Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to novel compounds which are naphthalene diimides, more particularly tri- and tetra- substituted naphthalene diimides.
• the invention also concerns pharmaceutical compositions comprising the novel compounds and their use in therapy, particularly in cancer treatment.
• Telomeres are highly specialised DNA-protein structures that form the end of chromosomes. Telomere integrity is required for a cell to remain viable. The enzyme telomerase maintains telomere length and is over-expressed in around 90% of all cancer types, and therefore in recent years this enzyme has become a common target in cancer therapeutics. Telomerase can be inhibited by inducing telomeric DNA to form G-quadruplex structures, which cannot be accessed by the enzyme.
• G-quadruplexes are also present in the promoter regions of some genes and can regulate gene expression by disrupting the transcription mechanism.
• NDs Naphthalene imide and diimide derivatives
• DNA duplex
• Disubstituted NDs have been screened as G- quadruplex ligands, but showed limited affinity [2].
• the molecular structure of the majority of text compounds was as follows:
• R is an amine such as (CH 2 ) 2 N(CH 3 ) 2 .
• naphthalimides as anticancer agents.
• the naphthalimide nitrogen is substituted with a wide range of groups including tertiary amines. However, no naphthalene diimide compounds are synthesised.
• R 1 and R 2 are each independently divalent radicals derived from C 1-2 O alkyl, C 2 - 2 0 alkenyl, C 7-20 alkaryl, C 2-10 alkynyl, C 7-20 aralkyl, C 2-20 heteroaralkyl, C 3-30 heterocyclylalkyl, 0 3 . 30 alkylheterocyclyl, C 3-20 cycloalkyl, C 3-20 heterocyclyl, C 2-20 heteroaryl, C 5-20 aryl or C 1-10 alkoxy;
• R is H or A 2 X 4 ; wherein X 1 -X 4 are each independently selected from halo, OH, OR 3 , COH, NH 2 , NHR 3 , NR 3 R 4 , COOH, CONH 2 , COOR 3 , CONHR 3 , CONR 3 R 4 , SH, SR 3 , COR 3 or cyano; wherein R 3 and R 4 are independently selected from C 1-6 alkyl, C 6-20 aryl, C 7-20 aralkyl or R 3 and R 4 together with the nitrogen atom to which they are attached form a 3-8 membered ring, which is optionally substituted and optionally comprises other hetero atoms;
• a 1 and A 2 are each independently selected from NHR 5 ; wherein R 5 is a divalent radical derived from C 1-20 alkyl, C 6-20 aryl, C 7-20 alkaryl or C 7-20 aralkyl; wherein any of the groups R 1 -R 5 , A 1 and A 2 may be substituted with C 1-20 alkyl, C 2-20 alkenyl, C 7-20 alkaryl, C 2-10 alkynyl, C 7-20 aralkyl, C 2-20 heteroaralkyl, C 3-30 heterocyclylalkyl, C 3-30 alkylheterocyclyl, C 3-20 cycloalkyl, C 3-20 heterocyclyl, C 2-2O heteroaryl, C 5-20 aryl or C 1-10 alkoxy, halo, OH, OR 3 , COH, NH 2 , NHR 3 , NR 3 R 4 , COOH, CONH 2 , COOR 3 , CONHR 3 , CONR 3 R 4 , SH,
• the third aspect of the invention provides a compound of general formula (I) 1 or a salt, solvate or pro-drug of general formula (I), for use in therapy.
• the final aspect of the invention provides use of a compound of general formula (I), or a salt, solvate or pro-drug thereof, or a pharmaceutical composition as defined above, in the manufacture of a medicament for prophylaxis or treatment of cancer.
• substituted naphthalene diimides as dyes is well known.
• the compounds have extensively conjugated structures which makes them excellent fluorescent colorants.
• [4] discloses compounds which may be substituted on the aryl rings as well as on the nitrogen atoms of the imide groups.
• the compounds in this reference have simple alkyl or aryl chains attached to the imide nitrogens, and do not have groups corresponding to X 1 -X 4 of the present invention.
• [5] discloses naphthalene 1 ,4,5,8 tetracarboxylic bisimides which may be substituted on the ring (see substituents X and Y on the structure below):
• the compounds of the present invention have been shown to be able to stabilise G-quadruplex regions in DNA to a greater extent than the anti-cancer agents of the prior art.
• the compounds have also been shown to have better selectivity towards the G-quadruplex rather than duplex DNA, in comparison to the disubtituted naphthalene diimides already tested and reported in the literature.
• novel compounds of this invention therefore have great potential as anti- cancer drugs.
• Compounds according to the first aspect of the invention, of general formula (I), may be either tri- or tetrasubstituted. Accordingly, when the compounds are tri- substituted, R is hydrogen. Tetrasubstituted compounds wherein R is A 2 X 4 , are preferred, however, since these have been found to have a greater stabilisation effect on quadruplex DNA.
• R 1 X 1 , A 1 X 3 , R 2 X 2 and A 2 X 4 may be the same or different.
• R 1 X 1 is the same as R 2 X 2 .
• a 1 X 3 is the same as A 2 X 4 .
• X 1 -X 4 may be halo.
• halo is meant a halogen radical such as fluoro, chloro, bromo or iodo.
• the halo is chloro or bromo.
• the groups X 1 -X 4 comprise a group which is ionisable. Accordingly, particularly preferred groups for X 1 -X 4 are NH 2 , NR 3 R 4 , OH, OR 3 and NHR 3 . In particular, groups that are protonated at physiological pH are preferred, for instance, primary, secondary or tertiary amines. Typically, at least one of the groups X 1 -X 4 is a tertiary amine, for instance dimethylamine or diethylamine.
• any or all of X 1 -X 4 are NR 3 R 4 , wherein R 3 and R 4 together with the nitrogen to which they are attached form a 5-8 membered ring, typically a 5 or 6 membered ring.
• the ring may contain atoms other than carbon (and the nitrogen) atoms, for instance, it may contain an oxygen atom.
• NR 3 R 4 is preferably pyrrolidine, piperidine or morpholine.
• R 1 and R 2 are preferably linkers which act to space the groups X 1 and X 2 from the naphthalene diimide core.
• R 1 and R 2 are each, independently divalent radicals (generated by removal of H from C-H) selected from Ci -2 o alkyl, preferably C 2 - A alkyl.
• a 1 and A 2 are groups which link the naphthalene diimide core with groups X 3 and X 4 .
• these groups A 1 and A 2 are of general formula NHR 5 , wherein R 5 is a divalent radical derived from C-M O alkyl, preferably C 2 _ 4 alkyl.
• R 5 is a divalent radical derived from C-M O alkyl, preferably C 2 _ 4 alkyl.
• a 2 X 4 have structure:
• n 1-10, preferably 1-4, and p is 2-6.
• R 5 when present is identical to R 1 and R 2 .
• R 5 when present is identical to R 1 and R 2 .
• a particularly preferred group of compounds of this invention has general formula:
• n is independently, 1-10, preferably 1-4.
• Particularly preferred compounds are as follows:
• the present invention also provides the use of a compound of formula (I), or a salt, solvate or pro-drug thereof, substantially as described herein before, in the manufacture of a medicament for the prophylaxis or treatment of cancer.
• the compounds of the present invention may be present in the form of pharmaceutical acceptable salts.
• Pharmaceutically acceptable salts of the acidic or basic compounds of the invention can of course be made by conventional procedures, such as by reacting the free base or acid with at least a stoichiometric amount of the desired salt-forming acid or base.
• Pharmaceutically acceptable salts of the acidic compounds of the invention include salts with inorganic cations such as sodium, potassium, calcium, magnesium, zinc, and ammonium, and salts with organic bases.
• Suitable organic bases include N-methyl-D-glucamine, arginine, benzathine, diolamine, olamine, procaine and tromethamine.
• Pharmaceutically acceptable salts of the basic compounds of the invention include salts derived from organic or inorganic acids.
• Suitable anions include acetate, adipate, besylate, bromide, camsylate, chloride, citrate, edisylate, estolate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, maleate, mesylate, methylbromide, methylsulfate, napsylate, nitrate, oleate, pamoate, phosphate, polygalacturonate, stearate, succinate, sulfate, subsalicylate, tannate, tartrate, terephthalate, tosylate and triethiodide.
• Hydrochloride salts of compound (I) are particularly preferred.
• the compounds of general formula (I) may be prodrugs, that is compounds which are converted into the active drug at an appropriate target location in the human or animal body.
• prodrugs that is compounds which are converted into the active drug at an appropriate target location in the human or animal body.
• the amine may be oxidised to form an N-oxide, N + (-O " ) R 3 R 4 , which is a prodrug and can be bioreduced in hypoxic tissue.
• Pro-drug forms of the pharmacologically-active compounds of the invention may be compounds according to formula (I) having an acid group which is esterified or amidated. Included in such esterified acid groups are groups of the form -C(O)OR a , wherein R a is Ci -6 alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, or one of the following:
• Amidated acid groups include groups of the formula -CONR b R c , wherein R b is H, Ci -5 alkyl, phenyl, substituted phenyl, benzyl, or substituted benzyl, and R c is -OH or one of the groups just recited for R b .
• Compounds of formula (I) having an amino group may be derivatised with a ketone or an aldehyde such as formaldehyde to form a Mannich base. This will hydrolyse with first order kinetics in aqueous solution.
• the compounds of the invention can be administered to a patient in need thereof by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical administration, and inhalation.
• the compounds of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension.
• Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives.
• suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose.
• Corn starch and alginic acid are suitable disintegrating agents.
• Binding agents may include starch and gelatine.
• the lubricating agent if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
• Capsules for oral use include hard gelatine capsules in which the active ingredient is mixed with a solid diluent and soft gelatine capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
• the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride.
• Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin.
• suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth
• a wetting agent such as lecithin.
• Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
• the compounds of the present invention may be used in the treatment of a disease.
• the disease may be selected from the group consisting of cardiovascular diseases, disorders of the peripheral and central nervous system, inflammation, urological diseases, developmental disorders, cancer, metabolic diseases, viral, bacterial and endocrinological diseases and disorders of the gastroenterology system in a mammal.
• the disease may be a parathyroid gland adenoma, parathyroid gland hyperplasia, parathyroid gland carcinoma, squamous carcinoma, renal carcinoma, breast carcinoma, prostate carcinoma, lung carcinomas, osteosarcomas, clear cell renal carcinoma, prostate cancer, lung cancer, breast cancer, gastric cancer, ovarian cancer, bladder cancer, leukaemias, melanomas, lymphomas or gliomas.
• the compounds of the invention are used to treat gastric cancer.
• a therapeutically effective amount of a compound of general formula (I), or a salt, solvate or producing thereof, is administered to a patient in need thereof.
• side chains we mean the groups R 1 X 1 , R 2 X 2 , A 1 X 3 and A 2 X 2 .
• side chains we mean the groups R 1 X 1 , R 2 X 2 , A 1 X 3 and A 2 X 2 .
• the compounds are synthesised in one or two steps from key intermediates, 2,6-dibromo-1 ,4,5,8-naphthalene tetracarboxylic acid dianhydride [A] or 2,6-dichloro-1 ,4,5,8-naphthalene tetra-carboxylic acid dianhydride [B]. See scheme 1 below:
• the tetrasubstituted analogues containing four identical side chains may be synthesised from either A or B using neat amine as a solvent and heating at 15O 0 C for around 10 minutes, for instance in a microwave.
• the more economical dibromo compound A is preferentially used as a starting material.
• Trisubstituted (and occasionally disubstituted) analogues are typically obtained as subproducts in the reactions using A. It is thought that the occurrence of these subproducts is due to radical debromination of the starting materials or intermediates caused by traces of DBI (dibromoisocyanuric acid) used in the previous synthetic step.
• Compound B is preferably used for the synthesis of compounds which have different side chains. For this two-step synthesis compound B can be treated with a first amine in acetic acid and subsequently with a second amine, which substitutes the chlorine radicals.
• FIG 1 shows the synthetic route to compounds 8 and 9
• Figure 2 shows the synthetic route to compounds 12 to 31 ;
• FIG. 3 shows the synthetic route to compounds 32 and 33;
• Figure 4 shows the competition FRET results;
• Figure 5 shows cell uptake detection under fluorescent confocal microscopy: (1) - Transmission/fluorescence composite image of compound 24 localised in the nucleus of MCF7 cells after 30 min exposure at 0.5 ⁇ M; (2) - Unspecific and low uptake of compound 30, 50 ⁇ M after 30 min; (3) - Compound 12 localisation in the nucleolus, 0.5 ⁇ M after 30 min.
• Reagents, solvents and chemicals were purchased from Sigma-Aldrich, Alfa Aesar, Lancaster Synthesis, GOSS or Avocado Organics and were used as supplied without further purification. All organic solvents were anhydrous.
• Microwave irradiation was performed with an Initiator microwave from Personal
• the analytical column was C18 5 ⁇ (100 x 4.6 mm) (41622271 (W), YMC, Japan).
• the preparative column was C18 5 ⁇ (100 x 20 mm) (201022272(W), YMC, Japan). Flows were 1 ml/min for analytical and
• Preparative HPLC were performed using method C (Aqueous solvent: 0.1 % formic acid in water; Organic solvent: 0.1% formic acid in acetonitrile; Gradient: 10% aqueous for 5 minutes after injection, gradually to 60% aqueous over 25 minutes and gradually to 40% aqueous over 10 minutes). Compound isolation was achieved by fraction basification with ammonia followed by chloroform extraction and work up in the usual manner. Melting points (mp) were recorded on a Stuart Scientific SMP1 melting point apparatus and are uncorrected. "mp d250" (e.g.) refers to decomposition observed at 250 0 C.
• NMR spectra were recorded at 400 MHz ( 1 H NMR) and 100 MHz ( 13 C NMR) on a Bruker spectrometer in CDCI 3 , MeOD or DMSO-d 6 using the residual solvent peaks as internal standards.
• Coupling constant J values are given in hertz (Hz) designated as s (singlet), br s (broad singlet), d (doublet), t (triplet), dd (doublet of doublets), td (triple of doublets), tt (triplet of triplets), 4q (quartet), 5q (quintuplet) and m (multiplet).
• HRMS High resolution mass spectra
• CHN elemental analysis
• ESI electrospray ionisation
• MALDI matrix assisted laser desorption/ionisation
• LC/MS were performed using a Waters system combining a 2695 separation module, a Micromass ZQ spectrometer and a 2996 photodiode array detector (Mobile Phase: 50:50 (0.1 % formic acid in water):(0.1% formic acid in acetonitrile); Run Time: 3 minutes isocratic; Mode: Electrospray positive (ES+); MS running conditions: 3 min run time; Cone: 25. Offset: 1 ; Skimmer: 1.5; RF lens: 0.1 ; Source Heater: 150 (degrees Celsius); Gas: 400 l/hr). Synthetic Methods
• the solid in the mixture was filtered, washed with toluene (2 x 50 ml) and dried under vacuum. A first crop (6.93 g) of the product was obtained as a pale green powder. The filtrate was left to stand at RT for 48 h. The solid formed was filtered and washed with toluene (2 x 50 ml) and dried to give a second crop of the desired product (5.28 g).
• Fuming HNO 3 (12.7 ml) was added to a two-necked flask equipped with a thermometer in a -5 0 C bath.
• the mixture of isomers 3 and 4 (4.33 g, 10.6 mmol) was added portionwise over a 30 min period with good stirring and maintaining the temperature under 5 0 C. After completion of the addition the mixture was stirred for another 15 min at O 0 C and then filtered to obtain a dark orange solid that was washed with acetic acid (5 x 10 ml) and water (2 x 10ml).
• the product was purified by sublimation (1-2 mbar, 25O 0 C) to obtain an orange solid.
• thermometer compound 5 500 mg, 1 .35 mmol was dissolved in cone sulfuric acid (7 ml). The flask was heated at 100 0 C and fuming nitric acid (0.775 ml) was added dropwise maintaining the temperature around 12O 0 C. The mixture was then cooled down to 7O 0 C and poured onto ice (50 ml). The yellow solid formed was filtered and washed with cold acetic acid (5 ml). The product was purified by crystallisation from acetic acid.
• Naphthalene dianhydride (100 mg, 0.373 mmol) was suspended in N 1 N- dimethyl-1 ,3-propanediamine (2 ml) in a microwave reaction vessel. The tube was flushed with nitrogen, sealed and treated at 12O 0 C for 10 min in the microwave. Water (20 ml) was added to the mixture. The crystalline solid was taken by filtration and washed with water (2 x 10 ml), ethanol (2 x 10 ml) and ether (2 x 10 ml). The solid was then dissolved in chloroform and treated in the usual manner to afford a yellow solid.
• the appropriate tagged DNA 5'-F/W-d(GGG[TTAGGG] 3 -L4MR/4-3' for the telomeric G-quadruplex; 5'-F/W-dTATAGCTATA-HEG-TATAGCTATA- TAMRA-Z' (HEG linker: [(-CH2-CH2-O-)6]) for the duplex DNA; 5'-FAM- AGAGGGAGGGCGCTGGGAGGAGGGGCT-7/ ⁇ MR/ ⁇ -3' for the ckiti G- quadruplex; or 5'-F/ ⁇ M-CCCGGGCGGGCGCGAGGGAGGGGAGG-7VWR,4-3' for the ckit2 G-quadruplex (all purchased from Eurogentec, Southhampton, UK); was diluted to 400 nM using FRET buffer (50 mM potassium cacodylate pH 7.4) and annealed by heating at 85 0 C for 5 min and cooling down to RT over 5h.
• FRET buffer 50 mM potassium cacody
• Compound dilutions were prepared in a concentration of twice the final concentration using FRET buffer from the 1 mM stock solutions.
• 50 ⁇ l_ of annealed DNA and 50 ⁇ l_ of compound per well were put into 96-well plates and processed in a DNA Engine Opticon (MJ Research). Fluorescence readings were taken at intervals of 0.5 0 C over the range 30-100 0 C, with a constant temperature being maintained for 30 seconds prior to each reading. Irradiation was at 450-495 nm and detection at 515-545 nm.
• the raw data were imported into Origin 7.0 (OriginLab Corp.) and the graphs smoothed using a 10-point running average and subsequently normalised.
• the first derivative of the smoothed melting curve was calculated.
• the difference between the melting temperature at 0.5 ⁇ M of the compound and the melting temperature for the blank ( ⁇ T m (0.5 ⁇ M)) was used for comparison.
• the TRAP assay was carried out in three steps with an initial primer elongation step by telomerase, a subsequent removal of the primer bound ligand and a final PCR amplification of the telomerase products.
• the first step of the TRAP assay was carried out by preparing a master mix containing the TS forward primer (0.1 ⁇ g; 5'-AAT CCG TCG AGC AGA GTT- 3'), TRAP buffer (2OmM Tris-HCI [pH 8.3], 68mM KCI, 1.5mM MgCI 2 , 1 mM EGTA, 0.05% v/v Tween-20), bovine serum albumin (0.05 ⁇ g), and dNTPs (125 ⁇ M each), protein extract (500 ⁇ g/sample) diluted in lysis buffer (10 mM Tris- HCI, pH 7.5, 1 mM MgCI 2 , 1 mM EGTA, 0.5% CHAPS, 10% Glycerol, 5mM ⁇ - mercaptoethanol, 0.1 mM AEBSF).
• lysis buffer 10 mM Tris- HCI, pH 7.5, 1 mM MgCI 2 , 1 mM EGTA, 0.5% CHAPS, 10% G
• the PCR master mix was added to tubes containing freshly prepared compounds at various concentrations and to the negative control containing no drug.
• the initial elongation step was carried out for 10 min at 3O 0 C, followed by a 94 0 C for 5 min and a final maintenance of the mixture at 20 0 C.
• the QIAquick nucleotide purification kit (Qiagen) was used according to manufacturer's instructions.
• the kit is especially designed for the purification of both double and single-stranded oligonucleotides from 17 bases in length.
• the kit employs a high salt concentration buffer to bind the negatively charged oligos to the positively charged spin-tube membrane through centrifugation. An ethanol based buffer is then used to wash any impurities away before elution of the DNA using a low salt concentration solution. This was substituted with PCR-grade water in our experiments.
• telomere extended samples were then subject to PCR amplification.
• a second PCR master mix was prepared consisting of ACX reverse primer (1 ⁇ M; 5'-GCG CGG [CTTACC]3 CTA ACC-3'), TS forward primer (0.1 ⁇ g; 5'-AAT CCG TCG AGC AGA GTT-3'), TRAP buffer, BSA (5 ⁇ g), 0.5mM dNTPs and 2U of taq polymerase.
• An aliquot of 10 ⁇ l of the master mix was added to the purified telomerase extended samples and amplified at: 35 cycles of 94°C for 30 sec, 61 0 C for 1 min and 72°C for1 min. samples were separated on a 12% PAGE and visualised with Sybregreen staining. Fluorescence from drug samples were normalised against positive control containing protein only. All samples were corrected for background by subtracting the fluorescence reading of negative controls.
• HGC-7 Human cancer cell lines, breast (MCF7), lung (A549), colon (HT-29), gastric (HGC-27) and normal human lung fibroblast lines (WI-38) were purchased from American Type Cell Culture (ATCC).
• ATCC American Type Cell Culture
• the GIST882 line was a gift from Dr Jonathan Fletcher. All cell lines except HGC27 and WI38 were maintained in Dulbecco's Modified Eagles Media containing 10% foetal bovine serum (Invitrogen, UK), 0.5 mg/ml hydrocortisone (Acros Chemicals, Loughborough, UK), 2mM L-glutamine (Invitrogen, Netherlands), and nonessential amino-acids (Invitrogen, Netherlands), and incubated at 37°C, 5%CO 2 .
• the WI38 and HGC27 lines were maintained in minimum essential medium, prepared as above. All cell lines were routinely passaged at a ratio of 1 :6. SRB toxicity assay
• Short-term growth inhibition was measured using the SRB assay as described previously. Briefly, cells were seeded (4000 cells/wells) into the wells of 96 well-plates in appropriate medium and incubated overnight to allow the cells to attach. Subsequently cells were exposed to freshly-made solutions of drugs and incubated for a further 96 h. Following this the cells were fixed with ice cold trichlo-acetic acid (TCA) (10%, w/v) for 30 min and stained with 0.4% SRB dissolved in 1 % acetic acid for 15 min. All incubations were carried out at room temperature. The IC 50 value, concentration required to inhibit cell growth by 50%, was determined from the mean absorbance at 540 nm for each drug concentration expressed as a percentage of the control untreated well absorbance.
• TCA ice cold trichlo-acetic acid
• Senescence detection and quantification experiments were carried out using a commercially available kit (Senescence ⁇ -galactosidase staining kit, Cell Signalling Technology, MA, USA).
• 1x10 5 cells were seeded in a 35 mm well of a 6-well plate (Nunc, Denmark) in 2 ml of medium and compound to test. The cells were incubated for 24 hours. The medium was then removed and the cells were washed with PBS (1 x 2 ml). The cells were fixed by treating them with 1 ml of fixative solution (2% formaldehyde and 2% glutaraldehyde in PBS) for 15 minutes at RT. The fixative solution was then removed and the wells washed with PBS (2 x 2 ml).
• fixative solution 2% formaldehyde and 2% glutaraldehyde in PBS
• Freshly prepared staining solution (a mixture of 930 ⁇ l_ of 40 mM citric acid/sodium phosphate (pH 6.0), 0.15 M NaCI and 2 mM MgCI 2 , 10 ⁇ l_ of 500 mM potassium ferrocyanide, 10 ⁇ L of 500 mM potassium ferricyanide and 50 ⁇ L of 20 mg/ml 5-bromo-4-chloro-3-indolyl- ⁇ D-galactopyranoside in DMF) (1 ml) was added and the cells were incubated overnight. The senescent cells, detected by their blue pigmentation, were quantified under a light microscope.
• the stabilisation ability of the compounds towards the G-quadruplex DNA sequences was excellent. For some, concentrations of 0.5 ⁇ M were sufficient to increase the melting temperature by 35 0 C. This level of stabilisation with BRACO19 (a conventional G-quadruplex ligand) requires a concentration of 2.5 ⁇ M. The values of ⁇ Tm at 0.5 ⁇ M ( ⁇ Tm(0.5 ⁇ M)) were used for comparison as for some compounds the 1 ⁇ M concentration normally used was enough to completely avoid melting of the DNA. Consistently for the whole series, the tetrasubstituted ligands performed better than the tri- and these better than the disubstituted counterparts.
• the toxicity of the compounds against the panel of cell lines was assessed using the SRB assay.
• the compounds showed a very strong potency, especially towards the cancer cell lines.
• the results are shown in Table 2.
• Senescence phenotype was investigated in MCF7 cells treated for 1 week with sub-cytotoxic concentrations of a selection of compounds. All the compounds used in this study showed a significant increase in % of senescence cells after treatment.
• G-quadruplex interacting agents may cause telomere uncapping which may result in the fusion of two independent chromosomes.
• the tri- and tetrasubstituted compounds like other naphthalene diimide compounds, presented fluorescent properties. This allowed us to use the natural occurring florescence of a selected group of compounds to detect their localisation within MCF7 cells using confocal microscopy. Compounds 12, 17 and 24 were shown to localise exclusively in the nucleus upon exposure of 30 minutes at 0.5 ⁇ M concentrations (Figure 5-1). For compound 30 however a less intense and homogeneously distributed signal was observed upon treatment with up to 50 ⁇ M ( Figure 5-2). The compounds that concentrated in the nucleus showed preference for the nucleolus (Figure 5-3).

Landscapes

• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Nitrogen Condensed Heterocyclic Rings (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Publications (1)

Family

ID=38962374

Family Applications (1)

Country Status (5)

Cited By (5)

Families Citing this family (16)

Citations (3)

• 2007
  • 2007-11-29 GB GBGB0723439.6A patent/GB0723439D0/en not_active Ceased
• 2008
  • 2008-11-28 WO PCT/GB2008/051131 patent/WO2009068916A1/en not_active Ceased
  • 2008-11-28 US US12/745,431 patent/US8796456B2/en active Active
  • 2008-11-28 EP EP08853446.6A patent/EP2227470B1/en not_active Ceased
  • 2008-11-28 JP JP2010535460A patent/JP5366968B2/ja not_active Expired - Fee Related

Patent Citations (3)

Non-Patent Citations (8)

Also Published As

Similar Documents

Legal Events