EP2004618A1 - Inhibiteurs de télomérase - Google Patents

Inhibiteurs de télomérase

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Publication number
EP2004618A1
EP2004618A1 EP07732304A EP07732304A EP2004618A1 EP 2004618 A1 EP2004618 A1 EP 2004618A1 EP 07732304 A EP07732304 A EP 07732304A EP 07732304 A EP07732304 A EP 07732304A EP 2004618 A1 EP2004618 A1 EP 2004618A1
Authority
EP
European Patent Office
Prior art keywords
compound
formula
carbocyclic
heterocyclic
group
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Withdrawn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
EP07732304A
Other languages
German (de)
English (en)
Inventor
Ramon Vilar
Anna Arola Arnal
Stephen Neidle
Julie Reed
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.)
Institut Catala dInvestigacio Quimica ICIQ
Cancer Research Technology Ltd
School of Pharmacy University of London
Ip2ipo Innovations Ltd
Original Assignee
Institut Catala dInvestigacio Quimica ICIQ
Imperial Innovations Ltd
Cancer Research Technology Ltd
School of Pharmacy University of London
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Institut Catala dInvestigacio Quimica ICIQ, Imperial Innovations Ltd, Cancer Research Technology Ltd, School of Pharmacy University of London filed Critical Institut Catala dInvestigacio Quimica ICIQ
Publication of EP2004618A1 publication Critical patent/EP2004618A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C251/02—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups
    • C07C251/24—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups having carbon atoms of imino groups bound to carbon atoms of six-membered aromatic rings

Definitions

  • the first aspect of the invention therefore provides a compound of formula (I)
  • R 1 and R 2 are independently hydrogen, C 1-2O alkyl, C 3-12 carbocyclic, halo, C 1- 20 haloalkyl, OR 13 , CN, NO 2 , NR 13 R 13 , COR 13 , CO 2 R 13 , O-(CH 2 ) n -N(R 14 ) 3 or (CH 2 ) n -R 15 , CONR 13 R 13 , C 3-12 heterocyclic, C 1-20 alkylC 3 , 12 carbocyclic, C 1- i 2 alkylC 3-12 heterocyclic, or wherein R 1 and R 2 together form a carbocyclic or heterocyclic group having 5 to 18 members, fused to ring A, optionally substituted with one or more of group R 13 , or R 1 and R 2 are independently
  • R 3 , R 4 , R 5 and R 6 are independently hydrogen, halides, C 1-20 alkyl, OR 13 or CN, or R 3 and R 5 and/or R 4 and R 6 together form a 5-6 membered carbocyclic or heterocyclic ring,
  • R 1 and R 3 and/or R 2 and R 4 together form a carbocyclic or heterocyclic group having 5 to 18 members fused to ring A, optionally substituted with one or more of group R ,
  • X 1 and X 2 are independently O, S or NR 13 ,
  • R 7 , R 8 , R 9 , R 10 , R 11 or R 12 are independently hydrogen, halide, OR 14 , 0-(CH 2 V N(R 14 ) 3 or O-(CH 2 ) n -R 15 , or where one or more of R 7 and R 8 or R 10 and R 11 together form a carbocyclic or heterocyclic ring having 5 to 12 members,
  • G 1 and G 2 can together form
  • n 1 to 6
  • R 13 is independently hydrogen, C 1-12 alkyl, C 3-12 carbocyclic, C 3-12 heterocyclic, C 1-6 alkylC 3-12 carbocyclic, C 1-6 alkylC 3-12 heterocyclic, halo, CO 2 H, OH, NH 2 or CONH 2 ; and R 30 , R 31 , R 32 , R 33 and R 34 are independently C 1-12 alkyl, C 3-12 carbocyclic, C 3-12 heterocyclic, C 1-6 alkylC 3-12 carbocyclic, C 1-6 alkylC 3- i 2 heterocyclic, halo, CO 2 H, OH, NH 2 or CONH 2 .
  • a metal ion M can be co-ordinated between the two N atoms and the two X 1 atoms of the groups R 1 and R 2 .
  • any of groups R 7 , R 8 , R 9 , R 10 , R 11 or R 12 can combine with any of groups R 30 , R 31 , R 32 or R 33 to form a bridged structure.
  • the groups A, B or C are independently preferably a six membered aryl or heteroaryl group, more preferably selected from phenyl or pyridine.
  • the groups A, B or C independently are a six membered aryl or heteroaryl group fused to an aromatic ring, for example naphthyl.
  • R 1 and R 2 are preferably selected from hydrogen, F, Cl, CO 2 H, O-(CH 2 ) q -N(CH 3 ) 3 , O-(CH 2 ) q -C 5-6 -carbocyclic, O-(CH 2 ) q -C 5-6 -heterocyclic, CONH-(CH 2 ) m -carbocyclic or CONH(CH 2 ) m -heterocyclic, wherein m is 1 to 6. wherein q is 1 to 6 preferably 2, 3, 4 or 5.
  • R 1 and R 2 , or R 1 and R 3 or R 2 and R 4 together form a carbocyclic or heterocyclic group said carbocyclic or heterocyclic group preferably has 5 to 14 members fused to ring A, more preferably 5 to 10 members fused to ring A.
  • G 1 or G 2 are preferably selected from
  • n is 1 to 6, preferably 2, 3, 4 or 5.
  • the first aspect of the invention preferably relates to a compound of formula (Ia)
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are hydrogen and R 1 , X 1 , X 2 , M, G 1 and G 2 are as defined above.
  • ring A is absent from the compound of formula (I).
  • the resulting compound has the formula (Ib) or (Ic) as set out below:
  • R 3 and R 5 together form a 5-6 membered carbocyclic or heterocyclic ring and R 4 and R 6 together form a 5-6 membered carbocyclic or heterocyclic ring
  • ring B is absent and ring C is replaced with wherein Y is a group -CH 2 -, CR 26 or CO, X is NH, N, or O, D is a carbocycyl or heterocycyl group having 5 to 10 members
  • R 16 , R 17 , R 18 and R 19 are hydrogen, C 1-6 alkyl, C 3-12 carbocyclic, C 3-12 heterocyclic, C 1-6 alkylC 3- i 2 carbocyclic, C 1-6 alkylC 3-12 heterocyclic, halo, CO 2 H, OH, NH 2 or CONH 2 and
  • G 3 is H, OH, NH 2 , OR 20 , O-(CH 2 ) n -N(R 20 ) 3 or O-(CH 2 ) n -R 21 wherein R 20 is C 1-12 alkyl, R 21 is a 5 or 6 membered carbocyclic or heterocyclic ring and R 26 is hydrogen or C 1-12 alkyl.
  • the compound is a compound of formula (Ha) as illustrated below:
  • E and F are independently a 5-6 membered carbocyclic or heterocyclic ring and R >22 , T R-.23 , R r»2 z 4 q and J R r>2 / 5 D are hydrogen, halides, OH, OR 20 , O-(CH 2 ) n - N(R 20 ) 3 , O-(CH 2 ) n -R 21 , or CN,
  • P is OR 13 , CO 2 R 13 , CN, NO 2 , CN, halide, SCN, H 2 O, NO 3 , OH, CH 3 CN or OCN, and
  • the compounds of the invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms.
  • the compounds of the invention may exist in trans or cis form.
  • the first aspect of the invention covers all of these compounds.
  • ⁇ O formula (III) can be incubated with an excess of L-G or L-G to form a compound of formula (I). However, where G 1 and G 2 are different, the groups L-G 1 and L-G 2 are added separately. For example, a compound of formula (III) can be incubated with a group L-G 1 to form a compound of formula (IV),
  • a compound of formula (III) can be prepared by the reaction of a compound of formula (VI) with a metal, a compound of formula (VII) and a compound of formula (VIII)
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , G 1 , G 2 , X 1 , X 2 and M are as defined in the first aspect of the invention.
  • the third aspect of the invention provides a composition comprising a compound according to the first aspect of the invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.
  • the composition may contain from 0.1% to 99% (w/w) preferably from 0.1- 60% (w/w), more preferably 0.2-12% by weight and most preferably 0.25 to 8% (w/w) of a compound of the first aspect depending on the method of administration.
  • Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile).
  • the composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration).
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.
  • suitable pharmaceutical carrier(s) include magnesium stearate, starch, lactose, sucrose and microcrystalline cellulose.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures.
  • powders, granules or pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatine capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatine capsule.
  • compositions for nasal or oral administration may conveniently be formulated as aerosols, drops, gels and powders.
  • Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device.
  • the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve, which is intended for disposal once the contents of the container have been exhausted.
  • the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant.
  • the aerosol dosage forms can also take the form of a pump-atomiser.
  • compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.
  • a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.
  • compositions for rectal or vaginal administration are conveniently in the form of suppositories (containing a conventional suppository base such as cocoa butter), pessaries, vaginal tabs, foams or enemas.
  • Compositions suitable for transdermal administration include ointments, gels, patches and injections including powder injections.
  • the fifth aspect of the present invention relates to a compound of the first aspect, or a composition of the third aspect, for use in medicine.
  • the fifth aspect of the invention particularly provides a compound of the first aspect or a composition of the third aspect for use in the treatment of cancer.
  • the compound of the first aspect or the composition of the third aspect can be provided for the treatment of adrenal cancer, AIDS-related lymphoma, anal cancer, ataxia-telangiectasia, bladder cancer, brain tumours, breast cancer, carcinoma, cervical cancer, chronic lymphocytic leukaemia, chronic myelogenous leukaemia, colorectal cancer, crainiopharyngioma, cutaneous T-cell lymphoma/mycosis fungoides, endometrial and uterine cancer, espophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, getational trophoblastic disease and choriocarcinoma, hairy cell leukaemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukaemia, LiFraumeni syndrome, liver cancer, lung cancer, lymphomas, medulloblastoma,
  • the compounds of the present invention interact with and stabilize the guanine- rich quadraplex structures of the telomere.
  • the present invention therefore provides a compound of the first aspect of the invention or a composition of the third aspect of the invention for the stabilization of the guanine-rich quadruplex structure of a telomere.
  • the present invention further provides a compound of the first aspect of the invention or a composition of the third aspect of the invention for use in the inhibition of telomerase.
  • the present invention further provides a compound of the first aspect of the invention or a composition of the third aspect of the invention for use in promoting senescence or apoptosis of a cancer cell.
  • treatment means any amelioration, reduction in severity or reduction in the progress of the condition or a reduction in the symptoms of the condition. It will be appreciated that in some cases, the degree of the disease will be such that it is not possible to cure the patient. In this case, the term “treatment” means preventing the condition from deteriorating or getting worse for example by halting the progress of the disease without necessary ameliorating the condition or slowing the progress of the disease such that the life span and/or quality of life of the patient is improved.
  • a compound of the present invention may be administered simultaneously, subsequently or sequentially with one or more other active agent, such as a chemotherapeutic agent or an antiproliferative agent.
  • the compound of the present invention can be administered with one or more of an alkylating agent (such as cyclophosphamide, Ifosphamide, Melphalan, Chlorambucil, BCNU, CCNU, Decarbazine, Procarbazine, Busulfan or Thiotepa), an antimetabolite (such as Cytarabine, Gemcitabine, 6- mercaptopurine, 6-thioguanine, Fludarabine and Cladribine), an anthracycline (such as Idarubicin, Epirubicin), an antibiotic (such as Bleomycin), a camptothecin, a etoposide, a vinca alkaloid, a taxane (such as taxol, paclitaxel and docetaxel),and/or a platinium (such as
  • the compounds of the invention will normally be administered in a daily dosage regimen (for an adult patient) of, for example, an oral dose of between 1 mg and 2000 mg, preferably between 30 mg and 1000 mg, e.g. between 10 and 250 mg or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 50 mg, e.g. between 1 and 25 mg of the compound of the formula (I) or (II) or a physiologically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day.
  • the compounds will be administered for a period of continuous therapy, for example for a week or more.
  • the sixth aspect of the invention relates to the use of a compound of the first aspect of the invention in the manufacture of a medicament for the treatment of cancer.
  • the medicament of the sixth aspect of the invention may further comprise one or more other active agent, such as a chemotherapeutic agent or an antiproliferative agent.
  • the medicament of the sixth aspect may comprise one or more of an alkylating agent (such as cyclophosphamide, Ifosphamide, Melphalan, Chlorambucil, BCNU, CCNU, Decarbazine, Procarbazine, Busulfan or Thiotepa), an antimetabolite (such as Cytarabine, Gemcitabine, 6-mercaptopurine, 6-thioguanine, Fludarabine and Cladribine), an anthracycline (such as Idarubicin, Epirubicin), an antibiotic (such as Bleomycin), a camptothecin, a etoposide, a vinca alkaloid, a taxane (such as taxol, paclitaxel and docetaxel),and/or a platinium (such as cisplatin or o
  • the medicament may contain from 0.1% to 99% (w/w) preferably from 0.1- 60% (w/w), more preferably 0.2-12% by weight and most preferably 0.25 to 8% (w/w) of a compound of the first aspect, depending on the method of administration.
  • the seventh aspect of the invention relates to a method of treating cancer comprising administering to a person in need therefore a compound as defined in the first aspect of the invention or a composition of the third aspect of the invention.
  • the compound of the first aspect of the invention is preferably provided in a therapeutically effective amount.
  • the amount of the compound of the first aspect of the invention effective to treat a disorder as set out above depends on the nature and severity of the disorder being treated and the weight of the patient in need thereof.
  • a single unit dose for a 70kg adult will normally contain. 0.01 to lOOmg, for example 0.1 to 50mg, preferably 0.5 to 6mg of the compound of the invention per day.
  • Unit doses may be administered once or more than once a day, for example, 2, 3 or 4 times a day, usually 1 to 3 times a day, more preferably 1 or 2 times per day.
  • the total daily dosage can be in range of approximately 0.0001 to 0.2mg per kg per day, more usually 0.001 to O.lmg per kg per day, preferably 0.01 to O.lmg per kg per day.
  • the unit dose is preferably provided in the form of a capsule or a tablet.
  • the compound of the first aspect or composition of the third aspect may be provided prior to, in combination with and/or subsequent to a different cancer treatment.
  • the compound and composition of the invention therefore may be administered prior to, in combination with and/or subsequent to chemotherapy, radiotherapy, surgery etc.
  • the compound or composition of the invention may be administered in combination with one or more active agents for use in the treatment of side effects of cancer treatment such as antiemetics, antibiotics etc.
  • the compound or composition of the invention may be provided as a single course of treatment or repeated courses of treatment over a period of time to be determined by a physician.
  • Figure 1 shows the docking of the nickel(II) complex 3 with the human parallel intramolecular quadruplex formed from four repeats of telomeric DNA.
  • the model shows very good stacking between the rings of the metal complex and three of the guanine rings of the quadruplex DNA;
  • Figure 2 shows the TRAP gel for compound 3 showing the characteristic ladders produced by PCR amplification of the oligonucleotides generated by the activity of telomerase on a TS primer;
  • Figure 3 show the observed changes in quadruplex and duplex melting temperature ( ⁇ Tm) with changes in ligand concentrations
  • Figure 4 shows two views (a) and (b) of the docking of the nickel(II) complex 3 with the human parallel intramolecular quadruplex formed from four repeats of telomeric DNA.
  • the model shows very good stacking of the metal complex with the quadruplex DNA and also a good interaction between the side chains and the grooves of DNA;
  • Figure 5 shows the effects of compound 3 on MACl 5 A tumours.
  • Telomerase activity in the presence of the compounds was assessed using a modified version of standard published TRAP protocols, with cell extract from exponentially growing A2780 human ovarian carcinoma cells used as the enzyme source.
  • the TRAP assay was carried out in two steps with an initial primer-elongation step and subsequent PCR amplification of the telomerase products to enable detection.
  • a master reaction mix (40 ⁇ l) was prepared containing the TS forward primer (0.1 ⁇ g; 5- AATCCGTCGAGCAGAGTT-3), TRAP buffer (20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween- 20), BSA (0.05 ⁇ g), and dNTPs (125 ⁇ M each). Protein (1 ⁇ g) was then incubated with the reaction mixture with or without drug (made up in solution as the HCl salt) for 10 min at 30 0 C.
  • telomerase Following heat inactivation of telomerase at 94 0 C for 4 min and cooling to 20 0 C, 10 ⁇ l of a PCR reaction mix containing ACX primer (0.1 ⁇ g; 5-GTG[CCCTTA]3CCCTAA-3) and 2U Taq polymerase (RedHot, Surrey, UK) was added to each tube to start the PCR protocol for part 2, with thermal cycling being carried out in 3 parts following an initial 5 min denaturing period at 94 0 C (30 cycles of 94 0 C for 30 s, 65 0 C for 60 s, 72 0 C for 60 s).
  • PCR- amplified reaction products were then run out on a 10% w/v non-denaturing PAGE gel and visualised by staining with SYBR Green I (Sigma). 161 EC 50 values were subsequently calculated by quantitating the TRAP product using a gel scanner and GeneTools software (Syngene, Cambridge, UK), Measurements were made with respect to a negative control run using the equivalent TRAP-PCR conditions but omitting the protein extract, thus ensuring that the ladders observed were not due to artefacts of the PCR reaction.
  • telomerase activity in the presence of the compounds was assessed using a modified version of previously published TRAP protocols, with cell extract from exponentially growing A2780 human ovarian carcinoma cells used as the enzyme source.
  • the TRAP assay was carried out in two main steps with an initial primer-elongation step and subsequent PCR amplification of the telomerase products to enable detection.
  • Step 1 a master reaction mixture (40 ⁇ l) was prepared containing lhe TS forward primer (0.1 ⁇ g; 5'- AATCCGTCGAGCAGAGTT-3'), TRAP buffer (20 niM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween-20), BSA (0.05 ⁇ g), and dNTPs (125 ⁇ M each). Protein (1 ⁇ g) was then incubated with the reaction mixture with or without the compound to be tested (made up in solution as the HCl salt) for 10 min at 30 0 C.
  • TRAP buffer 20 niM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween-20
  • BSA 0.05 ⁇ g
  • dNTPs 125 ⁇ M each
  • Step 2 Following heat inactivation of tclomerase at 94 0 C for 4 min and cooling to 20 0 C, purification of the telomerase products was performed using QIAquick nucleotide removal spintubes, following the protocol described for their use with the exception that the elution stage was performed with 40 ⁇ L PCR-grade water. Samples were then dried using a SpeedVac centrifuge.
  • Step 3 A master mix (50 ⁇ L) of (TS forward primer (0.1 ⁇ g; 5'-AATCCGTCGAGCAGAGTT-S'), TRAP buffer (20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween-20), BSA (0.05 ⁇ g), and dNTPs (125 ⁇ M each) ACX primer (0.1 ⁇ g; 5'-GTG[CCCTTA] 3 CCCTAA-3') and 2 ⁇ M Taq polymerase (RedHot, AB gene, Surrey, UK) were added to each tube to start the PCR protocol.
  • TRAP buffer 20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween-20
  • BSA 0.05 ⁇ g
  • Thermal cycling was carried out in three parts following an initial 5 min denaturing period at 94 0 C (30 cycles of 94 0 C for 30 s, 61 0 C for 60 s, 72 0 C for 60 s). PCR-amplified reaction products were then run out on a 10% w/v non- denaturing PAGE gel and visualised by staining with SYBR Green I (Sigma).
  • tcl EC 5 o values were calculated by quantitating the TRAP product using a gel scanner and GeneTools software (Syngene, Cambridge, UK), Measurements were made with respect to a negative control run using the equivalent TRAP- PCR conditions but omitting the protein extract, thus ensuring that the ladders observed were not due to artefacts of the PCR reaction.
  • a qualitative computer model to investigate the stacking of the nickel-salphen complexes and DNA was carried out. This was done using Maestro, Jaguar.
  • the metal complex was optimized using DFT with a LAV3P basis set and a HF initial guess LFT + dd methods.
  • the optimized metal complex was used to do the docking shown in figure 5.
  • the structure of quadraplex DNA used for the qualitative modeling corresponds to the Human Telomere Repeat Sequence 5'- DCApGpGpGpTpTpApGpGpGpTpTpApGpGpGpTpTpApGpGpGpTpTpAp GpGpG)-3 (PDB code: IKFl).
  • sulforhodamine B assay was measured using the sulforhodamine B assay.
  • Cells are exposed to a single dose of drug and the percentage of cell viability is measured after four days exposure. This is performed over a wide range of drug concentrations in order to establish the IC 50 value (the concentration where 50% of cell growth is inhibited).
  • IC 50 value the concentration where 50% of cell growth is inhibited.
  • 4,000 cells were seeded into the wells of 96- well microtiter plates and allowed to attach overnight.
  • Compounds dissolved as the HCl or KOH salt were dissolved to 1 mM solutions in sterilised water before being dissolved in media to the final concentrations of 0.05, 0.25, 1, 5, and 25 ⁇ M, which was added to wells containing cells, in 8-fold replication.
  • Group 2 - Complex 3 20mg/Kg, days 0-3, i.p.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne des complexes métalliques de formules (I), (IIa) ou (IIb) qui interagissent avec de l'ADN quadruplex et qui agissent en tant qu'inhibiteurs d'activité de télomérase, la synthèse de ces complexes et leur utilisation dans le traitement du cancer.
EP07732304A 2006-04-07 2007-04-05 Inhibiteurs de télomérase Withdrawn EP2004618A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB0607096.5A GB0607096D0 (en) 2006-04-07 2006-04-07 Compound
PCT/GB2007/001256 WO2007128968A1 (fr) 2006-04-07 2007-04-05 Inhibiteurs de télomérase

Publications (1)

Publication Number Publication Date
EP2004618A1 true EP2004618A1 (fr) 2008-12-24

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EP07732304A Withdrawn EP2004618A1 (fr) 2006-04-07 2007-04-05 Inhibiteurs de télomérase

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Country Link
EP (1) EP2004618A1 (fr)
JP (1) JP2009533336A (fr)
GB (1) GB0607096D0 (fr)
WO (1) WO2007128968A1 (fr)

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RU2468030C2 (ru) * 2010-04-09 2012-11-27 Государственное учебно-научное учреждение Химический факультет Московского государственного университета им. М.В. Ломоносова Ингибиторы теломеразы и способ их получения
US9593203B2 (en) 2011-07-18 2017-03-14 Novomer, Inc. Metal complexes
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