, tribromomethoxy, t
  • arylalkyl and cycloalkylalkyl refer to an alkyl group (preferably straight chain) substituted (preferably terminally) by an aryl and a cycloalkyl group, respectively.
  • arylacyl and cycloalkylacyl refer to an acyl group (preferably where R is straight chain alkyl) substituted (for example, terminally substituted) by an aryl and a cycloalkyl group, respectively.
  • each of R 4 -R 7 and R-t-R 7 respectively may independently be selected from the group consisting of hydrogen, methyl, ethyl, ..-propyl, /-propyl, rz-butyl, -fee-butyl, t-butyl, cyclopropylmethyl (or cyclopropylethyl), cyclobutylmethyl (or -ethyl), cyclopentylmethyl (or -ethyl), cyclohexylmethyl (or -ethyl), phenyl, benzyl, acetyl and C-l linked saccharide.
  • Glycosidic formation may be effected chemically, eg by reacting the starting compound with a protected sugar compound in which C-l has been activated by halogenation for coupling with the hydroxyl or carboxyl groups and the sugar hydroxyls have been blocked by protecting groups.
  • glycoside formation may be effected enzymatically using an appropriate glycosyltransferase such as UDP-galactose dependent galactocyltransferase and UDP-glucose dependent glycocyltransferase (SIGMA).
  • Methods for the conversion of a carboxylic acid or ester group; ie. where X is OR 8 to an amide (X is NR 9 R 10 ) are known to the skilled person and may include treatment of a carboxylic acid with an appropriate amine in the presence of a coupling reagent such as DCC or treatment of an acid halide with the appropriate amine.
  • a coupling reagent such as DCC
  • Other methods which may be suitable are described in Larock, R.E, Comprehensive Organic Transformations pp 963- 995, VCH Publishers (1989).
  • protecting group refers to an introduced functionality which may temporarily render a particular functional group, eg hydroxy or carboxylic acid, inactive under certain conditions in which the group might otherwise be reactive.
  • Suitable protecting groups are known to those skilled in the art, for example as described in Protective Groups in Organic Synthesis (supra).
  • Suitable protecting groups for hydroxy include alkyl, (such as C ⁇ -C 6 alkyl), acyl (such as C(O)C ⁇ -C 6 alkyl, benzoyl and the like), benzyl, and silyl groups (such as trimethylsilyl, t-butyldimethyl silyl, t-butyldiphenylsilyl and the like).
  • alkyl, acyl or arylalkyl may serve as either a temporary protecting group or as a non-hydrogen R ⁇ R 8 group in Formula (I).
  • R 3 -R 5 are methyl, ethyl or propyl, preferably methyl.
  • R and R are both hydrogen.
  • -X is NR 9 R 10 where R 9 and R 10 are both hydrogen or methyl; or R 9 and R 10 are different but at least one of R 9 or R 10 is hydrogen and the other is C ⁇ - 6 alkyl, such as methyl, ethyl or propyl.
  • -Y is an optionally substituted 5-6 membered heterocyclyl group or an optionally substituted C 5 -C 6 cycloalkyl group.
  • the compounds of the invention may have use in the treatment of cancerous conditions, or other conditions associated with cellular hyperproliferation, in a subject.
  • Cancerous conditions which may be treated by the compounds of the present invention include conditions wherein the cancers or tumours may be simple (monoclonal, ie composed of a single neoplastic cell type), mixed (polyclonal, ie. composed of more than one neoplastic cell type) or compound (ie. composed of more than one neoplastic cell type and derived from more than one germ layer) and may include benign and malignant neoplasia/hyperplasia.
  • cancerous conditions which may be treated by the present invention include leukemia and breast, colon, bladder, pancreatic, endometrial, head and neck, mesothelioma, myeloma, oesophagal/oral, testicular, thyroid, uterine, prostate, renal, lung, ovarian, cervical brain, skin, liver, bone, bowel and stomach cancers, sarcomas, tumours and melanomas.
  • benign hyperplasias include those of vascular (eg hemangioma), prostate, renal, adrenal, hepatic, colon (eg colonic crypt), parathyroid gland and other tissues.
  • another aspect of the invention relates to a method for the treatment of cancer or a cancerous condition comprising the administration of an effective amount of a compound of Formula (I) and a further therapeutic agent to a subject in need thereof, and the use of said compound in the manufacture of a medicament for use in conjunction with other therapeutic agents.
  • the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 ⁇ g to 100 mg per kg of body weight per dosage, such as up to 50 mg per kg body weight per dosage.
  • the dosing regime for each subject may be dependent upon the age, weight, health and medical history of the subject and the extent and progress of the condition to be treated, and can be determined by the attending physician.
  • the active ingredient may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition.
  • compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parental (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • compositions for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter, gelatin, polyethylene glycol.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage compositions are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.
  • compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents.
  • suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine.
  • Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.
  • Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
  • Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
  • Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
  • Suitable lubricants may include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
  • Suitable time delay agents may include glyceryl monostearate or glyceryl distearate.
  • One or more embodiments of the present invention may also provide methods, compositions agents or compounds which have an advantage over (or avoid a disadvantage) associated with known methods, compositions, agents or compounds used in the chemotherapeutic treatment of cancerous conditions or conditions associated with the hyperproliferation of cells.
  • Such advantages may include one or more of: increased therapeutic activity, reduced side effects, reduced cytotoxicity to non-cancerous or non- proliferative cells, improved solubility or dispersibilty for formulation into pharmaceutical compositions, improved stability or a more readily available means of obtaining said compounds, eg. by simpler, cheaper or higher yielding synthetic or isolation processes.
  • step (f) Chromatograph the concentrate obtained under step (e) on a C-l 8 preparative column (WATERS, Nova-Pak C-l 8, 6 micron, 2.5 x 25 cm) at a flow rate of 20 mL/min using a linear gradient from 25%) to 45% acetonitrile in water in 30 minutes with 0.1%> formic acid.
  • a C-l 8 preparative column WATERS, Nova-Pak C-l 8, 6 micron, 2.5 x 25 cm
  • step (g) Collect and concentrate the eluates with the chromatographic and spectroscopic characteristics outlined in step (d) at approximately 22 minutes.
  • step (h) Chromatograph each eluate obtained under (g) on a Sephadex LH20 column using methanol as a solvent. Collect and concentrate the fractions with spectral characteristics outlined in (d). These samples were used for the structural elucidation of Compounds A and B.
  • the methanol extract obtained under (a) may be partitioned with equal volumes of water and dichloromethane. The dichloromethane phase is then processed according to steps (e) to (h).
  • MS Mass spectra were obtained on a Finnigan LCQ iontrap mass spectrometer using the ESI source in the positive ion mode. The sample was dissolved in 0.1 %FA in MeOH and introduced into the source by infusion with a syringe pump at rate of 3 ⁇ L/min.
  • the objective of this experiment was to unambiguously determine the attachment position of the dioxanyl sidechain to the cyclopentabenzofuran core in Compounds A and B.
  • Compounds A and B are cytostatic and cytotoxic for human tumour cell lines
  • Compounds A and B potently inhibited TNF- ⁇ production at broadly similar concentrations that were active in the WST-1 reduction, DNA and protein synthesis assays.
  • the effects of Compounds A and B on A549 lung epithelial carcinoma cells were also measured and the data is also included in Table 4.
  • Compounds A and B are significantly less potent for inhibition of interleukin-1 (IL-l)-induced Intercellular Adhesion Molecule-1 (ICAM-1) expression by A549 cells even though in these cells the protein and DNA synthesis inhibition occur at broadly similar concentrations as for THP-1 cells.
  • IL-l interleukin-1
  • IAM-1 Intercellular Adhesion Molecule-1
  • TNF ⁇ TNF ⁇ by THP-1 cells was measured as that released into the culture supernatant over 18 hours by sandwich enzyme-linked immunosorbent assay (ELISA) using the following mouse anti-TNF ⁇ monoclonal antibodies (capture antibody, MAB610; detection antibody, biotinylated MAB210; both from R & D Systems, Minneapolis MN, USA).
  • ICAM-1 Surface expression of ICAM-1 by A549 cells was assayed after 24 hours of culture by direct antibody binding using a europium-labelled mouse anti- ICAM-1 monoclonal antibody (R&D Systems Cat No. BBA3) and measured by time- resolved fluorescence using Delfia assay (EG&G Wallac, Turku, Finland). Reduction of WST-1 (Roche, Cat. No. 1644807) by THP-1 cells was measured after 18 hours of culture according to the manufacturer's instructions.
  • Protein synthesis was measured as the uptake of [ 14 C]-leucine (0.5 ⁇ Ci/mL) after 48 hours for THP-1 cells and 72 hours for A549 cells cultured in growth medium (RPMI-1640, 10%) FBS) containing 10%) the usual L-leucine concentration (5 mg/mL).
  • DNA synthesis was measured as the uptake of [ 14 C]-thymidine (0.5 ⁇ Ci/mL) after 48 hours for THP-1 cells and 72 hours for A549 cells in normal growth medium.
  • Compound A was assessed for cytotoxic and cytostatic activity against a panel of cell lines derived from a variety of human tumour types in addition to THP-1 and A549 cells (Table 5). These included K562 leukemic cells (Lozzio and Lozzio, 1975, Blood 45:321-34), PC3 prostate tumour cells (Kaighn et al, 1979, Invest. Urol. 17:16-23) and SF268 glioblastoma cells (Westphal et al, 1985, Biochem. Biophys. Res. Commun., 132:284-9). Compound A exhibited potent cytostatic activity in nearly all cell lines tested with GI 5 o values ranging between 1-7 nM.
  • Cell death was measured as the proportion of dead cells exhibiting sub-diploid DNA content determined by flow cytometry after staining with propidium iodide (Nicoletti et al, J. Immunol. Methods, 1991, 139:271-79).
  • Compound A was tested for activity in the National Cancer Institute in vitro anticancer drug discovery screen. For this Compound A was tested at five 10-fold dilutions ranging from 10 "4 M to 10 "8 M against a panel of different human tumour cell lines representing major types of cancer as described by Boyd and Paull, Drug Development Research, 1995, 34:91- 109. Briefly, this involved a 48 hr incubation of the cells with Compound A prior to measuring the relative cell number by staining with sulforhodamine B. GI 50 values represent the concentration of Compound A that inhibited net growth of the cells by 50% compared to untreated controls. LC 50 values represent the concentration of Compound A that resulted in a net 50%) loss (killing) of the cells relative to the start of the experiment. The data represent the average values from two such experiments conducted.
  • Cytotoxic activity of Compound A is not shared by other known related compounds lacking dioxanyloxy substitution.
  • Compound A ' displays cytotoxic activity.
  • Table 8 compares the cytostatic and cytotoxic effects of Compound A to three previously identified lH-cyclopenta[b]benzofuran lignans that lack the dioxanyloxy group at the C6-position.
  • the reference compounds are: Rocaglaol (Reference Compound 1) (Ohse et al, JNat Prod, 1996, 59(7):650-52); 4'-Demethoxy-3',4'-methylenedioxyrocaglaol (Reference Compound 2) and Methyl 4'-demethoxy-3',4'-methylenedioxyrocaglate (Reference Compound 3) (Lee et al, Chem Biol Interact, 1998, 115(3):215-28). All four compounds exhibited detectable cytostatic activity in A549 cells with Compound A being the most potent followed in decreasing order by Reference Compounds 3, 2 and 1 respectively. Importantly, of the compounds tested, other than Compound A none of the Reference
  • Table 9 shows that acetylation of the dioxanyl side chain of Compounds A and B did not reduce their biological activity since Compounds A' and B' inhibited WST-1 reduction of THP-1 leukemic with at least similar potencies to the unmodified compounds.
  • the lower IC 5 o values for all the compounds depicted in this WST-1 reduction experiment compared to the values shown in Table 4 reflects the enhanced sensitivity of the cells when treated for 3 days compared to the 18 hr treatment used in the latter assay.
  • THP-1 cells and A549 cells were pretreated with the indicated concentrations of Compound A for 1 hour prior to the addition of (1 ⁇ Ci/mL) [ 14 C] leucine (protein synthesis) or [ 14 C] thymidine (DNA synthesis) for a further 2 hours.
  • the IC 5 o values represent the concentration of Compound A required to inhibit incorporation of isotope by 50%) relative to untreated control cell cultures.
  • Compound A induces differentiation of human leukemic cell lines.
  • This widely used cell line is well characterised as a model of human myelomonocytic differentiation (Collins, Blood, 1987, 70(5): 1233-44).
  • monocytic differentiation was quantitated by measuring CD 14 surface antigen expression by flow cytometric analysis.
  • CD14 an LPS-binding protein, is expressed on the surface of cells of the myelomonocytic lineage and is normally expressed at very low levels in undifferentiated HL60 cells (Ferrero et al., R/ooi,1983,61(l):171-9).
  • Table 10 shows that Compound A at doses greater than 10 nM significantly enlianced CD 14 expression in the viable HL60 cells remaining after 4 days of culture. Taken together these data strongly indicate that Compound A has the ability to induce differentiation of human leukemic cell lines.
  • HL60 cells were cultured for 4 days with the indicated concentration of Compound A then collected and fixed in 70% ethanol.
  • Cells were then stained with mouse monoclonal anti CD14 antibody (OKM1) and this was measured using FITC-conjugated goat anti-mouse IgGl as a secondary antibody. Stained cells were visualised by flow cytometry and analysis was restricted to cells judged viable at the time of fixing based on their forward and side light-scatter characteristics. Non specific staining of cells was controlled for by incubating with secondary antibody only.
  • Cytostatic activity of Compound A is associated with a general inhibition of cell cycle progression in A549 cells
  • FIG. 2 shows that the microtubule destabilising drug paclitaxel (Sorger et al, Curr Opin Cell Biol, 1997, 9(6):807-14) which also induced THP-1 cell death, caused cells to accumulate in the G2/M phases of the cell cycle.
  • Compound A inhibits cell cycle-dependent cytotoxicity elicited by various anti-cancer agents
  • Camptothecin is an inhibitor of DNA topoisomerase 1, an enzyme required for DNA replication, and results in pertubation of the S phase of the cell cycle with subsequent cell death due to activation of an S phase checkpoint (Darzynkiewicz et al, Ann N Y Acad Sci, 1996, 803:93-100).
  • Paclitaxel inhibits microtubule function required for formation of the mitotic spindle thereby resulting in activation of an M phase checkpoint and subsequent cell death (Sorger et al, Curr Opin Cell Biol, 1997 9(6): 807- 14).
  • Figure 7 shows that 10 nM Compound A significantly reduced the cytotoxic effects of both camptothecin and paclitaxel even when these drugs were added at up to a 2000-fold excess. Compound A may, in a dominant manner, prevent the cell cycle-dependent cytotoxic effects of camptothecin and paclitaxel.
  • camptothecin resulted in accumulation of cells in S phase of the cell cycle and also increased the level of dead cells detected as those with a sub-diploid DNA content.
  • both vinblastin and paclitaxel resulted in the majority of cells arresting in the G2/M phases of the cell cycle and increased appearance of sub-diploid dead cells.
  • the presence of 10 nM Compound A prevented their characteristic cell cycle arrest and significantly inhibited their cytotoxic effects, dramatically reducing the appearance of sub-diploid dead cells.
  • Compound A had little effect on the cytotoxic effects of staurosporine, an agent which appears capable of killing cells at all active phases of the cell cycle.
  • Cytostatic effects of Compound A do not correlate with a biomarkerfor replicative senescence.
  • SA- ⁇ -gal activity a biomarker previously described to correlate well with senescence of human cells.
  • SA- ⁇ -gal activity a biomarker previously described to correlate well with senescence of human cells.
  • some anti-cancer agents that act by diverse mechanisms including doxorubicin, cisplatin, cytarabine, etoposide and paclitaxel, can all induce SA- ⁇ -gal activity in a variety of tumour cell lines (Chang et al, Cancer Res 1999, 59(15):3761-7).
  • Compound A inhibits cell proliferation but not increased cell size
  • Compound A inhibits growth of human tumour cell lines in a mouse xenograft tumour model.

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WO2014092514A1 (ko) 2012-12-14 2014-06-19 한국화학연구원 신규한 화합물, 이의 약학적으로 허용가능한 염 또는 이의 광학 이성질체, 이의 제조방법 및 이를 유효성분으로 함유하는 바이러스성 질환의 예방 또는 치료용 약학적 조성물
US10639378B2 (en) 2016-06-06 2020-05-05 Genentech, Inc. Silvestrol antibody-drug conjugates and methods of use
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