WO2008122667A2 - Dérivés d'uréylène - Google Patents

Dérivés d'uréylène Download PDF

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WO2008122667A2
WO2008122667A2 PCT/EP2008/054362 EP2008054362W WO2008122667A2 WO 2008122667 A2 WO2008122667 A2 WO 2008122667A2 EP 2008054362 W EP2008054362 W EP 2008054362W WO 2008122667 A2 WO2008122667 A2 WO 2008122667A2
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nhc
mmol
alkyl
aryl
2arch
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PCT/EP2008/054362
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WO2008122667A3 (fr
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John Edward Moses
Stephen Neidle
Adam Donald Moorhouse
Michael Moore
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School Of Pharmacy
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    • 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
    • C07D295/14—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 carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—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 carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—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 carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/38—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by doubly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/40—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C275/42—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/95—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in positions 2 and 4
    • C07D239/96—Two oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms

Definitions

  • the present invention is concerned with ureylene derivatives, their intermediates, uses thereof and processes for their production.
  • the present invention relates to compounds which may be used as chemotherapeutic agents in the treatment of cancer.
  • Anti-cancer agents prevent the uncontrolled proliferation of cells which is characteristic of many cancers.
  • the concepts behind cell cycle regulation are described in detail in WO02/08193. When the tight controls regulating cell replication break down, uncontrolled proliferation ensues. This typically leads to tumour growth and cancer.
  • telomeres In many cancerous cells, the telomeric sequences of chromosomes have become shortened. In these cells, a specialised DNA polymerase, telomerase, often appears and synthesises new telomeric DNA, preventing further shortening of the telomeres. This stabilises the cells and contributes to their immortalisation. Telomerase is not usually active in normal mammalian somatic cells. However, telomerase activity has been detected in up to 80-90% of all human cancers examined.
  • telomerase inhibitors can selectively target tumour cells and cause tumour cell death well before damage to regenerative tissues occurs, thereby minimising undesirable side-effects.
  • polycyclic compounds including polycyclic acridines, anthraquinones, and fluorenones have been shown to inhibit telomerase and/or have anti- tumour effects in vitro. These are described, for instance, in Bostock-Smith et al (1998) Antitumour Polycyclic Acridines, Part 6, Biochemistry, 38(21): 6723-6731. Improved therapeutic acridone and acridine compounds are described in WO02/08193.
  • G-quadruplexes may be formed in human telomeres at regions of single stranded G-rich DNA found at the ends of chromosomes.
  • a range of G-quadruplex structures have been reported. Stabilisation of these G-quadruplex structures with small molecules has been shown to inhibit the action of telomerase [Neidle, S. et ah, (2002) Telomere maintenance as a target for anticancer drug discovery. Nat. Rev. Drug Disc. 1(5): 383- 393].
  • Thousands of molecules have been screened for G-quadruplex binding in recent years.
  • BRACO- 19 fused polycyclic intercalators, exemplified by BRACO- 19; macrocyclic compounds, including the natural product telemostatin; and polyaromatic unfused systems, for example the peptide hemi- cyanide ligand described in Arthanari, H. et ah, (1998) Fluorescent dyes specific for quadruplex DNA. Nucleic Acids Res. 26(16): 3724-3728.
  • the c-kit gene encodes a receptor tyrosine kinase. Engagement by this kinase's ligand triggers the signals leading to cell proliferation.
  • C-kit activity is elevated in many human malignancies, particularly in gastrointestinal tumours. It is thought that G- quadruplexes are formed within the human c-kit oncogene [Phan, A, et al., (2007) Structure of an unprecedented G-Quadruplex scaffold in the human c-kit promoter. J ⁇ m.Chem.Soc. 129, 4386-4392].
  • the present invention provides a new class of G-quadruplex binding agents which are thought to inhibit telomerase and stabilise the c-kit gene.
  • the novel agents are based on a diphenyl urea core, and have structural features that are more drug-like than the polycyclic moieties typical of most current G- quadruplex binding agents.
  • R 1 and R 2 are independently selected from H, NH 2 , NH(Ci_ 6 alkyl), N(Ci_ 6 alkyl) 2 ,
  • R 3 , R 4 , R 5 and R 6 are independently selected from the group consisting of H, C(O)OH, C(O)O(C 1-6 alkyl), C(O)O(C 6 _ 20 aryl), C(O)O(C 7 _ 20 aralkyl), C(O)O(C 7 _ 20 alkaryl), halo, OH, O(Ci_ 6 alkyl), NH 2 , NH(Ci_ 6 alkyl), N(Ci_ 6 alkyl) 2 , NH(C 6 _ 20 aryl), NH(C 7 _ 2 o alkaryl), NH(C 7 _ 20 aralkyl), NHC(O)(C 1-6 alkyl), NHC(O)(C 6 _ 20 aryl), NHC(0)(C 7 _2o alkaryl), NHC(O)(C 7 _ 20 aralkyl), NHC(0)(Ci_ 2 o heteroaryl), NHC(O)
  • R 7 and R 8 are independently selected from the group consisting of H and Ci_ 6 alkyl; and at least one of R 3 , R 4 , R 5 and R 6 is not H.
  • At least two of R 3 , R 4 , R 5 and R 6 are not H.
  • R 7 and R 8 are independently selected from the group consisting of H and Ci-3 alkyl, more preferably both R 7 and R 8 are H.
  • R 1 and R 2 are independently selected from H, NHC(O)(Ci_ 6 alkyl), NHC(0)(C 6 _2o aryl), NHC(0)(C 7 -2o alkaryl), NHC(0)(C 7 -2o aralkyl), NHC(O)(C 1-20 heteroaryl), NHC(0)(C 2 -2o heterocyclyl), NHC(0)(C 2 -2o heteroaralkyl), NHC(0)(C 3 -2o heterocyclylalkyl), and NHC(O)(C3_ 20 alkylheterocyclyl).
  • R 1 and R 2 are independently selected from H and NHC(O)(C 342 heterocyclylalkyl) .
  • R 1 and R 2 are independently selected from NHC(O)Ci_6 alkyl(C 3 - 6 heterocyclyl).
  • R 1 and R 2 are independently selected from the group consisting of NHC(O)Ci_6 alkyl(C 3 _ 6 heterocyclyl)
  • the Ci_ 6 alkyl is preferably selected from 1,1- methanediyl, 1,2-ethanediyl, 1,3-propanediyl and 1,4-butanediyl.
  • the C3-6 heterocyclyl group is selected from the group consisting of pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, morpholinyl, imidazolinyl, imidazolidinyl, pyrazolidinyl, piperidinyl and piperazinyl, most preferably pyrrolidinyl.
  • R 1 and R 2 are not H, they have the structure independently selected from structure (II):
  • n 0, 1, 2, 3, 4 or 5, preferably 1, 2 or 3.
  • both R 1 and R 2 are H.
  • R 3 , R 4 , R 5 and R 6 are independently selected from the group consisting of H, C(O)OH, C(O)NH(Ci_ 6 alkyl), C(O)NH(C 6 _i 0 aryl), C(O)NH(C 7 _i 0 alkaryl), C(O)NH(C 7 -I 0 aralkyl), C(O)NH(C 140 heteroaryl), C(O)NH(C 240 heterocyclyl), C(O)NH(C 240 heteroaralkyl), C(O)NH(C 340 heterocyclylalkyl), C(O)NH(C 340 alkylheterocyclyl), NHC(O)(C 640 aryl), NHC(O)NH(C 640 aryl), NHC(O)NH(C 1-6 alkyl), NHC(O)NH(C 640 aryl), NHC(O)NH(C 740 alkaryl), NHC(O)NH(C
  • R 3 and R 4 are both H.
  • R 5 and R 6 are both H.
  • R 3 and R 4 are both H.
  • R 1 and R 2 are not H
  • R 3 and R 4 are both H.
  • R 1 and R 2 are not H
  • R 5 and R 6 are not H.
  • R 3 , R 4 , R 5 and R 6 are independently selected from the group consisting of (a), (b), (c), (d), (e) and (f):
  • R 9 is selected from the group consisting of -(CH 2 ) n -X, and n is 0, 1, 2, 3, 4 or 5, preferably 1, 2 or 3;
  • X is selected from N(CH 3 ) 2 , pyrrolidinyl, piperidinyl or morpholinyl (attached to the rest of the molecule via the nitrogen atoms).
  • one or more of these groups may comprise at least one terminal group independently selected from the group consisting of C 1-10 heteroaryl, C 2-10 heterocyclyl, C 2-1 O heteroaralkyl, C 3-10 heterocyclylalkyl and C3_i 0 alkylheterocyclyl containing at least one nitrogen atom, more preferably selected from the group consisting of C3-6 heteroaryl, C3-6 heterocyclyl, C3-7 heteroaralkyl, C 3 _ 7 heterocyclylalkyl and C 3 _ 7 alkylheterocyclyl containing at least one mtrogen atom, more preferably a C 3 5 heterocyclyl group containing at least one nitrogen atom, most preferably a pyrrolidinyl group
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are not H
  • one or more of these groups may comprise at least one terminal group having the structure (III)
  • n 0, 1, 2, 3, 4 or 5, preferably 1, 2 or 3, and X is as defined above
  • the compound (I) contains at least one terminal group having the structure (III), preferably at least 2 such groups
  • R 1 and R 2 have the same structure
  • R 4 and R 4 have the same structure
  • R 5 and R 6 have the same structure
  • R 7 and R 8 have the same structure
  • the present invention also provides a method of making compounds according to formula (I).
  • Another aspect of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), substantially as described herein before, with a pharmaceutically acceptable diluent or carrier.
  • Yet another aspect of the present invention is a method of making a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) substantially as described herein before, comprising mixing said compound with a pharmaceutically acceptable diluent or carrier.
  • the present invention provides a compound of formula (I), or a salt, solvate or pro-drug thereof, substantially as described herein before, for use in therapy.
  • Some diseases that may be treated according to the present invention include 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, particularly cancer.
  • the present invention provides a method for the treatment of a disease by administration to a subject of a compound of formula (I), or a salt, solvate or pro-drug thereof, substantially as described herein before.
  • the present invention provides a method for the prophylaxis or treatment of cancer, by administration to a subject of a compound of formula (I), or a salt, solvate or pro-drug thereof, substantially as described herein before.
  • the present invention provides a method for the prophylaxis or treatment of metastases, by administration to a subject of a compound of formula (I), or a salt, solvate or pro-drug thereof, substantially as described herein before.
  • Specific diseases that may be treated or prevented according to the present invention include 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 and gliomas.
  • 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 any of the diseases described herein before.
  • the compounds of the present invention may also be present in the form of pharmaceutical acceptable salts.
  • the salts of the compounds of this invention refer to non-toxic "pharmaceutically acceptable salts.”
  • FDA approved pharmaceutical acceptable salt forms International J. Pharm. 1986, 33,201-217; J. Pharm. ScL, 1977, Jan, 66 (1), pi
  • pharmaceutically acceptable acidic/anionic or basic/cationic salts include pharmaceutically acceptable acidic/anionic or basic/cationic 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 invention also comprehends derivative compounds ("pro-drugs") which are degraded in vivo to yield the species of formula (I).
  • Pro-drugs are usually (but not always) of lower potency at the target receptor than the species to which they are degraded.
  • Pro-drugs are particularly useful when the desired species has chemical or physical properties which make its administration difficult or inefficient. For example, the desired species may be only poorly soluble, it may be poorly transported across the mucosal epithelium, or it may have an undesirably short plasma half-life. Further discussion of pro-drugs may be found in Stella, V. J. et al, "Prodrugs", Drug Delivery Systems, 1985, pp. 112-176, Drugs, 1985, 29, pp. 455-473 and "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.
  • Pro-drug forms of the pharmacologically-active compounds of the invention will generally 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.
  • administering shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the subject.
  • ester derivatives in which one or more free hydroxy groups are esterified in the form of a pharmaceutically acceptable ester are particularly pro-drug esters that may be convertible by solvolysis under physiological conditions to the compounds of the present invention having free hydroxy groups.
  • the compounds of the invention can be administered 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.
  • 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, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin.
  • Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
  • Effective doses of the compounds of the present invention may be ascertained be conventional methods.
  • the specific dosage level required for any particular patient will depend on a number of factors, including severity of the condition being treated, the route of administration and the weight of the patient. In general, however, it is anticipated that the daily dose (whether administered as a single dose or as divided doses) will be in the range 0.001 to 5000 mg per day, more usually from 1 to 1000 mg per day, and most usually from 10 to 200 mg per day.
  • a typical dose will be expected to be between 0.01 ⁇ g/kg and 50 mg/kg, especially between 10 ⁇ g/kg and 10 mg/kg, between 100 ⁇ g/kg and 2 mg/kg.
  • dialkyl groups e.g. N(Ci_ 6 alkyl) 2
  • the two alkyl groups may be the same or different.
  • linking bonds may be on any suitable ring atom, subject to the normal rules of valency.
  • pyrrolyl substituted on the backbone contemplates all possible isomeric forms.
  • pyrrolyl substituted on the backbone includes all of the following permutations:
  • halogen or "halo" is used herein to refer to any of fluorine, chlorine, bromine and iodine. Most usually, however, halogen substituents in the compounds of the invention are chlorine, bromine and fluorine substituents.
  • composition means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
  • the compounds according to this invention may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. Where the processes for the preparation of the compounds according to the invention give rise to mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in racemic form or individual enantiomers may be prepared by standard techniques known to those skilled in the art, for example, by enantiospecific synthesis or resolution, formation of diastereomeric pairs by salt formation with an optically active acid, followed by fractional crystallization and regeneration of the free base.
  • the compounds may also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.
  • solvate means a compound of as defined herein, or a pharmaceutically acceptable salt of a compound of structure (I), wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a hydrate.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • backbone preferably means the carbon backbone of the group being referred to.
  • backbone includes the possibility for substitution on a heteroatom, such as nitrogen, which is located in the carbon backbone.
  • on the backbone when referring to a substitution, means that one or more hydrogen atoms on the backbone is replaced by one or more of the groups indicated. Where more than one substitution occurs, they may be on the same, adjacent or remote carbon atoms, i.e., located on carbon atoms that are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more carbon atoms apart. Where a group comprises two or more moieties defined by a single carbon atom number, for example, C 2 - 20 aralkyl, the carbon atom number indicates the total number of carbon atoms in the group.
  • heteroatom includes N, O, S, P, Si and halogen (including F, Cl, Br and I).
  • alkyl refers to a straight or branched saturated monovalent hydrocarbon radical, having the number of carbon atoms as indicated.
  • suitable alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, dodecyl and eicosyl.
  • alkenyl refers to a straight or branched unsaturated monovalent hydrocarbon radical, having the number of carbon atoms as indicated, and the distinguishing feature of a carbon-carbon double bond.
  • suitable alkenyl groups include ethenyl, propenyl, butenyl, penentyl, hexenyl, octenyl, nonenyl, dodecenyl and eicosenyl, wherein the double bond may be located any where in the carbon backbone.
  • alkynyl refers to a straight or branched unsaturated monovalent hydrocarbon radical, having the number of carbon atoms as indicated, and the distinguishing feature of a carbon-carbon triple bond.
  • suitable alkynyl groups include ethynyl, propynyl, butynyl, penynyl, hexynyl, octynyl, nonynyl, dodycenyl and eicosynyl, wherein the triple bond may be located any where in the carbon backbone.
  • cycloalkyl refers to a cyclic saturated monovalent hydrocarbon radical, having the number of carbon atoms as indicated.
  • suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclohexyl, dimethylcyclohexyl, trimethylcyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclododecyl, spiroundecyl, bicyclooctyl and adamantyl.
  • (cycloalkyl)alkyl refers to an alkyl group with a cycloalkyl substituent. Binding is through the alkyl group. Such groups have the number of carbon atoms as indicated.
  • suitable (cycloalkyl)alkyl groups include cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, methylcyclohexylmethyl, dimethylcyclohexylmethyl, trimethylcyclohexylmethyl, cycloheptylmethyl, cycloheptylethyl, cycloheptylpropyl, cycloheptylbutyl and adam
  • Aryloxy refers to the group "aryl-O-", where aryl is as defined herein.
  • suitable aryloxy groups include phenoxy, tolyloxy and xylyloxy.
  • alkoxyalkyl refers to an alkyl group having an alkoxy substituent. Binding is through the alkyl group.
  • the alkyl group and/or the alkoxy group has the number of carbon atoms as indicated.
  • the alkyl moiety may be straight or branched.
  • the alk and alkyl moieties of such a group may be substituted as defined above, with regard to the definition of alkyl.
  • suitable alkoxyalkyl groups include methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, methoxypropyl and ethoxypropyl.
  • alkoxyaryl refers to an aryl group having an alkoxy substituent. Binding is through the aryl group.
  • the aryl group and/or the alkoxy group have the number of carbon atoms as indicated.
  • the alkoxy and aryl moieties of such a group may be substituted as defined herein, with regard to the definitions of alkoxy and aryl.
  • the alkyl moiety may be straight or branched.
  • suitable alkoxyaryl groups include methoxyphenyl, ethoxyphenyl, dimethoxyphenyl and trimethoxyphenyl .
  • aryl refers to monovalent unsaturated aromatic carbocyclic radical having one, two, three, four, five or six rings, preferably one, two or three rings, which may be fused or bicyclic.
  • aryl refers to an aromatic monocyclic ring containing 6 carbon atoms, which may be substituted on the ring with 1, 2, 3, 4 or 5 substituents as defined herein; an aromatic bicyclic or fused ring system containing 7, 8, 9 or 10 carbon atoms, which may be substituted on the ring with 1, 2, 3, 4, 5, 6, 7, 8 or 9 substituents as defined herein; or an aromatic tricyclic ring system containing 10, 11, 12, 13 or 14 carbon atoms, which may be substituted on the ring with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 substituents as defined herein.
  • suitable aryl groups include phenyl, biphenyl, binaphthyl, indanyl, phenanthryl, fluoryl, flourenyl, stilbyl, benzphenanthryl, acenaphthyl, azulenyl, phenylnaphthyl, benzfluoryl, tetrahydronaphthyl, perylenyl, picenyl, chrysyl, pyrenyl, tolyl, chlorophenyl, dichlorophenyl, trichlorophenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl, fluorophenyl, difluorophenyl, trifluorophenyl, nitrophenyl, dinitrophenyl, trinitrophenyl, aminophenyl, diaminophenyl, triaminophenyl, cyanophenyl,
  • heteroaryl refers to a monovalent unsaturated aromatic heterocyclic radical having one, two, three, four, five or six rings, preferably one, two or three rings, which may be fused or bicyclic.
  • heteroaryl refers to an aromatic monocyclic ring system containing five members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms, an aromatic monocyclic ring having six members of which one, two or three members are a N atom, an aromatic bicyclic or fused ring having nine members of which at least one member is a N, O or S atom and which optionally contains one, two or three additional N atoms or an aromatic bicyclic ring having ten members of which one, two or three members are a N atom.
  • suitable heteroaryl groups include furanyl, pyranyl, pyridyl, phthalimido, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, pyronyl, pyrazinyl, tetrazolyl, thionaphthyl, benzofuranyl, isobenzofuryl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, azaindolyl, isoindazolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxadinyl,
  • heterocyclyl refers to a saturated or partially unsaturated ring having three members of which at least one member is a N, O or S atom and which optionally contains one additional O atom or additional N atom; a saturated or partially unsaturated ring having four members of which at least one member is a N, O or S atom and which optionally contains one additional O atom or one or two additional N atoms; a saturated or partially unsaturated ring having five members of which at least one member is a N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms; a saturated or partially unsaturated ring having six members of which one, two or three members are an N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atoms; a saturated or partially unsaturated ring having seven members of which one, two or three members are an N, O or S atom and which optionally contains one additional O atom or one, two or three additional N atom
  • heterocycles comprising peroxide groups are excluded from the definition of hetercyclyl.
  • suitable heterocyclyl groups include pyrrolinyl, pyrrolidinyl, dioxolanyl, tetrahydrofuranyl, morpholinyl, imidazolinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrothiopyranyl and piperazinyl.
  • heterocyclylalkyl refers to an alkyl group with a heterocyclyl substituent. Binding is through the alkyl group. Such groups have the number of carbon atoms as indicated.
  • the heterocyclyl and alkyl moieties of such a group may be substituted as defined herein, with regard to the definitions of heterocyclyl and alkyl.
  • the alkyl moiety may be straight or branched.
  • suitable heterocyclylalkyl groups include methyl, ethyl, propyl, butyl, pentyl and hexyl substituted with one or more of the heterocyclyl groups indicated immediately above.
  • alkaryl refers to an aryl group with an alkyl substituent. Binding is through the aryl group. Such groups have the number of carbon atoms as indicated.
  • the alkyl and aryl moieties of such a group may be substituted as defined herein, with regard to the definitions of alkyl and aryl.
  • the alkyl moiety may be straight or branched.
  • alkaryl include tolyl, xylyl, butylphenyl, mesityl, ethyltolyl, methylindanyl, methylnaphthyl, methyltetrahydronaphthyl, ethylnaphthyl, dimethylnaphthyl, propylnaphthyl, butylnaphthyl, methylfluoryl and methylchrysyl.
  • aralkyl refers to an alkyl group with an aryl substituent. Binding is through the alkyl group. Such groups have the number of carbon atoms as indicated.
  • the aryl and alkyl moieties of such a group may be substituted as defined herein, with regard to the definitions of aryl and alkyl.
  • the alkyl moiety may be straight or branched.
  • aralkyl include benzyl, methylbenzyl, ethylbenzyl, dimethylbenzyl, diethylbenzyl, methylethylbenzyl, methoxybenzyl, chlorobenzyl, dichlorobenzyl, trichlorobenzyl, phenethyl, phenylpropyl, diphenylpropyl, phenylbutyl, biphenylmethyl, fluorobenzyl, difluorobenzyl, trifluorobenzyl, phenyltolylmethyl, trifluoromethylbenzyl, bis(trifluoromethyl)benzyl, propylbenzyl, tolylmethyl, fluorophenethyl, fluorenylmethyl, methoxyphenethyl, dimethoxybenzyl, dichlorophenethyl, phenylethylbenzyl, isopropylbenzyl, diphenylmethyl, propylbenzy
  • heteroarylkyl refers to an alkyl group with a heteroaryl substituent. Binding is through the alkyl group. Such groups have the number of carbon atoms as indicated.
  • the heteroaryl and alkyl moieties of such a group may be substituted as defined herein, with regard to the definitions of heteroaryl and alkyl.
  • the alkyl moiety may be straight or branched.
  • suitable heteroaralkyl groups include methyl, ethyl, propyl, butyl, pentyl and hexyl substituted with one or more of the specific heteroaryl groups indicated above.
  • arylamino refers to an amine group with an aryl substituent.
  • Binding is through the amine group.
  • Such groups have the number of carbon atoms as indicated.
  • the aryl moiety of such a group may be substituted as defined herein, with regard to the definition of aryl.
  • suitable arylamino groups include phenylamino, biphenylamino, methylphenylamino, methoxyphenylamino, tolylamino and chlorophenylamino .
  • heteroarylamino refers to an amine group with a heteroaryl substituent. Binding is through the amine group. Such groups have the number of carbon atoms as indicated.
  • the heteroaryl moiety of such a group may be substituted as defined herein, with regard to the definition of heteroaryl.
  • substituents which are referred to as being on the carbon backbone of a group with a compound definition for example, "alkaryl”
  • the substituent may be on either or both of the component moieties, e.g., on the alkyl and/or aryl moieties.
  • cyclic systems e.g., cycloalkyl, aryl, heteroaryl, etc.
  • Such systems comprise fused, non-fused and spiro conformations, such as bicyclooctyl, adamantyl, biphenyl and benzofuran.
  • Microwave reactions were conducted upon a Biotage Initiator Microwave, software version 1.1, in Biotage vials (0.5 ml - 2.0 ml or 2.0 ml - 5.0 ml) sealed with Biotage caps with septa. All chemistry was conducted in clean, oven dried glassware. Compound names were generated using ChemDraw Ultra 8.0. Physical Characterisation and Spectroscopic Techniques
  • NMR and 13 C NMR spectra were recorded at 295K upon on a Bruker Avance 400 spectrometer at 400 MHz and 100 MHz respectively using the specified deuterated solvent purchased from GOSS Scientific or Sigma-Aldrich.
  • NMR spectra were analysed in MestReC 4.5.6.0 with chemical shifts calibrated to the residual proton and carbon resonance of the solvent (d6-OMSO dH 2.50, dC 39.43; CDCl 3 dH 7.26, dC 77.0).
  • NMR multiplicity abbreviations are: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
  • Multiplicity and coupling constants are reported as observed in hertz (Hz). All compounds were NMR pure unless stated. Melting points (Mp.) were conducted upon a Bibby Stuart Scientific SMP3 melting point apparatus, where 'dec' indicates decomposition. Mass Spectra (MS) were recorded upon either a ThermoQuest Navigator Mass Spectrometer, or a Waters Alliance 2695 LCMS eluting with 0.1% Formic Acid (FA) in aqueous Acetonitrile, with a Waters 2996 photodiode array detector and a Waters Micromass ZQ Mass Spectrometer, both operated under Electrospray Ionisation in positive ([M+ 1]) or negative ([M-I]) modes.
  • FA Formic Acid
  • High resolution accurate mass spectra were conducted upon a Micromass Q-TTOF Ultima Global Tandem Mass Spectrometer. All samples were run under Electrospray Ionization mode using 50% Acetonitrile in Water and 0.1% FA as solvent, and manipulated using the MassLab 3.2 software. Infrared spectra (IR) were recorded from neat samples upon a Nicolet Smart Golden Gate spectrometer (Avatar 360 FT-IR E. S. P.) and manipulated using the software OMNIC E.S.P. 5.1. Elemental analysis (Anal. CHN) were conducted upon a Carlo Erba CHNl 108 Elemental Analyser.
  • Analytical-TLC was performed upon Merck silica gel 60 F 2 5 4 TLC plates and were visualised under UV light or with ninhydrin (10% in EtOH) followed by heating. Flash chromatography was conducted on Silica gel (partial size 33 - 70 ⁇ m; BDH) eluting with the specified solvent.
  • Analytical HPLC HPLC were conducted upon a Gilson Chromatograph with a YMC C18 5 ⁇ (100 x 4.6 mm) column and a Aglient 1100 series Photo Diode array detector. Spectra were manipulated in the Unipoint 5.11 software and compound purity and retention time (RT) were accessed at 254 nm unless stated.
  • Several HPLC solvent systems and gradients were employed as specified:
  • Method A 0.1% TFA in MeOH and 0.1% aqueous TFA, 25% - 75% organic over 28 minutes;
  • Method B 0.1% TFA in MeOH and 0.1% aqueous TFA, 25% - 50% organic over
  • Method C 0.1% TFA in MeOH and 0.1% aqueous TFA, 25% - 75% organic over 120 minutes;
  • Method D 0.1% FA in Acetonitrile and 0.1% aqueous FA, 5% - 50% organic over 28 minutes;
  • Method E 0.1% FA in Acetonitrile and 0.1% aqueous FA, 5% - 50% organic over 18 minutes;
  • Method F 0.1% FA in MeOH and 0.1% aqueous FA, 25% - 75% organic over 18 minutes.
  • Semi-Preparative reversed-phase HPLC (Semi-Prep HPLC) were conducted upon a Gilson Chromatograph with a Gilson 215 Liquid Handler, a Gilson 845Z injection module coupled to a Gilson UV/VIS 155 detector and a YMC C18 5 ⁇ (100 x 20 mm) column. Spectra were manipulated in the Unipoint 5.11 software.
  • Method G 0.1% TFA in MeOH and 0.1% aqueous TFA, 25% - 75% organic over
  • Method H 0.1% TFA in MeOH and 0.1% aqueous TFA, 25% - 50% organic over 60 minutes, 50% - 75% organic over 5 minutes, injected from 25% MeOH in 0.1% aqueous TFA (3 ml).
  • Method I 0.1% TFA in MeOH and 0.1% aqueous TFA, 25% - 50% organic over 15 minutes, 50% - 75% organic over 2 minutes, injected from 25% MeOH in 0.1% aqueous TFA (3 ml). Compound isolation by reduction of fraction volumes (5 ml), precipitation with 5% NH 3 (aq.) and filtration;
  • Method J 0.1% FA in Acetonitrile and 0.1% aqueous FA, 5% - 50% organic over
  • N-(4-nitrophenyl)-2-(pyrrolidin-l-yl)acetamide (2) (1.50 g, 6.018 mmol) was dissolved in MeOH (30 ml) and stirred under N 2 . To this was added ammonium formate (3.79 g, 60.176 mmol, 10 equiv.) and Pd/C catalyst (150 mg, 0.1 equiv. w/w), and the mixture stirred overnight at room temperature.
  • 3-nitroaniline (6.37 g, 46.12 mmol) was dissolved in THF (150 ml) and cooled to 0 0 C prior to TEA (12.86 ml, 92.232 mmol, 2 equiv.) being added and the drop wise addition of 2-chloroacetyl chloride (5.50 ml, 69.174 mmol, 1.5 equiv.). The mixture was stirred overnight at room temperature under N 2 . The precipitate was filtered, THF removed in vacuo and the resulting oil suspended in sat. NaHCO 3 (aq.) (200 ml) and the solid product filtered and oven dried as a brown solid (7.68 g, 35.952 mmol, 78%).
  • N-(3-nitrophenyl)-2-(pyrrolidin-l-yl)acetamide (5) (1.5 g, 6.018 mmol) was dissolved in MeOH (30 ml) and stirred under N 2 . To this was added ammonium formate (3.79 g, 60.176 mmol, 10 equiv.) and Pd/C catalyst (150 mg, 0.1 equiv. w/w), and the mixture stirred overnight at room temperature.
  • N-(4-nitrophenyl)-3-(pyrrolidin-l-yl)propanamide (8) (5.00 g, 18.990 mmol) was dissolved in MeOH (100 ml) and stirred under N 2 . To this was added ammonium formate (11.98 g, 189.90 mmol, 10 equiv.) and Pd/C catalyst (500 mg, 0.1 equiv. w/w) and the mixture stirred at room temperature overnight.
  • N-(4-aminophenyl)-3-(pyrrolidin-l-yl)propanamide (9) (1.7002 g, 7.287 mmol) was dissolved in THF (100 ml) and cooled to 0 0 C prior to the addition of cone.
  • HCl (12 M, 3.34 ml, 40.08 mmol, 5.5 equiv.) followed by 1 BuONO (2.16 ml, 18.218 mmol, 2.5 equiv.) and the mixture stirred at 0 0 C for 1 hour.
  • N-(3-nitrophenyl)-3-(pyrrolidin-l-yl)propanamide (12) (5.06 g, 19.218 mmol) was dissolved in MeOH (100 ml) and stirred under N 2 . To this was added ammonium formate (11.98 g, 189.90 mmol, 10 equiv.) and Pd/C catalyst (500 mg, 0.1 equiv. w/w) and the mixture stirred at room temperature overnight.
  • N-(3-aminophenyl)-3-(pyrrolidin-l-yl)propanamide (13) (1.70 g, 7.286 mmol) was dissolved in THF (100 ml) and cooled to 0 0 C prior to the addition of cone.
  • HCl (12 M, 3.34 ml, 40.08 mmol, 5.5 equiv.) followed by 1 BuONO (2.16 ml, 18.218 mmol, 2.5 equiv.) and the mixture stirred at 0 0 C for 1 hour.
  • N-(4-nitrophenyl)-4-(pyrrolidin- 1 -yl)butanamide (16) (5.0 g, 20.215 mmol) was dissolved in MeOH (100 ml) and stirred under N 2 . To this was added ammonium formate (12.75 g, 202.15 mmol, 10 equiv.) and Pd/C catalyst (500 mg, 0.1 equiv. w/w) and the mixture stirred at room temperature overnight.
  • N-(4-aminophenyl)-4-(pyrrolidin-l-yl)propanamide (17) (1.89 g, 7.641 mmol) was dissolved in THF (100 ml) and cooled to 0 0 C prior to the addition of cone.
  • HCl (12 M, 3.50 ml, 42.03 mmol, 5.5 equiv.) followed by 1 BuONO (2.27 ml, 19.103 mmol, 2.5 equiv.) and the mixture stirred at 0 0 C for 1 hour.
  • N-(3-nitrophenyl)-4-(pyrrolidin-l-yl)butanamide (20) (5.0 g, 20.215 mmol) was dissolved in MeOH (100 ml) and stirred under N 2 . To this was added ammonium formate (12.75 g, 202.15 mmol, 10 equiv.) and Pd/C catalyst (500 mg, 0.1 equiv. w/w) and the mixture stirred at room temperature overnight.
  • N-(3-aminophenyl)-4-(pyrrolidin-l-yl)propanamide (21) (2.0524 g, 8.298 mmol) was dissolved in THF (100 ml) and cooled to 0 0 C prior to the addition of cone.
  • HCl (12 M, 3.80 ml, 45.64 mmol, 5.5 equiv.) followed by 1 BuONO (2.46 ml, 20.745 mmol, 2.5 equiv.) and the mixture stirred at 0 0 C for 1 hour.
  • 2-nitrobenzoic acid (5.0 g, 29.919 mmol) was dissolved in anhydrous DCM (150 ml) and anhydrous DMF (2 ml) and to this was added DMAP (500 mg, 0.1 equiv. w/w) and tert-butanol (4.29 ml, 44.879 mmol, 1.5 equiv.) prior to cooling the mixture to 0 0 C and the addition of DCC (6.79 g, 32.910 mmol, 1.1 equiv.) in two portions. The mixture was warmed to room temperature and stirred for 4 hours until shown to be complete.
  • 2,2'-ureylene-di-(tert-butyl benzoate) (33) (3.1103 g, 7.546 mmol) was suspended in TFA (30 ml) and stirred vigorously at room temperature for 1 hour. The mixture was cooled to 0 0 C on ice and flooded with ice cold ether (50 ml) inducing precipitation which was isolated by filtration, washed with ice cold ether (3 x 10 ml) and oven dried to yield a white powder (1.0457 g, 3.483 mmol, 46%). Mp. 192 - 194 0 C.
  • 2,2'-ureylene-di-benzoic acid (34) (150.0 mg, 0.500 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(4-aminophenyl)-2-(pyrrolidin-l- yl)acetamide (3) (438.1 mg, 1.998 mmol, 4 equiv.) and PyBOP (779.9 mg, 1.499 mmol, 3 equiv.) and the mixture stirred at room temperature under N 2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl 3 (20 ml) with sonication, and the organic solvent decanted from the oily residue.
  • 2,2'-ureylene-di-benzoic acid (34) (150.0 mg, 0.500 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(4-aminophenyl)-3-(pyrrolidin-l- yl)propanamide (9) (466.17 mg, 1.998 mmol, 4 equiv.) and PyBOP (779.9 mg, 1.499 mmol, 3 equiv.) and the mixture stirred at room temperature under N 2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication, and the organic solvent decanted from the oily residue.
  • 2,2'-ureylene-di-benzoic acid (34) (150.0 mg, 0.500 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(4-aminophenyl)-4-(pyrrolidin- 1 - yl)butanamide (17) (494.2 mg, 1.998 mmol, 4 equiv.) and PyBOP (779.9 mg, 1.499 mmol, 3 equiv.) and the mixture stirred at room temperature under N 2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl 3 (20 ml) with sonication, and the organic solvent decanted from the oily residue.
  • 2,2'-ureylene-di-benzoic acid (34) (150.0 mg, 0.500 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(3-aminophenyl)-2-(pyrrolidin-l- yl)acetamide (6) (438.1 mg, 1.998 mmol, 4 equiv.) and PyBOP (779.9 mg, 1.499 mmol, 3 equiv.) and the mixture stirred at room temperature under N 2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication, and the organic solvent decanted from the oily residue.
  • 2,2'-ureylene-di-benzoic acid (34) (150.0 mg, 0.500 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(3-aminophenyl)-3-(pyrrolidin-l- yl)propanamide (13) (466.17 mg, 1.998 mmol, 4 equiv.) and PyBOP (779.9 mg, 1.499 mmol, 3 equiv.) and the mixture stirred at room temperature under N 2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl 3 (20 ml) with sonication, and the organic solvent decanted from the oily residue.
  • 2,2'-ureylene-di-benzoic acid (34) (150.0 mg, 0.500 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(3-aminophenyl)-4-(pyrrolidin-l- yl)butanamide (21) (494.2 mg, 1.998 mmol, 4 equiv.) and PyBOP (779.9 mg, 1.499 mmol, 3 equiv.) and the mixture stirred at room temperature under N 2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl 3 (20 ml) with sonication, and the organic solvent decanted from the oily residue.
  • 3-amino-5-nitrobenzoic acid (1.0 g, 5.490 mmol) was dissolved in DMF (25 ml) and to this was added aniline (0.55 ml, 6.039 mmol, 1.1 equiv.), HOBt (816.05 mg, 6.039 mmol, 1.1 equiv.) and DCC (1.246 g, 6.039 mmol, 1.1 equiv.) and the mixture stirred under N 2 at room temperature for 24 hours.
  • 3-amino-5-nitrobenzoic acid (3.5 g, 19.216 mmol) was suspended in anhydrous toluene (20 ml) in a sealed tube and added under N 2 was TEA (2.95 ml, 21.14 mmol, 1.1 equiv.) followed by DPPA (4.14 ml, 19.216 mmol, 1 equiv.) and the mixture stirred at room temperature for 30 minutes prior to the addition of aniline (3.50 ml, 38.432 mmol, 2 equiv.). The mixture was heated at reflux for 16 hours. The toluene was removed in vacuo and the residue dissolved in DCM (100 ml) and washed with sat.
  • 3-nitrobenzoic acid (346.0 mg, 2.070 mmol) was suspended in anhydrous toluene (5 ml) under nitrogen in a sealed tube and to this was added under N 2 TEA (0.32 ml, 2.277 mmol, 1.1 equiv.) prior to the addition of DPPA (0.45 ml, 2.070 mmol, 1 equiv.) and the mixture stirred at room temperature for 30 minutes.
  • N-(4-aminophenyl)-3- (pyrrolidin-l-yl)propanamide (9) (724.6 mg, 3.106 mmol, 1.5 equiv.) was dissolved in CHCI 3 (2 ml) and toluene (2 ml) and added to the reaction prior to heating the reaction at 115 0 C for 16 hours. The brown solution was cooled to room temperature and quenched with sat. NaHCO 3 (aq.) (20 ml) and the reaction mixture extracted into CHCl 3 (30 ml), washing with sat.
  • 3-nitrobenzoic acid (400.0 mg, 2.393 mmol) was suspended in anhydrous toluene (5 ml) under nitrogen in a sealed tube and to this was added under N 2 TEA (0.37 ml, 2.632 mmol, 1.1 equiv.) prior to the addition of DPPA (0.52 ml, 2.393 mmol, 1 equiv.) and the mixture stirred at room temperature for 30 minutes.
  • N-(3-aminophenyl)-3- (pyrrolidin-l-yl)propanamide (13) (837.6 mg, 3.590 mmol, 1.5 equiv.) was dissolved in CHCI 3 (2 ml) and added to the reaction prior to heating the reaction at 115 0 C for 16 hours.
  • the brown solution was cooled to room temperature and quenched with sat. NaHCO 3 (aq.) (20 ml) and the reaction mixture extracted into CHCI3 (30 ml) which was washed with sat. NaHCO 3 (aq.) (3 x 20 ml), brine (20 ml), dried over MgSO 4 .
  • N-(4-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (10) (149.5 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and dH 2 O (2.5 ml) and to this was added 1,3- bis(3-ethynylphenyl)urea (77) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated to 130 0 C by microwave irradiation for 30 minutes.
  • N-(3-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (14) 149.5 mg, 0.577 mmol, 3 equiv. was dissolved in 1 BuOH (2.5 ml) and distilled water (2.5 ml) and to this was added l,3-bis(3-ethynylphenyl)urea (77) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated to 130 0 C under microwave irradiation for 30 minutes.
  • N-(4-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (18) (157.4 mg, 0.576 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and distilled water (2.5 ml) and to this was added l,3-bis(3-ethynylphenyl)urea (77) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated to 130 0 C under microwave irradiation for 30 minutes.
  • N-(3-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (22) (157.4 mg, 0.576 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and distilled water (2.5 ml) and to this was added l,3-bis(3-ethynylphenyl)urea (77) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated to 130 0 C under microwave irradiation for 30 minutes.
  • N-(4-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (10) (149.5 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and dH 2 O (2.5 ml) and to this was added 1,3- bis(4-ethynylphenyl)urea (78) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated at 130 0 C under microwave irradiation for 30 minutes.
  • N-(3-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (14) 149.5 mg, 0.577 mmol, 3 equiv. was dissolved in 1 BuOH (2.5 ml) and distilled water (2.5 ml) and to this was added l,3-bis(4-ethynylphenyl)urea (78) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated at 130 0 C under microwave irradiation for 30 minutes.
  • N-(4-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (18) (157.4 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and distilled water (2.5 ml) and to this was added l,3-bis(4-ethynylphenyl)urea (78) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated at 130 0 C under microwave irradiation for 30 minutes.
  • N-(3-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (22) (157.4 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and distilled water (2.5 ml) and to this was added l,3-bis(4-ethynylphenyl)urea (78) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated at 130 0 C under microwave irradiation for 30 minutes.
  • N-(4-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (18) (157.4 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and distilled water (2.5 ml) and to this was added l,3-bis(2-ethynylphenyl)urea (79) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated at 130 0 C under microwave irradiation for 30 minutes.
  • N-(3-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (22) (157.4 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and distilled water (2.5 ml) and to this was added l,3-bis(2-ethynylphenyl)urea (79) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (2.4 mg, 9.6 ⁇ mol, 5 mol %) and the mixture heated at 130 0 C under microwave irradiation for 30 minutes.
  • N-(4-aminophenyl)-3-(dimethylamino)propanamide (93) (1.5867 g, 7.6552 mmol) was dissolved in THF (100 ml) and cooled to 0 0 C prior to the addition of cone.
  • HCl (12 M, 3.51 ml, 42.1038 mmol, 5.5 equiv.) followed by 1 BuONO (2.27 ml, 19.1380 mmol, 2.5 equiv.) and the mixture stirred at 0 0 C for 1 hour.
  • N-(4-nitrophenyl)-3-(piperidin-l-yl)propanamide (95) (5.0584 g, 18.240 mmol) was dissolved in MeOH (100 ml) and stirred under N 2 . To this was added ammonium formate (11.50 g, 182.40 mmol, 10 equiv.) and Pd/C catalyst (500 mg, 0.1 equiv. w/w) and the mixture stirred at room temperature overnight.
  • N-(4-aminophenyl)-3-(piperidin-l-yl)propanamide (96) (1.7529 g, 7.087 mmol) was dissolved in THF (100 ml) and cooled to 0 0 C prior to the addition of cone.
  • HCl (12 M, 3.25 ml, 38.979 mmol, 5.5 equiv.) followed by 1 BuONO (2.10 ml, 17.7175 mmol, 2.5 equiv.) and the mixture stirred at 0 0 C for 1 hour.
  • N-(4-aminophenyl)-3-morpholinopropanamide (99) (1.7669 g, 7.087 mmol) was dissolved in THF (100 ml) and cooled to 0 0 C prior to the addition of cone.
  • HCl (12 M, 3.25 ml, 38.979 mmol, 5.5 equiv.) followed by 1 BuONO (2.10 ml, 17.7175 mmol, 2.5 equiv.) and the mixture stirred at 0 0 C for 1 hour.
  • 3,3'-ureylene-di-benzoic acid (26) (100.0 mg, 0.333 mmol) was dissolved in anhydrous DMF (6 ml) and to this was added N-(4-aminophenyl)-3-(piperidin-l- yl)propanamide (96) (329.8 mg, 1.333 mmol, 4 equiv.) and PyBOP (519.9 mg, 1.665 mmol, 3 equiv.) and the mixture stirred at room temperature under nitrogen for 22 hours.
  • N-(4-azidophenyl)-3-(dimethylamino)propanamide (134) (134.6 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and dH 2 O (2.5 ml) and to this was added 1,3- bis(3-ethynylphenyl)urea (77) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (4.8 mg, 19.2 ⁇ mol, 10 mol %) and the mixture stirred at room temperature overnight.
  • N-(4-azidophenyl)-3-(piperidin-l-yl)propanamide (97) (157.7 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and dH 2 O (2.5 ml) and to this was added 1,3- bis(3-ethynylphenyl)urea (77) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (4.8 mg, 19.2 ⁇ mol, 10 mol %) and the mixture stirred at room temperature overnight.
  • N-(4-azidophenyl)-3-morpholinopropanamide (100) (158.9 mg, 0.577 mmol, 3 equiv.) was dissolved in 1 BuOH (2.5 ml) and dH 2 O (2.5 ml) and to this was added 1,3- bis(3-ethynylphenyl)urea (77) (50 mg, 0.192 mmol), sodium ascorbate (19.0 mg, 0.096 mmol, 0.5 equiv.) and CuSO 4 .5H 2 O (4.8 mg, 19.2 ⁇ mol, 10 mol %) and the mixture stirred at room temperature overnight. The reaction was flooded with distilled water (5 ml) and cooled on ice for 10 minutes.
  • Ligands 101-106 were analysed by HPLC purity analysis. The results are shown in Table 1 below.
  • F21T 5 FAM-GGG TTA GGG TTA GGG TTA GGG-TAMRA 3' ; t-loop: 5 FAM-TAT AGC TATA TTT TTT TATA GCT ATA-TAMRA 3' ; c-kitl: 5 FAM-AGA GGG AGG GCG CTG GGA GGA GGG GCT-
  • TAMRA 3' TAMRA 3' ;
  • c-kit2 FAM-CCC GGG CGG GCG CGA GGG AGG GGA GG-
  • the required oligonucleotide (stock solution 120 ⁇ l, 20 ⁇ M) was suspended in FRET buffer (60 rnM KCl, KCacodilate, pH 7.4, 5340 ⁇ l, 400 nM DNA) and heated to 85 0 C for 10 minuets prior to cooling to room temperature overnight. DNA was distributed (50 ⁇ l) across a 96 well RT-PCR plate (Bio-Rad) to which ligand was added to afford the required concentration. FRET buffer was used as a negative control. DNA melting was assessed upon a MJ Research Opticon DNA Engine Continuous Fluorescence Detector exciting at 450 - 495 nm.
  • FRET buffer 60 rnM KCl, KCacodilate, pH 7.4, 5340 ⁇ l, 400 nM DNA
  • Fluorescence values were recorded at 515 - 545 nm at 0.5 0 C intervals as the plate was heated from 30 - 100 0 C.
  • the raw data was smoothed, normalised and interpolated in the Orgin 7.0 software package, prior to assessing the maximum derivative of the sigmoid melting curve.
  • the non-sigmoid melting curves were fitted to sigmoid curves and their maximum derivative assessed. All values were normalised relative to the negative control to afford the change in melting temperature induced by a given ligand concentration ( ⁇ Tm), with all datasets averaged ⁇ s.d.
  • BRACO- 19 was prepared in-house to a HPLC purity of 98% (HPLC method 0.1% TFA in acetonitrile and 0.1% aqueous TFA, 20% - 40% organic over 28 minutes) and stored as a 10 mM DMSO stock solution. TMPyP4 was used at its supplied purity of 97% (Sigma-Aldrich) as a 10 mM distilled water stock solution. FRET Assay
  • the aim of the FRET assay is to assess the thermal stabilisation of a DNA oligonucleotide by a ligand.
  • a G-quadruplex forming sequence F21T - telomeric, c-kitl, c-kit2
  • FAM donor fluorophore
  • TAMRA acceptor
  • the FRET assay measures the ability of a ligand to thermally stabilise the DNA so that it unfolds at an elevated temperature relative to DNA control.
  • the t-loop duplex DNA sequence is used to assess duplex DNA interaction.
  • the required ligand (20 mM stock solution in d6-OMSO, stored at -20 0 C) was diluted to 1 mM in HPLC grade DMSO (Sigma-Aldrich). This stock was used to make four independent dilutions of ligand at the concentrations 0.1, 0.2, 0.5, 1.0, 2.5, 5.0 and 10.0 ⁇ M in the wells of the RT-PCR plate as required (50 ⁇ M). A buffer negative control was also screened.
  • the aim of the competition assay is to challenge the ability of a ligand to thermally stabilise the G-quadruplex DNA structure (as in the FRET assay), by introducing elevated concentrations of a duplex DNA competitor.
  • a ligand is seen to be selective if there is no loss of G-quadruplex DNA FRET affinity upon introduction of the duplex DNA competitor.
  • CT-DNA Sigma-Aldrich; 25 ⁇ l; 533.3 ⁇ M DNA bp stock in 0.5 mM EDTA/30mM Kcacodalate buffer
  • CT-DNA bp concentrations 0.0 (buffer; ⁇ Tm l ⁇ M control), 0.6, 6.0, 60.0 and 120.0 ⁇ M in the wells of the RT-PCR plate as required.
  • Buffer ⁇ Tm l ⁇ M control
  • the required ligand (20 mM stock solution in d6- DMSO, stored at -20 0 C) was diluted to 1 mM in HPLC grade DMSO (Sigma- Aldrich). This stock was used to make three independent dilutions of concentration 1.0 ⁇ M in the wells of the RT-PCR plate as required. A buffer negative control was also screened. The data was normalised against the ⁇ Tmi ⁇ M control taken as 100% F21T stabilisation, and expressed as the % reduction in F21T ⁇ Tnii ⁇ M at a given CT-DNA bp concentration. SPR Assay (courtesy of the W. D. Wilson group, Georgia State University, USA)
  • the aim of the SPR assay is to measure the dynamic and steady-state binding kinetics of a ligand for a G-quadruplex (hTel - telomeric, c-kitl , c-myc) or duplex DNA sequence.
  • a favourable ligand interaction is indicated by a large equilibrium binding constant (K A ).
  • Biosensor experiments were conducted in filtered, degassed HEPES buffer (10 mM HEPES, 100 mM KCl, 3 mM EDTA, 0.00005 v/v of 10% P20 BIACORE surfactant, pH 7.3) at 25°C.
  • the 5'-biotin labeled DNA sequences (Midland Certified Reagent Company or IDT) were HPLC purified and of the sequence: hTel: 5' biotin - d[AGGG(TTAGGG) 3 ] 3' c-kitl : 5' biotin - d[(AG3)2CGCTG 3 AG 2 AG3] 3' c-myc: 5' biotin - d[(AG 3 TG 4 ) 2 A] 3'
  • RU directly proportional to the amount of bound ligand as a series of sensorgrams from which the response (RU) in the steady-state region was averaged over a selected time period.
  • the predicted maximum response in the steady-state region (RU ma ⁇ ) was determined from the DNA molecular weight and the refractive index gradient ratio of the compound and DNA.
  • the number of binding sites and equilibrium constant were obtained by fitting plots of RU vs C f r ee , the concentration of free ligand in equilibrium with the complex to a two-site equilibrium model using Kaleidagraph for nonlinear least squares optimization of the binding parameters: where K 1 and K 2 are the macroscopic binding constants for a two-site binding mode.
  • TRAP-LIG Modified Telomerase Repeat Amplification Protocol
  • Telomerase was extracted as total cellular protein into lysis buffer (10 mM Tris- HcI, pH 7.5, 1 mM MgCl 2 , 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 5 mM ⁇ - mercaptoehtanol, 0.1 mM AEBSF) from exponentially growing A2780 cells (ATCC-LGC Promochem) maintained in Dulbecco's Modified Eagles Media (DMEM) as per the general cell culture experimental. Protein concentration was calculated by the Bradford assay.
  • the required ligand (20 mM stock solution in d6-DMSO, stored at -20 0 C) was diluted to 1 mM in HPLC grade water (Fisher Scientific) containing 1% HCl (Fisher Scientific). This stock was in turn used to screen the ligand concentrations of 1.0, 10.0, 25.0 and 50.0 ⁇ M, as well as a positive (PCR-grade water) and telomerase negative (lysis buffer) control.
  • TS primer elongation was assessed at the required ligand concentration from the master mix 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), bovine serium albumin (0.05 ⁇ g) dNTPs (125 ⁇ M each) and protein extract (500 ⁇ g/sample), which was added (40 ⁇ l) to the required ligand concentration or control (10 ⁇ l) at 4°C.
  • 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
  • bovine serium albumin 0.05 ⁇ g
  • dNTPs 125 ⁇ M each
  • the elongation step was conducted for 30 min at 30 0 C, followed by 5 min at 94°C and final maintenance of the mixture at 20 0 C.
  • the elongation reaction mixture was introduced to a QIA quick nucleotide purification tube (Qiagen) as per the manufacturer's instructions.
  • Qiagen Qiagen
  • a high-salt buffer was used to elute the ligand while retaining the DNA on the membrane of the purification tube, prior to DNA elution in PCR-grade water and sample freeze drying.
  • PCR-grade water 40 ⁇ l
  • the sample was resuspended in PCR-grade water (40 ⁇ l) prior to the addition (10 ⁇ l) of the PCR amplification mix containing the ACX reverse primer (1 ⁇ M; 5'-GCGCGG(CTTACC) 3 CTAACC-3'), the TS forward primer (0.1 ⁇ g), TRAP buffer (5 ⁇ l), BSA (5 ⁇ g), dNTPs (0.5 mM) and TAQ polymerase (2 units; RedHot, ABgene, U.K.).
  • PCR amplification was conducted for 34 cycles of 94°C for 30 sec, 61 0 C for 1 min and 72°C for 1 min.
  • Dulbecco's Modified Eagles Media (DMEM; Invitrogen) supplemented with foetal bovine serium (10% v/v; Invitrogen), hydrocortisone (0.5 ⁇ g/ml; Acros Organics), L-glutamine (2 mM; Invitrogen) and non-essential amino acids (1 x; Invitrogen) was used for the MCF7, A549 and A2780 cell lines, and Minimal Essential Medium (MEM; Sigma-Aldrich) supplemented with foetal bovine serum (10% v/v; Invitrogen), L-glutamine (2 mM; Invitrogen) and non-essential amino acids (1 x; Invitrogen) was used for the WI38 cell line.
  • DMEM Dulbecco's Modified Eagles Media
  • foetal bovine serium 10% v/v; Invitrogen
  • hydrocortisone 0.5 ⁇ g/ml; Acros Organics
  • L-glutamine
  • SRB Sulforhodamine B Cytotoxicity Assay
  • MCF7 breast
  • A549 lung
  • WI38 somatic control cell line
  • the required cell line in logarithmic growth phase was counted upon a Neybauer haemocytometer (Assistant, Germany) to allow dilutions to be made to afford media (20 ml) containing the required number of cells (2.5 x 10 4 cells/ml MCF7 and WI38; 6.25 x 10 3 cells/ml A549). This was distributed across a 96 well plate as required (160 ⁇ l/well; 4000 cells/well MCF7 and WI38; 1000 cells/well A549; Fisher Scientific) and the plate incubated overnight (37 0 C, 5% CO 2 ).
  • the required ligand (20 mM stock solution in d6- DMSO, stored at -20 0 C) was diluted to 1 mM in HPLC grade water (Fisher Scientific) containing 1% HCl (Fisher Scientific). This stock was in turn used to screen the ligand concentration ranges of 0.1 - 25.0 ⁇ M or 0.25 - 50 ⁇ M as stated, distributed across the wells of a 96 well plate (40 ⁇ l) to afford eight repeats of each exposure. Eight positive and negative controls (media, 40 ⁇ l) were also screened and the plate incubated for 96 hours (37 0 C, 5% CO 2 ).
  • TCA aqueous trichloroacetic acid
  • SRB solution (0.4% in 1% acetic acid, 80 ⁇ l; Acros Organics) was added to each well except the negative control, and incubated at room temperature (15 min), prior to the removal of SRB, the washing of wells with 1% acetic acid (160 ⁇ l) and oven drying (> 1 hour, 60 0 C).
  • Tris-Base (1OmM, 100 ⁇ l) was introduced to each well and plates shaken (5 min) prior to reading the absorbance of each well at 540 nm on an Anthos 2010 plate reader using the software ADAP 1.1.
  • the data was analysed considering the most consistent 6 - 8 data sets in Origin 7.0, and expressed as the mean % cell viability relative to the positive (100% viability) and negative (no SRB staining) controls ⁇ s.d.
  • the IC50 value is the ligand concentration required for 50% cell survival.
  • Sub-Cytotoxic Induction of Cellular Senescence ( ⁇ -Galactosidase Assay)
  • the aim of the ⁇ -galactosidase assay is to assess the ability of the ligands to induce cellular senescence upon exposure of MCF7 cells to sub-cytotoxic ligand concentrations over a 1 week period. Induction of cellular senescence can be indicative of telomere dysfunction.
  • Cells were stained for senescence using the ⁇ -galacotosidase staining kit (Cell Signalling Technology) according to the manufacturer's instructions.
  • cells were seeded (1 x 10 5 , 2 ml) in a 6 well plate (Fisher- Scientific) with the required ligand concentration and incubated overnight (37 0 C, 5% CO 2 ).
  • the medium was removed, the well washed with PBS (2 ml) prior to fixing (Ix fixative solution, 10 min).
  • the fixative was removed and the well washed with PBS (2 x 2 ml) prior to the addition of the staining solution (ImI) and the plates incubated overnight (37 0 C, 5% CO 2 ).
  • Three independent fields of cells were visualised (20Ox magnification) from both repeats, with the mean percentage of blue senescing cells reported ⁇ s.d.
  • the aim of metaphase spread assessment is to establish if the ligands are able to induce the telomeric regions of the chromosomes of MCF7 cells to fuse upon exposure to sub-cytotoxic ligand concentrations over a 1 week period. Observation of telomere end - end fusion can be indicative of telomere dysfunction.
  • MCF7 Cells (1 x 10 5 , 10 ml media, ATCC-LGC Promochem) were exposed to two independent sub-cytotoxic concentrations of the required ligand over a 1 week period (37 0 C, 5% CO 2 ), with a biweekly treatment. A 0.0 ⁇ M (media) negative control was also screened. After 1 week exposure, colcemid (lOO ⁇ l, GibcoBRL) was added prior to 1 hour incubation (37 0 C, 5% CO2). The cells were harvested (1200 rpm, 15 min) discarding the supernatant.
  • Pellets were resuspended in potassium chloride (12ml, 75 mM) and incubated for 20 min at room temperature, prior to the addition of fixative (5 drops, 3:1 methanol: acetic acid, freshly prepared) and incubation for 15 min at room temperature. Centrifugation (1200 rpm, 15 min) and media disposal afforded a pellet which was added to and resuspended in fixative (9ml) prior to centrifugation (1200 rpm, 15 min). The supernatant was discarded, and fresh fixative added dropwise while vortexing (5 ml) prior to centrifugation (1200 rpm, 6 min). This was repeated adding less fixative (3ml), and then a minimum amount.
  • the optimal ligands for telomeric G-quadruplex DNA interaction based upon the results from the telomeric FRET F21T DNA model are 80 and 84.
  • the optimal ligands for c-kit G-quadruplex DNA interaction based upon the results from the c-kit 1 and c-kit2 FRET DNA models are 84 and 105.
  • the optimal ligands with a potent and selective G-quadruplex DNA interaction over duplex DNA are 36, 37, 90, 91 and 101. These ligands were assessed by the FRET- based t-loop duplex DNA model, as well as the FRET -based competition assay.
  • the SPR assay was also used to highlight G-quadruplex DNA selectivity.
  • the optimal ligands with a potent and cancer cell line selective cytotoxic response identified by the SRB assay are 36, 45, 102, 73, 76, 80 and 105.
  • telomeres synthesised are able to strongly interact with G-quadruplex DNA structures formed by telomeric DNA, as well as those formed in the promotor regions of the c-kit and c-myc proto-oncogenes. This potentially allows therapeutic intervention against telomerase and cancer cell telomere integrity, as well as specific down-regulation of the c-kit and c-myc.
  • SRB Sulforhodamine B
  • ligands were observed to induce short-term cellular growth arrest upon exposure to sub- cytotoxic concentrations of ligands, correlated with low-level induction of cellular senescence and telomere end - end fusions in chromosomal metaphase spreads.

Abstract

L'invention concerne des composés de la formule (I) ou un sel, un solvate ou un promédicament de ces derniers. Les composés précités peuvent être utilisés en thérapie, en particulier dans la thérapie anticancéreuse.
PCT/EP2008/054362 2007-04-10 2008-04-10 Dérivés d'uréylène WO2008122667A2 (fr)

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JP2010138079A (ja) * 2008-12-09 2010-06-24 Mitsui Chemicals Inc アミド誘導体および殺虫剤
WO2012136997A1 (fr) * 2011-04-05 2012-10-11 University College London Agent de stabilisation de g-quadruplexe
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JP2010138079A (ja) * 2008-12-09 2010-06-24 Mitsui Chemicals Inc アミド誘導体および殺虫剤
WO2012136997A1 (fr) * 2011-04-05 2012-10-11 University College London Agent de stabilisation de g-quadruplexe
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