Patent Application
20110201648
The present invention relates to poly-heteroaryl derivatives that specifically bind and stabilise quadruplex DNA. The invention also relates to the pharmaceutically acceptable salts of such compounds, processes for the preparation of pharmaceutical compositions containing and the uses of such compounds in treating cancer and infectious diseases.
“Polyheteroaryl” as used in the description and the claims means a sequence/chain consisting of aromatic heterocyclic moieties, optionally including phenylene moiety(ies) and/or having end terminal phenyl groups.
G-quadruplex DNA is currently considered as a structural element able to regulate the function of G-rich sequences. In particular, many lines of evidence suggest that formation of this peculiar DNA structure at telomeres or in specific gene promoters in a variety of human oncogenes, including c-Myc, Bcl-2, VEGF, Hif-1a, Ret, c-Kit, PDGF-A, KRAS and c-Myb (1) may inhibit cancer cell proliferation.
Telomeric DNA of human cells comprises tandem repeats of sequence 5′-TTAGGG-3′, which terminates on their 3′ side in a single-stranded overhang that was demonstrated to fold into G-quadruplex in vitro. The formation of such quadruplex-structure in vivo is hypothesized to lead to the displacement of protective proteins normally associated with telomeres (shelterin complex), therefore disrupting telomere structure leading to genomic instability.
For oncogenes promoters, stabilisation of quadruplex is expected to disrupt the local environment of proteins factors that regulates gene expression and has been well documented for c-myc (1). Hence chemical intervention able to induce or stabilize quadruplex-DNA formation has been thoroughly investigated via small molecules quadruplex binders, with the goal to control DNA-related functions (telomere elongation, oncogene transcription) in particular in cancer cells.
Compounds that interact with G-quadruplex-DNA are currently numerous in the literature, as illustrated by the number of recent reviews dedicated to them (2-9) However only few compounds present a high degree of selectivity for quadruplex- vs duplex-DNA; additionally, only a few of these compounds have been thoroughly investigated as anti-cancer agents. The leading compound of this family is telomestatin, a naturally-occurring macrocycle (isolated from Streptomyces anulatus 3533-SV4) comprised of five oxazole rings, two methyl oxazole rings and a thiazoline ring. Telomestatin represents a paradigm in terms of quadruplex-selective interactions, and shows exceptional telomerase-inhibiting properties in vitro (evaluated through either TRAP or Direct assay, with IC50-TRAP=0.6 nM and IC50-Direct Assay=58 nM (10).Additionally, telomestatin is also deeply investigated for its cellular effects and presents antiproliferative and apoptotic properties in various tumor cell lines (11 and 12). Telomestatin alters telomere integrity (13) and triggers a DNA-damage response at telomeres (14). Telomestatin induced POT1 uncapping in vitro (IC50-POT1=500 nM) and removes GFP-POT1 from telomeres in tumor cells (15). Given the harsh synthetic access of telomestatin (16), analogues have been reported (17, 18 and 19) that present interesting quadruplex-interacting properties.
However, none of these compounds show the exceptional properties of telomestatin. Additionally, none of these compounds, including telomestatin itself, has been investigated for their fluorescence properties. Given the chemical instability, the poor water-solubility and in particular, the arduous synthesis of telomestatin, its large-scale therapeutic use is questionable.
In this context, the inventors have found that new non-macrocyclic polyheteroaryl derivatives exhibit an exquisite selectivity for quadruplex-over duplex-DNA and that the association of these derivatives with their DNA targets leads to i) deep modifications of their spectroscopic properties, ii) uncap human POT1 (protection of telomere 1) protein from the telomeric G-overhang, iii) inhibit cell proliferation of human tumour cell lines. The stability and solubility in physiological media of this new family of compounds are compatible with their use in therapeutic treatments. Moreover, the inventors have found an efficient and original process for obtaining these compounds.
An object of the present invention is then to provide, as new products, such non-macrocyclic polyheteroaryl derivatives.
It also relates to a method for their synthesis.
According to another object, the invention, taking advantage of the quadruplex-interacting properties of said compounds, further relates to the use of said derivatives as quadruplex-specific probes. In particular, in view of the high fluorescence quantum yield quantum of the disclosed compounds, such compounds could be used as fluorescent probes for the detection and/or purification of quadruplex DNA or related nucleic acid structure including but not limited to quadruplex RNA.
In still another object, the invention provides pharmaceutical compositions containing said derivatives as active principles and also relates to the use of said derivatives in the manufacture of drugs for a wide range of disorders, particularly for the treatment of patients suffering from, for example, cancer or infectious diseases such as malaria.
The invention also relates to a method for treating cancer and infections diseases comprising administering an efficient amount of said derivatives to a patient in need thereof.
The polyheteroaryl derivatives of the invention are penta-, hexa-, hepta-, octa-, nona- and deca-heteroaryl derivatives (abbreviated hereafter as penta- to deca-heteroaryl) comprising
a combination of heterocycle 1 (Het-1)a and/or heterocycle 2 (Het-2)b and/or heterocycle 3 (Het-3)c and/or heterocycle 4 (Het-4)d of formulae I, II, III and IV respectively,
the N-oxides, the pharmaceutically acceptable addition salts,
said combination comprising at least two different heterocyclic moieties, and optionally comprising an (Aryl-5)e moiety of formula (V)
a, b and e, being integers from 0 to 6, c and d being integers from 0 to 2, the sum a+b+c+d+e being ≦10
Y is O or S;
Het-1 is a class of nitrogen heterocyclic-diyl ring selected in the group comprising 2,6-pyridin-diyl or 2,4-pyrimidin-diyl or 3,5-pyrazin-diyl or 2,4-(1,3,5-triazin)diyl or 3,5-(1,2,4-triazin)diyl or 2,4-oxazolin-diyl or 2,4-thiazolin-diyl;
Het-2 is a class of five-membered heterocyclic-diyl ring selected in the group comprising 2,5-oxazolin-diyl or 2,5-thiazolin-diyl, or 2,5-thiophen-diyl or 2,5-furan-diyl;
Het-3 and/or Het-4 constitutes the endings of the said penta-hexa-, hepta-, octa-, nona- and deca-heteroaryl derivatives, wherein
Het-3 is a class of nitrogen heterocyclic-yl ring selected from 2-pyridyl or 2-pyrimidyl or 4-pyrimidyl or 2-pyrazyl or 2-(1,3,5)triazyl or 3-(1,2,4)triazyl or 5-(1,2,4)triazyl or 4-oxazolyl or 4-thiazolyl;
Het-4 is a class of five-membered heterocyclic-yl selected from 2-oxazolyl or 5-oxazolyl or 2-thiazolyl or 5-thiazolyl or 2-thienyl or 2-furanyl 1;
R1 and R2, are each independently selected from hydrogen; C1-6alkyl; C1-4alkyloxy; halo; hydroxy; hydroxymethyl; nitro; amino; mono- or di(C1-4alkyl)amino; C1-4alkylmethylamino; mono- or di(C1-4alkyl)amino(C2-4alkyl)amino methyl; morpholin-4-yl C2-4alkyloxy; piperazin-1-ylC2-4alkyloxy; 4-C1-4alkylpiperazin-1-yl, monocyclic or bicyclic selected from phenyl, benzyl, naphthyl.
According to an embodiment of the invention, a, b, c and d are integers from 0 to 3, e=0, and the sum a+b+c+d being ≦10.
According to an embodiment of the invention, one or several of the above moieties are substituted with one, two or three substituents, each independently selected from C1-6alkyl; C1-4alkyloxy; halo; hydroxy; nitro; amino; mono- or di(C1-4alkyl)amino; C1-4alkylmethylamino; morpholin-4-ylC2-4alkyloxy; piperazin-1-ylC2-4alkyloxy; 4-C1-4alkylpiperazin-1-yl C2-4alkyloxy; monocyclic or bicyclic ring selected from phenyl, benzyl, naphthyl.
The invention particularly relates to the derivatives such as above defined, comprising at least 5, at least 7, at least 8, at least 9 or 10 heterocyclic moieties.
According to another aspect, the invention also particularly relates to the derivatives such as above defined, further comprising one or several, such as two, three or four, 1,3-phenylene-diyl moieties.
Particularly preferred derivatives of the invention have one of the following structures:
(Het-4 substituted by R2) -(Het-1)-(Het-4 substituted by R2)
(Het-3 substituted by R1) -(Het-2)-(Het-3 substituted by R1)
(Het-3 substituted by R1) -(Het-2)-(Het-1)-(Het-2)-(Het-3 substituted by R1)
(Het-3 substituted by R1) -(Het-2)-1,3-phenylene-diyl-(Het-2)-((Het-3 substituted by R1)
(1,3-phenylene-diyl-substituted by R1 or R2) -(Het-2)-(Het-1)-(Het-2)-(1,3-phenylene-diyl-substituted by R1 or R2)
(Het-3 substituted by R1) -(Het-2)-(Het-1) (Het-2)-((Het-3 substituted by R1)
(Het-3 substituted by R1) -(Het-2)-(Het-1)-(Het-1)-(Het-2)-((Het-3 substituted by R1).
In preferred derivatives,
In an advantageous group, in view of the G-quadruplex interacting properties of the derivatives, Het-1 is linked to two Het-2.
In preferred derivatives of said group, Het-1 is a pyridine-diyl and Het-4 is an oxazolyl, substituted by a pyridyl or a pyridyl-oxazolyl.
In a second advantageous group, Het-2 is linked to two Het-1.
In preferred derivatives, Het-2 is an oxazolyl and Het-1 is a pyridyl oxazolyl.
Specific preferred compounds according to the invention are those listed as the Example section below, especially compounds 3 and 4.
Pharmaceutically acceptable addition salts of the above defined derivatives include acid addition and base salts (including disalts) thereof.
Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate salts.
Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
Such pharmaceutically acceptable salt of an above defined derivative may be readily prepared by mixing together solutions of said derivative and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the salt may vary from completely ionised to almost non-ionised.
The present invention includes also isotopically-labelled derivatives as above-defined, preferably pharmaceutically acceptable isotopically-labelled derivatives, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2H and 3H, carbon, such as 11C, 13C and 14C, chlorine, such as 36Cl, fluorine, such as 18F, iodine, such as 123I and 125I, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 32P, and sulphur, such as 35S.
Certain isotopically-labelled derivatives according to the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
Substitution with heavier isotopes such as deuterium, i.e. 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
Substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
As shown in the Examples hereinafter, the derivatives of the invention specifically bind and stabilise quadruplex DNA and therefore are of great interest as quadruplex-specific probes. The ability of said derivatives to bind to quadruplex DNA as well as their selectivity may be measured using assays know to those skilled in the art, including the assays described in the Example section below.
They are also of great value for the treatment of various diseases.
The invention thus relates to the use of the above defined derivatives as drugs.
The invention also relates to pharmaceutical compositions comprising an effective amount of at least one derivative such as above defined in combination with a pharmaceutically acceptable carrier.
As used herein, “therapeutically efficient amount” or “efficient amount” is intended an amount of therapeutic agent such as an above-defined derivative administered to a patient that is sufficient to constitute a treatment of a disease.
As used herein, the term “treatment” of a disease refers to any act aimed at (1) slowing down or stopping the progression, aggravation, re-occurrence, dissemination or deterioration of the symptoms of the disease state or condition to which such term applies; (2) alleviating or bringing about ameliorations of the symptoms of the disease state or condition to which such term applies; and/or (3) reversing or curing the disease state or condition to which such term applies.
The derivatives of the invention intended for pharmaceutical use may indeed be administered alone or in combination with one or more other derivatives of the invention or in combination with one or more other drugs (or as any combination thereof), especially anti-cancer drugs or anti-infectious drugs. Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable carrier. The term “carrier” is used herein to describe any ingredient other than the derivative(s) of the invention. The choice of carrier will to a large extent depend on factors such as the particular mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form.
Pharmaceutical compositions suitable for the delivery of above-defined derivatives and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in ‘Remington's Pharmaceutical Sciences’, 19th Edition (Mack Publishing Company, 1995).
The pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension; for parenteral administration as a solution, suspension or emulsion; for topical administration as an ointment or cream; or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages.
Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents. The pharmaceutical compositions may, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus for oral administration, tablets containing various excipients, such as citric acid may be employed together with various disintegrants such as starch, alginic acid and certain complex silicates and with binding agents such as sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes. Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules. Preferred materials, therefor, include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration the above-defined derivative may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intradermal, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations as discussed below. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
The pharmaceutical compositions of the invention can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
For therapeutic uses an effective daily amount of active principle in the compositions will in general, be from 0.01 mg/kg to 50 mg/kg body weight, more preferably from 0.1 mg/kg to 10 mg/kg body weight. It will, of course, vary with the derivative employed, the mode of administration, the treatment desired, the disorder indicated as well as with physiological data of the patient (e.g. age, size, and weight). The total daily dose may be administered in single or divided doses. Determining appropriate dosages and regiments for administration of the therapeutic agents is are well-known in the relevant art and would be understood to be encompassed by the skilled artisan.
The above-defined derivatives and pharmaceutical compositions thereof are particularly useful to treat cancer and infectious diseases such as malaria.
The term “cancer”, as used herein, refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, and certain characteristic morphological features. This term refers to any type of malignancy (primary or metastases).
The term <<infectious disease>> as used herein, refers to a disease resulting from the infection of a mammal (human or animal) by micro-organisms such as bacteria, parasites, yeasts, fungi. Infections by a virus are also included.
The use of the above derivatives and pharmaceutical compositions thereof for making a drug for treating cancer or infectious diseases such as malaria enters into the scope of the invention.
The invention also encompasses a method for treating cancer or infectious diseases, comprising administering an effective amount of an above-defined derivative or pharmaceutical compositions thereof to a patient in need thereof.
The invention also covers a method for making the above defined derivatives.
Said method comprises
reacting X1-A-X2
with B-X3 or with X4-B-X3
wherein
Heteroaryl-1,4 being a combination of Het-1 and/or Het-2 and/or Het-3 and/or Het-4 which are as above defined,
Other characteristics and advantages of the invention are given in the following Examples, which refer to
The following examples are intended to illustrate the present invention. The compounds may also be obtained by other processes known by the one skilled in the art. 2,6-pyridine dicarboxaldehyde, 6-bromopyridine-2-carbaldehyde, p-toluenesulfonylmethyl isocyanide (TosMIC), 2-pyridylzinc-bromide, 1,3-benzene dicarboxaldehyde, 6-bromopyridine-2-carboxaldehyde, 3-bromobenzaldehyde and 5-bromo-2-furaldehyde used as starting materials, are commercially available products.
2,6-Pyridine dicarboxaldehyde (4.0 g; 29.2 mmol), TosMIC (11.4 g; 58.3 mmol) and potassium carbonate (16.3 g; 117.8 mmol) in 100 mL of methanol were heated at reflux during 3 hours. The solvent was evaporated in vacuo and the residue was poured into brine solution and extracted with dichloromethane (4×150 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel column with dichloromethane-ethanol (95-5) as the eluent to give the titled compound as yellow solid (5.6 g, 90%), m.p.=190-191° C.; 1H NMR (300 MHz, CDCl3) 7.56 (d; 2H; J=8.0); 7.74 (s; 2H); 7.80 (t; 1H; J=8.0 Hz); 7.96 (s; 2H); 13C NMR (75 MHz, CDCl3) 118.7, 125.7, 138.0, 147.4, 150.8, 151.0; SM m/z 214.1 (M+1); Anal. calcd for C11H7N3O2: C, 91.97; H, 3.31; N, 19.71. found: C, 61.51, H, 3.43, 19.48.
Under an argon atmosphere, the intermediate 1 (0.1 g; 0.5 mmol) and TMEDA (0.2 mL; 1.0 mmol) are dissolved in 5 mL of anhydrous THF and cooled at −78° C. A solution of LiHMDS 1M in THF (0.5 mL; 0.5 mmol) was added dropwise and stirred during 30 minutes at −78° C. and one hour at −40° C. The mixture was cooled again at −78° C. and 1,2-diiodoethane (0.5 g; 1.9 mmol) was added. After stirring over night at room temperature the mixture was poured into a sodium thiosulfate solution and extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 95-5) to give the intermediate 2 as beige solid (117 mg, 50%), 1H NMR (300 MHz, CDCl3) 7.58 (d; 2H; J=8 Hz); 7.69 (s; 2H); 7.85 (t; 1H; J=8 Hz); 13C NMR (75 MHz, CDCl3) 119.5, 129.7, 138.7, 147.0, 157.2, 151.5; SM m/z 465.8 (M+1), 487.8 (M+23).
Method 1: A mixture of intermediate 2 (65 mg; 0.14 mmol), 2-pyridylzinc-bromide (1.2 mL; 0.60 mmol) and Pd(PPh3)4 (12 mg; 0.01 mmol) were heated at reflux in 2 mL of anhydrous THF during 4 hours. Water was added and the mixture was extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 95-5) to give the expected compound 1 as beige solid (13 mg, 25%), m.p.=242-245° C.; 1H NMR (300 MHz, CDCl3) 7.40 (m; 2H); 7.83-7.88 (m; 5H); 7.97 (s; 2H); 8.23 (d; 2H; J=7.9 Hz); 8.77 (d; 2H; J=4.5 Hz); 13C NMR (75 MHz, CDCl3) 119.7, 123.3, 125.6, 128.6, 137.8, 138.5, 146.6, 147.9, 150.9, 152.3, 161.5; SM m/z 368.0 (M+1), 390.0 (M+23).
Method 2: A mixture of intermediate 1 (0.1 g; 0.5 mmol), 2-bromopyridine (91 μL; 0.9 mmol), palladium diacetate (11 mg; 0.05 mmol; 10 mol %), PCy3.HBF4 (35 mg; 0.1 mmol; 20 mol %), copper (I) iodide (0.18 g; 0.9 mmol), cesium carbonate (0.61 mg; 1.88 mmol) and 1.3 mL of anhydrous toluene were submitted under microwave irradiation conditions (130° C., 150 W) during 4 hours. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 95-5) to give the compound 1 (8 mg, 33%) which is identically with than that described in method 1 above.
6-Bromopyridine-2-carbaldehyde (4.0 g; 21.5 mmol), TosMIC (4.2 g; 21.5 mmol) and potassium carbonate (6.0 g; 43.4 mmol) were heated at reflux during 3 hours in 75 mL of methanol. The solvent was evaporated in vacuo and the residue was poured into brine solution and extracted with dichloromethane (4×100 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 95-5) to give the intermediate 3 as yellow solid (2.9 g, 60%); m.p.=90-91° C.; 1H NMR (300 MHz, CDCl3) 7.38 (dd; 1H; J=3.5 & 5.3 Hz); 7.57 (m; 2H); 7.73 (s; 1H); 7.97 (s; 1H); 13C NMR (75 MHz, CDCl3) 118.1, 126.4, 127.6, 139.3, 142.4, 148.0, 151.6; SM m/z 225.0 and 227.1 (M+1).
A mixture of intermediate 1 (0.10 g; 0.5 mmol), intermediate 3 (0.10 g; 0.5 mmol), palladium diacetate (11 mg; 0.05 mmol; 10 mol %), PCy3.HBF4 (35 mg; 0.09 mmol; 20 mol %), copper (I) iodide (0.1 g; 0.5 mmol), cesium carbonate (0.3 mg; 0.9 mmol) and 1.3 mL of anhydrous toluene were submitted under microwave irradiation conditions (130° C., 150W) during 6 hours. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 95-5) to give the expected compound 2 as beige solid (60 mg, 36%); 1H NMR (300 MHz, CDCl3) 7.70 (d; 2H; J=7.5 Hz); 7.80 (d; 1H; J=7.5 Hz); 7.85 (d; 2H; J=7.5 Hz); 7.90 (s; 1H); 7.93 (d; 2H; J=5 Hz); 8.00 (s; 1H); 8.04 (d; 1H; J=5 Hz); 8.20 (d; 1H; J=7.5 Hz); SM m/z 358.1 (M+1).
A mixture of intermediate 1 (0.10 g; 0.5 mmol), intermediate 3 (0.21 g; 0.9 mmol), palladium diacetate (11 mg; 0.05 mmoles; 10 mol %), PCy3.HBF4 (35 mg; 0.09 mmol; 20 mol %), copper (I) iodide (0.18 g; 0.9 mmol), cesium carbonate (0.61 mg; 1.9 mmol) and 1.3 mL of anhydrous toluene were submitted under microwave irradiation conditions (130° C., 150W) during 6 hours. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 95-5) to give the expected compound 3 as beige solid (52 mg, 22%) m.p. >250° C.; 1H NMR (300 MHz, CDCl3) 7.65 (m; 1H); 7.75 (m; 2H); 7.90-8.05 (m; 9H); 8.10 (s; 1H); 8.20 (m; 2H); SM m/z 524.2 (M+23).
A mixture of 1,3-di(oxazol-5-yl)benzene, obtained as described by Sambavisarao and Vrajesh (Synthsis, 2007, 3653), (0.21 g; 1 mmol), 6-bromopyridine-2-carboxaldehyde (0.24 g; 1.3 mmol; palladium diacetate (64 mg; 0.28 mmol, 28 mol %), PCy3.HBF4 (62 mg; 0.17 mmol; 17 mol %), copper (I) iodide (0.43 g; 2.3 mmol), cesium carbonate (1.4 g; 4.3 mmol) and 4 mL of anhydrous dioxane were heated in a sealed tube at 130° C. during 2 hours. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 95-5) to give the intermediate 4 as beige solid (83 mg, 30%); 1H NMR (300 MHz, CDCl3) 10.27 (s; 1H); 8.45-8.40 (m; 1H); 8.20 (s; 0.5H); 8.10-8.05 (m; 2H); 7.83 (dd; 1H; J=7.8 & 1.6 Hz); 7.71 (s; 1H); 7.61 (t; 0.5H; J=7.93 Hz); SM m/z 423.1 (M+1).
A mixture of dicarboxaldehyde intermediate 4 (75 mg; 0.18 mmol), TosMIC (86 mg; 0.44 mmol) and potassium carbonate (126 mg; 0.9 mmol) in 8 mL of absolute ethanol was heated at reflux during 2 hours. The solvent was evaporated in vacuo and the residue was poured into brine solution and extracted with dichloromethane (3×50 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel column with dichloromethane-ethanol (95-5) as the eluent to give the titled compound as off-white solid (55 mg, 62%), m.p.>250° C.; 1H NMR (300 MHz, CDCl3) 8.22 (br s; 0.5H); 8.17 (d; 1H; J=7.8 Hz); 8.03 (s; 1H); 7.99 (t; 1H, J=7.8 Hz), 7.93 (s; 1H); 7.85-7.76 (m; 2H); 7.68 (s; 1H); 7.64-7.57 (m; 0.5H); SM m/z 501.1 (M+1); Anal. calcd for C28H16N6O4′0.75 H2O: C, 65.43; H, 3.40; N, 16.36, found: C, 65.92, H, 3.37, 15.92.
A mixture of intermediate 1 (0.18 g; 0.84 mmol), 3-bromobenzaldehyde (0.50 g; 2.7 mmol), palladium diacetate (80 mg; 0.36 mmol; 43 mol %), PCy3.HBF4 (68 mg; 0.18 mmol; 20 mol %), copper (I) iodide (0.37 g; 1.9 mmol), cesium carbonate (1.2 g; 3.7 mmol) and 3.5 mL of anhydrous dioxane were heated in a sealed tube at 130° C. during 18 hours. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 98-2) to give the intermediate 5 as yellow solid (50 mg, 14%); 1H NMR (300 MHz, CDCl3) 10.15 (s; 1H); 8.66 (s; 1H); 8.45 (d; 1H; J=7.7 Hz); 8.04 (d; 1H; J=7.6 Hz); 7.98-7.92 (m; 1.5H); 7.76 (d; 1H; J=7.9 Hz); 7.72 (t; 1H; J=7.7 Hz); SM m/z 444.0 (M+Na); Anal. calcd for C25H15N3O4. 0.25 H2O: C, 70.50; H, 3.64; N, 9.87, found: C, 70.11, H, 3.51, 9.75.
A mixture of dicarboxaldehyde intermediate 5 (50 mg; 0.12 mmol), TosMIC (50 mg; 0.25 mmol) and potassium carbonate (70 mg; 0.5 mmol) in 6 mL of absolute ethanol was heated at reflux during 2 hours. The solvent was evaporated in vacuo and the residue was poured into brine solution and extracted with dichloromethane (3×50 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel column with dichloromethane-ethanol (94-6) as the eluent to give the titled compound as pale yellow solid (40 mg, 67%), m.p. 254-256° C.; 1H NMR (300 MHz, CDCl3) 8.45 (s; 1H); 8.14 (d; 1H; J=7.9 Hz); 7.99 (s; 1H); 7.96-7.88 (m; 1.5H), 7.79 (d; 1H; J=7.9 Hz); 7.74 (d; 1H; J=7.9 Hz); 7.60 (t; 1H; J=7.9 Hz); 7.51 (s; 1H); SM m/z 500.2 (M+1); Anal. calcd for C29H17N5O4.H2O: C, 67.31; H, 3.67; N, 13.53, found: C, 67.53, H, 3.82, 13.29.
6.1: 5,5′-(5,5′-(Pyridine-2,6-diyl)bis(oxazole-5,2-diyl))difuran-2-carbaldehyde (Intermediate 6).
A mixture of intermediate 1 (0.21 g; 1 mmol), 5-bromo-2-furaldehyde (0.21 g; 1.2 mmol), palladium diacetate (83 mg; 0.37 mmol; 37 mol %), PCy3.HBF4 (72 mg; 0.2 mmol; 20 mol %), copper (I) iodide (0.46 g; 2.4 mmol), cesium carbonate (1.4 g; 4.3 mmol) and 4 mL of anhydrous dioxane were heated in a sealed tube at 130° C. during 2 hours. The residue was purified by flash chromatography (SiO2, dichloromethane-ethanol, 98-2) to give the intermediate 6 as yellow solid (62 mg, 26%); 1H NMR (300 MHz, CDCl3) 9.83 (s; 1H); 7.97 (s; 1H); 7.95-7.90 (m; 0.5H); 7.79 (d; 1H; J=8.2 Hz); 7.39 (d; 1H; J=3.8 Hz); 7.31 (d; 1H; J=3.8 Hz); SM m/z 402.1 (M+1); Anal. calcd for C21H11N3O6.1.5 H2O: C, 58.87; H, 3.27; N, 9.81, found: C, 58.77, H, 2.85, 9.83.
A mixture of dicarboxaldehyde intermediate 6 (30 mg; 0.07 mmol), TosMIC (43 mg; 0.22 mmol) and potassium carbonate (63 mg; 0.45 mmol) in 3 mL of absolute ethanol was heated at reflux during 2 hours. The solvent was evaporated in vacuo and the residue was poured into brine solution and extracted with dichloromethane (3×50 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel column with dichloromethane-ethanol (96-4) as the eluent to give the titled compound as pale yellow solid (13 mg, 36%), m.p.>250° C.; 1H NMR (300 MHz, CDCl3) 7.95-7.91 (m; 2.5H); 7.71 (d; 1H; J=8.1 Hz); 7.53 (s; 1H); 7.34-7.29 (m; 2H); SM m/z 480.1 (M+1).
The protocol described above for the synthesis of compound 3 is applied, starting from the intermediate 1 and using 6-bromo-2,2′-bipyridine (U. Lehmann and A.D. Schlüter, Eur. J. Org. Chem. 2000, 3483-3487) in place of intermediate 3, to give the title compound 7.
The protocol described above for the synthesis of intermediate 1 is applied, starting from pyrazine-2,6-dicarbaldehyde (H. Schumann and H.-K. Luo, Zeitschrift fuer Naturforschung, B: Chemical Sciences, 2005, 60(1), 22-24) in place of 2,6-pyridine dicarboxaldehyde, to give the title intermediate 7.
The protocol described above for the synthesis of intermediate 5 is applied, starting from intermediate 7 and 6-bromopyridine-2-carbaldehyde in place of intermediate 1 and 3-bromobenzaldehyde, to give the title intermediate 8.
The protocol described above for the synthesis of compound 5 is applied, starting from intermediate 8 in place of intermediate 5, to give the title compound 8.
The protocol described above for the synthesis of intermediate 1 is applied, starting from 2,2′-bipyridine-6,6′-dicarbaldehyde (G. R. Newkome and H.-W. Lee; J. Am. Chem. Soc. 1983, 105(18), 5956-5957) in place of 2,6-pyridine dicarboxaldehyde, to give the title intermediate 10.
The protocol described above for the synthesis of intermediate 5 is applied, starting from intermediate 10 and 6-bromopyridine-2-carbaldehyde in place of intermediate 1 and 3-bromobenzaldehyde, to give the title intermediate 11.
The protocol described above for the synthesis of compound 5 is applied, starting from intermediate 11 in place of intermediate 5, to give the title compound 9.
The study of UV-vis properties of pyridine-based polyheteroaromatic compounds detailed in this study enable the determination of their solubility in various conditions (DMSO, H2O and cacodylate buffers (10 mM sodium cacodylate+100 mM NaCl (for Caco.Na) or KCl (for Caco.K)): the simplest method for this is to measure the absorption spectra of compound 3 at various concentrations (from 0 to 32 μM); solubility of compound 3 is thus evaluated through the reporting of its absorbance at a given wavelength (herein 338 nm (A338)) as a function of the concentration of compound 3 and applying the Beer-Lambert law. The results are given in
The various titrations are summarized in
These data indicated that compound 3:
Stabilization, and so interaction, of compounds with quadruplex-structure is monitored via FRET-melting assay, in a version that also enables the determination of the quadruplex-over duplex-DNA selectivity as well as the intra-quadruplex selectivity (17).
FRET assay is performed with oligonucleotides that mimic the human or plasmodium telomeric sequences, and equipped with FRET partners at each extremities: F21T (FAM-G3[T2AG3]3-Tamra,) FPf1T (FAM-G3[T3AG3]3-Tamra) and FPf8T (FAM-G3[T2CAG3]3-Tamra) with FAM: 6-carboxyfluorescein and Tamra: 6-carboxy-tetramethylrhodamine). Measurements were made with excitation at 492 nm and detection at 516 nm.
Fluorescence melting were carried out. The results are given on
The stabilisation effect induced by compound 3 with the three oligonucleotides is quantified by the increase in melting temperature (ΔT1/2). The values are indicated in the table below.
b) Quadruplex- Vs duplex-DNA Selectivity:
Fluorescence melting are carried out with 0.2 μM of F21T in a buffer containing 10 mM lithium cacodylate pH 7.2 and 100 mM NaCl; the melting of the G-quadruplex was monitored alone and in the presence of 1 μM of compound 3 without or with excess (1, 3 and 10 μM) of duplex-DNA competitor ds26 (a 26 base-pair duplex-DNA comprised of the self complementary sequence [5′-CAATCGGATCGAATTCGATCCGATTG-3′]). The results are given on
Fluorescence melting experiments are carried out with 0.2 μM of F21T in a buffer containing 10 mM lithium cacodylate pH 7.2 and 100 mM NaCl; the melting of the G-quadruplex was monitored alone and in the presence of 1 μM of compound 3 without or with excess (1, 3 and 10 μM) of quadruplex-DNA competitors, both TG5T ([(5′-TG5T-3′)4], a tetramolecular quadruplex-DNA used in previous study (21) or c-myc ([5′-GAGGGTGGGGAGGGTGGGGAAG-3′], a sequence present in the promoter region of the oncogene c-myc, highly suspected to fold into an intramolecular quadruplex-structure (22-25). The results are given on
The various FRET-melting experiments are summarized in the graphical bar representation of
These data indicate that compound 3:
An Increase in Melting Temperature, Poorly Affected by the Presence of ds26, is Obtained for Both Compounds.
Study II-3: Interaction with DNA Monitored by Fluorescence Studies
Pyridine-based polyheteroaromatic compounds detailed in this study are characterized by a strong fluorescence. Remarkably, the quantum yield is not affected by the nature of the solvent they are used in, from pure organic (e.g. DMSO) to physiological conditions (e.g. buffer: 10 mM sodium cacodylate+100 mM KCl, pH 7.2, see Table). The modification of the fluorescence properties of studied compounds upon interaction with DNA enables to compare their apparent binding affinity for several quadruplexes of biological relevance (27).
NB: the quantum yields (and standard deviations (s.d.)) of compound 3 were measured in CH2Cl2 (DCM) and water, with anthracene in ethanol as reference.
The quadruplex-DNA used herein is 22AG: it results from the folding of a 22 nt oligonucleotide that mimics the human telomeric sequence: 22AG is [5′-AG3(T2AG3)3-3′]. Quadruplex-structure from 22AG is prepared by heating the corresponding oligonucleotide at 90° C. for 5 min in a 10 mM sodium cacodylate buffer pH 7.2, 100 mM NaCl (for 22AG Na) or KCl (for 22AG K) and cooling in ice to favor the intramolecular folding by kinetic trapping. Concentrations are determined by UV-Vis measurements (after thermal denaturation, 5 min at 85° C.) at 260 nm before use. (see
Increasing amounts of 22AG Na (left) or 22AG K (right) are added onto a solution of 0.25 μM of compound 3 in cacodylate buffer (10 mM sodium cacodylate+100 mM NaCl (right) or KCl (left)), which result in a progressive quench of the fluorescence of compound 3 (λex=340 nm).
The quadruplex-over duplex-selectivity is evaluated through fluorescence titrations by comparison of experiments carried out with 22AG (see above) and with a short duplex-DNA: ds17, which is a 17 base-pair duplex-DNA that represents a biological sequence used in previous studies (28); the sequences of the two complementary strands are the following: [5′-CCAGTTCGTAGTAACCC-3′]/[5′-GGGTTACTACGAACTGG-3′]. Duplex-structure is prepared by heating the two corresponding complementary strands at 90° C. for 5 min in a 10 mM sodium cacodylate buffer pH 7.3, 100 mM KCl followed by a slow cooling over 6 hrs. Concentrations are determined by UV-Vis measurements (after thermal denaturation, 5 min at 85° C.) at 260 nm before use.
The results are given on
Increasing amounts of ds17 are added onto a solution of 0.25 μM of compound 3 in cacodylate buffer (10 mM sodium cacodylate+100 mM KCl) do not result in a progressive quench of the fluorescence of compound 3 (λex=340 nm).
The intra-quadruplex selectivity is evaluated through fluorescence titrations by comparison of experiments carried out with 22AG (see above) and with two other quadruplex-structures: c-myc and c-kit2. The formation of these two quadruplex-DNAs is currently highly suspected in the promoter region of c-myc (see above) and c-kit (22; 29 and 30)oncogenes. The sequences are the following: c-myc: [5′-TGAGGGTGGGTAGGGTGGGTAA-3′] and c-kit2: [(5′-CGGGCGGGCGCGAGGGAGGGG-3′]; Quadruplex-structures are prepared by heating the corresponding oligonucleotide at 90° C. for 5 min in a 10 mM sodium cacodylate buffer pH 7.2, 100 mM KCl and cooling in ice to favor the intramolecular folding by kinetic trapping. Concentrations are determined by UV-Vis measurements (after thermal denaturation, 5 min at 85° C.) at 260 nm before use.
The results are given on
Increasing amounts of c-myc (left) or c-kit2 (right) are added onto a solution of 0.25 μM of compound 3 in cacodylate buffer (10 mM sodium cacodylate+100 mM KCl), which result in a progressive quench of the fluorescence of compound 3 (λex=340 nm).
The various fluorescence titrations (λex=340 nm) are summarized in
These data indicated that compound 3:
Circular dichroism (CD) enables a deep study of the modification of the DNA structure upon the binding of a ligand; it thus reflects the affinity of the ligand to its target, and can also provide insight into its binding mode (Paramasivan et al, Methods, 2007, 43, 324).
The quadruplex-DNA used herein is 22AG (see above), annealed both in a 10 mM sodium cacodylate buffer pH 7.2, 100 mM NaCl (for 22AG Na) or KCl (for 22AG K). To a 3 μM solution of 22AG in both buffers is added an excess of compound 3 (30 μM, 10 equiv.). The results are given in
The intra-quadruplex selectivity is evaluated through CD by comparison of experiments carried out with 22AG (see above) and with two other quadruplex-structures: c-myc and c-kit2 (see above). The results are given on
The equilibrium between folded and unfolded forms of 22AG oligonucleotide is highly sensitive to the presence and the nature of the buffer it is used in. Corresponding results are given in
These data indicated that compound 3:
An electrophoretic mobility shift assay using hPOT1 was performed on the telomeric 22AG oligonucleotide (see above). 22AG was labeled at the 5′ end with [γ-32P]-ATP using T4 polynucletide kinase. Purified recombinant hPOT1 was produced in a baculovirus expression system. The POT1/22AG binding assay was performed in a total volume of 10 μl containing 50 mM HEPES, pH 7.9, 100 mM NaCl, 0.1 mM EDTA, 4% w/v sucrose, 2% v/v glycerol, 0.1 mg/ml BSA, 0.02% w/v bromophenol blue, 30 nM hPOT1, 20 nM [α-32P]-22AG. Different concentrations of compound 3 (10, 1, 0.1 and 0.01 μM) were added with hPOT1 to the solution and the mixture was incubated at room temperature for 30 min. Each individual sample was separated by electroporesis on 1% agarose gel in 0.5×Tris-Borate-EDTA buffer. The gel was run at 80V for 35 min, dried on whatman DE81 paper and radiaoactivity visualized by a phosphorimager (Typhoon 9210, Amersham). Analysis of the data was carried out by ImageQuant software (Amersham) and results were expressed as a percentage of the POT1-22AG complex obtained in the untreated control (defined as 100%) (see
These data indicated that compound 3 inhibits the binding of hPOT1 to telomeric 22AG sequence in a dose-dependent manner. In the experiment showed below the IC50-POT1 for compound 3 is equal to 300 nM.
The HT1080 human fibrosarcoma cell line stably transfected with pEGFP-POT1 vector (HT1080GFP-POT1) has been previously described (32) and U20S human osteosarcoma was from American Type Culture Collection (Rockville, USA). Cells were grown in Dublecco's modified Eagles's medium (Invitrogen) supplemented with 10% v/v fetal bovine serum and with 400 μg/ml geneticin for HT1080GFP-POT1. Cells were plated in 6-wells culture plates on day 0 at 4.5×104 cells/well in the presence of different concentrations of compound 3 (10, 3, 1, 0.3 and 0.1 μM), each concentration in duplicate, and cultured for further 72 hours. At day 3, cells were washed with 1×PBS and trypsinized. For each treated cell sample, the number of viable cells was determined in the presence of trypan blue. Results are given on
These data indicates that compound 3 efficiently hampers the proliferation of cancer cells with an IC50 comprised between 0.1 and 1 μM.
All publications, including but not limited to, issued patents, patent applications, books and journal articles, cited in this application are each herein incorporated by reference in their entirety. Although the invention has been described above with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08168104.1 | Oct 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2009/054856 | 11/2/2009 | WO | 00 | 4/14/2011 |
