WO2008122667A2 - Ureylene derivatives - Google Patents

Ureylene derivatives 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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French (fr)
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WO2008122667A3 (en
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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.

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Abstract

The invention concerns compounds of Formula (I) or a salt, solvate or pro-drug thereof. The compounds may be used in therapy, particularly anti-cancer therapy.

Description

UREYLENE DERIVATIVES Field of the Invention
The present invention is concerned with ureylene derivatives, their intermediates, uses thereof and processes for their production. In particular, the present invention relates to compounds which may be used as chemotherapeutic agents in the treatment of cancer. Background of the Invention
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.
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.
The targeting of the enzyme telomerase has been the subject of much active research in the field of drug discovery. Telomerase inhibitors can selectively target tumour cells and cause tumour cell death well before damage to regenerative tissues occurs, thereby minimising undesirable side-effects.
A number of 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. Those molecules found to have greatest activity so far are the 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.
There has also recently been focus on targeting the c-kit oncogene in cancer therapy. 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. JΛm.Chem.Soc. 129, 4386-4392].
In Leukemia (2007), 1-3 in Letter to the Editor, "Inhibition of myc promoter and telomerase activity and induction of delayed apoptosis by SYUIQ-5, a novel G- quadruplex interactive agent in leukemia cells", it was shown that a new class of potent G-quadruplex interactive agents - quindoline derivatives (especially SYUIQ-5), can inhibit c-myc gene promoter activity and telomerase activity. This demonstrated the feasibility of down-regulating a cancer-associated gene by targeting a quadruplex sequence in the promoter region of the gene.
Although advances have been made, there remains a great need for potent telomerase inhibitors and anti-tumour agents which selectively target tumour cells and have little or no cytotoxic effects on normal cells. 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. Summary of the Invention
In a first aspect of the present invention, there is provided a compound of formula
(I):
Figure imgf000003_0001
(I) or a salt, solvate or pro-drug thereof; wherein:
R1 and R2 are independently selected from H, NH2, NH(Ci_6 alkyl), N(Ci_6 alkyl)2,
NH(C6_2o aryl), NH(C7.20 alkaryl), NH(C7.20 aralkyl), NHC(O)(CL6 alkyl), NHC(O)(C6_20 aryl), NHC(O)(C7-20 alkaryl), NHC(O)(C7-20 aralkyl), NHC(O)(Ci_20 heteroaryl), NHC(0)(C2_2o heterocyclyl), NHC(O)(C2_20 heteroaralkyl), NHC(O)(C3-20 heterocyclylalkyl), NHC(O)(C3-20 alkylheterocyclyl), NO2, CN and C1-10 alkyl;
R3, R4, R5 and R6 are independently selected from the group consisting of H, C(O)OH, C(O)O(C1-6 alkyl), C(O)O(C6_20 aryl), C(O)O(C7_20 aralkyl), C(O)O(C7_20 alkaryl), halo, OH, O(Ci_6 alkyl), NH2, NH(Ci_6 alkyl), N(Ci_6 alkyl)2, NH(C6_20 aryl), NH(C7_2o alkaryl), NH(C7_20 aralkyl), NHC(O)(C1-6 alkyl), NHC(O)(C6_20 aryl), NHC(0)(C7_2o alkaryl), NHC(O)(C7_20 aralkyl), NHC(0)(Ci_2o heteroaryl), NHC(O)(C2_20 heterocyclyl), NHC(O)(C2_20 heteroaralkyl), NHC(O)(C3_20 heterocyclylalkyl), NHC(0)(C3-2o alkylheterocyclyl), NHC(0)(C6-2o arylamino), NHC(O)(C2-20 heteroarylamino), NHC(0)NH(Ci_6 alkyl), NHC(O)NH(C6_20 aryl), NHC(O)NH(C7_20 alkaryl), NHC(O)NH(C7_20 aralkyl), NHC(O)NH(C i_20 heteroaryl), NHC(O)NH(C2_20 heterocyclyl), NHC(O)NH(C2_20 heteroaralkyl), NHC(O)NH(C3_20 heterocyclylalkyl), NHC(0)NH(C3_2o alkylheterocyclyl), NHC(O)NH(C6-20 arylamino), NHC(O)NH(C2-20 heteroarylamino), NO2, CN, C(O)H, C(O)(C1-6 alkyl), C(O)(C6_20 aryl), C(O)(C7_20 alkaryl), C(O)(C7_20 aralkyl), C(O)NH(C1-6 alkyl), C(0)NH(C6-2o aryl), C(O)NH(C7_20 alkaryl), C(O)NH(C7_20 aralkyl), C(O)NH(Ci_20 heteroaryl), C(O)NH(C2_20 heterocyclyl), C(0)NH(C2_2o heteroaralkyl), C(O)NH(C3_20 heterocyclylalkyl), C(O)NH(C3_20 alkylheterocyclyl), C1-20 heteroaryl, C6-20 aryl(Ci_20 heteroaryl) and (C1-20 heteroaryl)C6-20 aryl, wherein any of the groups alkyl, aryl, arylamino, heteroarylamino, alkaryl, aralkyl, heteroaryl, heterocyclyl, heteroaralkyl, alkylheterocyclyl and heterocyclylalkyl are optionally independently substituted on the backbone with one or more of the groups, preferably 1, 2, 3, 4, 5 or 6 groups, independently selected from C(O)OH, C(0)0(Ci_6 alkyl), C(O)O(C6_20 aryl), C(O)O(C7_20 aralkyl), C(O)O(C7_20 alkaryl), NHC(O)-CH=CH2, -C≡C-H, halo, OH, O(Ci_6 alkyl), O(C6_20 aryl), O(C7_20 alkaryl), O(C7_20 aralkyl), =0, NH2, =NH, NH(C1-6 alkyl), N(C1-6 alkyl)2, =N(C1-6 alkyl), NH(C6_20 aryl), NH(C7_20 alkaryl), NH(C7_20 aralkyl), NHC(0)(Ci_6 alkyl), NHC(O)(C6_20 aryl), NHC(O)(C7_20 alkaryl), NHC(O)(C7_20 aralkyl), NHC(O)(C1-20 heteroaryl), NHC(O)(C2_20 heterocyclyl), NHC(0)(C2_2o heteroaralkyl), NHC(O)(C3_20 heterocyclylalkyl), NHC(O)(C3_20 alkylheterocyclyl), NO2, CN, C(O)H, C(O)(C1-6 alkyl), C(O)(C6_20 aryl), C(O)(C7_20 alkaryl), C(O)(C7_20 aralkyl), C1-10 alkyl, C2-10 alkoxyalkyl, C7_20 alkoxyaryl, C12_20 aryloxyaryl, C7_20 aryloxyalkyl, C1-10 alkoxy, C6_20 aryloxy, C2-10 alkenyl, C2-10 alkynyl, C3-20 cycloalkyl, C4-20 (cycloalkyl)alkyl, C7-20 aralkyl, C7-20 alkaryl, C1-20 heteroaryl and C6_20 aryl;
R7 and R8 are independently selected from the group consisting of H and Ci_6 alkyl; and at least one of R3, R4, R5 and R6 is not H.
Preferably, at least two of R3, R4, R5 and R6 are not H.
Preferably, R7 and R8 are independently selected from the group consisting of H and Ci-3 alkyl, more preferably both R7 and R8 are H.
Preferably, R1 and R2 are independently selected from H, NHC(O)(Ci_6 alkyl), NHC(0)(C6_2o aryl), NHC(0)(C7-2o alkaryl), NHC(0)(C7-2o aralkyl), NHC(O)(C1-20 heteroaryl), NHC(0)(C2-2o heterocyclyl), NHC(0)(C2-2o heteroaralkyl), NHC(0)(C3-2o heterocyclylalkyl), and NHC(O)(C3_20 alkylheterocyclyl).
Preferably, R1 and R2 are independently selected from H and NHC(O)(C342 heterocyclylalkyl) . Preferably, R1 and R2 are independently selected from NHC(O)Ci_6 alkyl(C3-6 heterocyclyl).
Preferably, where R1 and R2 are independently selected from the group consisting of NHC(O)Ci_6 alkyl(C3_6 heterocyclyl), the Ci_6 alkyl is preferably selected from 1,1- methanediyl, 1,2-ethanediyl, 1,3-propanediyl and 1,4-butanediyl. Preferably, 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.
Preferably, where R1 and R2 are not H, they have the structure independently selected from structure (II):
Figure imgf000005_0001
wherein, n is 0, 1, 2, 3, 4 or 5, preferably 1, 2 or 3. Preferably, both R1 and R2 are H.
Preferably, R3, R4, R5 and R6 are independently selected from the group consisting of H, C(O)OH, C(O)NH(Ci_6 alkyl), C(O)NH(C6_i0 aryl), C(O)NH(C7_i0 alkaryl), C(O)NH(C7-I0 aralkyl), C(O)NH(C140 heteroaryl), C(O)NH(C240 heterocyclyl), C(O)NH(C240 heteroaralkyl), C(O)NH(C340 heterocyclylalkyl), C(O)NH(C340 alkylheterocyclyl), NHC(O)(C640 aryl), NHC(O)NH(C640 aryl), NHC(O)NH(C1-6 alkyl), NHC(O)NH(C640 aryl), NHC(O)NH(C740 alkaryl), NHC(O)NH(C740 aralkyl), NHC(0)NH(Ci_io heteroaryl), NHC(O)NH(C2-I0 heterocyclyl), NHC(O)NH(C2_i0 heteroaralkyl), NHC(O)NH(C3-I0 heterocyclylalkyl), NHC(O)NH(C3-I0 alkylheterocyclyl), NHC(O)NH(C6-10 arylamino), NHC(O)NH(C2-10 heteroarylamino), C1-20 heteroaryl, (C1-20 heteroaryl)C6-20 aryl and (C6-20 aryl)Ci_20 heteroaryl; wherein any of the groups alkyl, aryl, arylamino, heteroarylamino, alkaryl, aralkyl, heteroaryl, heterocyclyl, heteroaralkyl, alkylheterocyclyl and heterocyclylalkyl are optionally independently substituted on the backbone with one or more of the groups, preferably 1, 2 or 3 groups, independently selected from NHC(O)-CH=CH2, halo, OH, NH2, NH(C1-6 alkyl), N(C1-6 alkyl)2, =N(C1-6 alkyl), NH(C6-2o aryl), NH(C7-20 alkaryl), NH(C7-20 aralkyl), NHC(O)(C1-6 alkyl), NHC(O)(C6-20 aryl), NHC(O)(C7-20 alkaryl), NHC(O)(C7-20 aralkyl), NHC(O)(CL20 heteroaryl), NHC(O)(C2-20 heterocyclyl), NHC(O)(C2-20 heteroaralkyl), NHC(O)(C3-20 heterocyclylalkyl), NHC(O)(C3-20 alkylheterocyclyl), NO2, CN, C1-10 alkyl, C2-I0 alkenyl, C2-I0 alkynyl, C3-20 cycloalkyl, C4-20 (cycloalkyl)alkyl, C7-20 aralkyl, C7-20 alkaryl, C1-20 heteroaryl and C6-20 aryl. Preferably, R3, R4, R5 and R6 are independently selected from the group consisting of H, C(O)OH, C(O)NH(C6-I0 aryl), NHC(O)(C6-I0 aryl), NHC(O)NH(C6-10 aryl), C1-10 heteroaryl, (C1-10 heteroaryl)C6_i0 aryl and (C6_i0 aryl)Ci_i0 heteroaryl; wherein the groups aryl and heteroaryl are optionally independently substituted on the backbone with one or more of the groups, preferably 1, 2 or 3 groups, independently selected from NHC(O)-CH=CH2, NH2, NHC(O)(C1-3 alkyl), NHC(O)(C6-I0 aryl), NHC(O)(C7-I0 alkaryl), NHC(O)(C7-10 aralkyl), NHC(O)(C3-8 heteroaryl), NHC(O)(C3-8 heterocyclyl), NHC(O)(C3-S heteroaralkyl), NHC(O)(C3-8 heterocyclylalkyl), NHC(O)(C3-8 alkylheterocyclyl), C1-4 alkyl, C2-4 alkenyl, C3-10 cycloalkyl, C4-1O (cycloalkyl)alkyl, C7-10 aralkyl, C7_i0 alkaryl, C1-10 heteroaryl and C6_i0 aryl. Preferably, R3, R4, R5 and R6 are independently selected from the group consisting of H, C(O)OH, C(O)NH(C6-S aryl), NHC(O)NH(C6-8 aryl), C1-5 heteroaryl, (C1-5 heteroaryl)C6_8 aryl and (C6-8 aryl)Ci_5 heteroaryl; wherein the groups aryl and heteroaryl are optionally independently substituted on the backbone with one or more of the groups, preferably 1, 2 or 3 groups, most preferably 1 group, independently selected from NHC(O)-CH=CH2, NH2, NHC(O)(C1-3 alkyl), NHC(O)(C6-I0 aryl), NHC(O)(C7-I0 alkaryl), NHC(O)(C7-10 aralkyl), NHC(O)(C3-8 heteroaryl), NHC(O)(C3-8 heterocyclyl), NHC(O)(C3-8 heteroaralkyl), NHC(O)(C3-8 heterocyclylalkyl), NHC(O)(C3-8 alkylheterocyclyl), C1-10 heteroaryl and C6-10 aryl.
Preferably, when R3 and R4 are not H, R5 and R6 are both H. Preferably, when R5 and R6 are not H, R3 and R4 are both H. Preferably, when R1 and R2 are not H, R3 and R4 are both H. Preferably, when R1 and R2 are not H, R5 and R6 are not H.
Preferably, where R3, R4, R5 and R6 are not H, they are are independently selected from the group consisting of (a), (b), (c), (d), (e) and (f):
Figure imgf000007_0001
wherein, R9 is selected from the group consisting of -(CH2)n-X, and n is 0, 1, 2, 3, 4 or 5, preferably 1, 2 or 3; R10 and R11 are independently selected from the group consisting of H, NHC(O)-(CH2)n-pyrrolidinyl and NHC(O)-(CH2)n-CH=CH2, and n is 0, 1, 2, 3, 4 or 5, preferably 0, 1, 2 or 3; and wherein the wavy line indicates the point of attachment of the group to the rest of the molecule, and X is selected from NH2, NH(CI-CO alkyl), N(C1-Ce alkyl)2,
Figure imgf000007_0002
Preferably, X is selected from N(CH3)2, pyrrolidinyl, piperidinyl or morpholinyl (attached to the rest of the molecule via the nitrogen atoms).
Preferably, when R1, R2, R3, R4, R5 and R6 are not H, one or more of these groups may comprise at least one terminal group independently selected from the group consisting of C1-10 heteroaryl, C2-10 heterocyclyl, C2-1O heteroaralkyl, C3-10 heterocyclylalkyl and C3_i0 alkylheterocyclyl containing at least one nitrogen atom, more preferably selected from the group consisting of C3-6 heteroaryl, C3-6 heterocyclyl, C3-7 heteroaralkyl, C3_7 heterocyclylalkyl and C3_7 alkylheterocyclyl containing at least one mtrogen atom, more preferably a C3 5 heterocyclyl group containing at least one nitrogen atom, most preferably a pyrrolidinyl group
Preferably, when R1, R2, R3, R4, R5 and R6 are not H, one or more of these groups may comprise at least one terminal group having the structure (III)
Figure imgf000008_0001
wherein, n is 0, 1, 2, 3, 4 or 5, preferably 1, 2 or 3, and X is as defined above
Preferably, the compound (I) contains at least one terminal group having the structure (III), preferably at least 2 such groups
Preferably, R1 and R2 have the same structure Preferably, R4 and R4 have the same structure
Preferably, R5 and R6 have the same structure
Preferably, R7 and R8 have the same structure
Certain compounds of the invention exist in various regioisomeπc, enantiomeric, tautomeric and diastereomeπc forms It will be understood that the invention comprehends the different regioisomers, enantiomers, tautomers and diastereomers in isolation from each other as well as mixtures
Compounds of the present invention, and their intermediates, are optionally prepared by the representative procedures shown in the following reaction schemes, or by combinations thereof The synthesis of N-(4-aminophenyl)-2-(pyrrolidin-l-yl)acetamide (3)
Figure imgf000008_0002
The synthesis of N-(3-aminophenyl)-2-(pyrrolidin-l-yl)acetamide (6)
Figure imgf000008_0003
The synthesis of N-(4-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (10)
Figure imgf000008_0004
The synthesis of N-(3-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (14)
Figure imgf000009_0001
The synthesis of N-(4-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (18)
Figure imgf000009_0002
The synthesis of N-(3-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (22)
Figure imgf000009_0003
Urea central cores 26, 30 and 34:
The synthesis of 3,3'-ureylene-di-benzoic acid (26)
Figure imgf000009_0004
The synthesis of 4,4'-ureylene-di-benzoic acid urea (30)
Figure imgf000009_0005
30 The synthesis of 2,2'-ureylene-di-benzoic acid (34)
Figure imgf000010_0001
34
Target Compounds 35-52, 57, 61 and 65:
The synthesis of l,3-bis(3-(4-(2-(pyrrolidin-l-yl)acetamido)phenylcarbamoyl)phenyl) urea (35)
Figure imgf000010_0002
35
The synthesis of l,3-bis(3-(4-(3-(pyrrolidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (36)
Figure imgf000011_0001
The synthesis of l,3-bis(3-(4-(4-(pyrrolidin-l-yl)butanamido)phenylcarbamoyl) phenyl)urea (37)
Figure imgf000011_0002
37
The synthesis of l,3-bis(3-(3-(2-(pyrrolidin-l-yl)acetamido)phenylcarbamoyl)phenyl) urea (38)
Figure imgf000011_0003
The synthesis of l,3-bis(3-(3-(3-(pyrrolidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (39)
Figure imgf000012_0001
The synthesis of l,3-bis(3-(3-(4-(pyrrolidin-l-yl)butanamido)phenylcarbamoyl) phenyl)urea (40)
Figure imgf000012_0002
The synthesis of l,3-bis(4-(4-(2-(pyrrolidin-l-yl)acetamido)phenylcarbamoyl) phenyl)urea (41)
Figure imgf000012_0003
The synthesis of l,3-bis(4-(4-(3-(pyrrolidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (42)
9 + 30
Figure imgf000012_0004
The synthesis of l,3-bis(4-(4-(4-(pyrrolidin-l-yl)butanamido)phenylcarbamoyl) phenyl)urea (43)
Figure imgf000013_0001
The synthesis of l,3-bis(4-(3-(2-(pyrrolidin-l-yl)acetamido)phenylcarbamoyl)phenyl) urea (44)
Figure imgf000013_0002
The synthesis of l,3-bis(4-(3-(3-(pyrrolidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (45)
Figure imgf000013_0003
The synthesis of l,3-bis(4-(3-(4-(pyrrolidin-l-yl)butanamido)phenylcarbamoyl) phenyl)urea (46)
Figure imgf000013_0004
The synthesis of N-(4-(2-(pyrrolidin-l-yl)acetamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3 (4H)-yl)benzamide (47)
Figure imgf000013_0005
47 The synthesis of N-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3 (4H)-yl)benzamide (48)
Figure imgf000014_0001
48
The synthesis of N-(4-(4-(pyrrolidin-l-yl)butanamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3(4H)-yl)benzamide (49)
Figure imgf000014_0002
49
The synthesis of N-(3-(2-(pyrrolidin-l-yl)acetamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3 (4H)-yl)benzamide (50)
Figure imgf000014_0003
50
The synthesis of N-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3(4H)-yl)benzamide (51)
Figure imgf000015_0001
51
The synthesis of N-(3-(4-(pyrrolidin-l-yl)butanamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3(4H)-yl)benzamide (52)
Figure imgf000015_0002
52
The Synthesis of l,3-bis(3-(phenylcarbamoyl)-5-(2-(pyrrolidin-l-yl)acetamido) phenyl)urea (57)
Figure imgf000015_0003
53 54 55
Figure imgf000015_0004
57 56 The synthesis of l,3-bis(3-(phenylcarbamoyl)-5-(3-(pyrrolidin-l-yl)propanamido) phenyl)urea (61)
Figure imgf000016_0001
61
The synthesis of l,3-bis(3-(phenylcarbamoyl)-5-(4-(pyrrolidin-l-yl)butanamido) phenyl)urea (65)
Figure imgf000016_0002
65 Target Compounds 70, 73 and 76:
The synthesis of l,3-bis(l-(3-phenylureido)-5-(3-(pyrrolidin-l-yl)propanamido) phenyl)urea (70)
Figure imgf000017_0001
70
The synthesis of 5,5'-ureylene-di-(l-(4-(3-(pyrrolidin-l-yl)propanamido)-phenyl)-3- phenylurea) (73)
Figure imgf000017_0002
71 72 73
The synthesis of 5,5'-ureylene-di-(l-(3-(3-(pyrrolidin-l-yl)propanamido)-phenyl)-3- phenylurea) (76)
Figure imgf000018_0001
Urea central cores 77, 78 and 79:
The synthesis of l,3-bis(3-ethynylphenyl)urea (77)
Figure imgf000018_0002
77
The synthesis of l,3-bis(4-ethynylphenyl)urea (78)
Figure imgf000018_0003
The synthesis of l,3-bis(2-ethynylphenyl)urea (79)
Figure imgf000018_0004
Targets 81-91:
The synthesis of l,3-bis(3-(l-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (80) and l-(3-(l-(4-(3-(pyrrolidin-l-yl)propanamido) phenyl)-lH-l,2,3-triazol-4-yl)phenyl)-3-(3-(l-(4-(acrylamido)phenyl
)-lH-l,2,3-triazol-4-yl)phenyl)urea (81)
Figure imgf000019_0001
The synthesis of l,3-bis(3-(l-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-lH-l,2,3 triazol-4-yl)phenyl) urea (82) and l-(3-(l-(3-(3-(pyrrolidin-l-yl)propanamido) phenyl)-lH-l,2,3-triazol-4-yl)phenyl)-3-(3-(l-(3-(acrylamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl)urea (83)
Figure imgf000019_0002
The synthesis of l,3-bis(3-(l-(4-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (84)
Figure imgf000019_0003
The synthesis of l,3-bis(3-(l-(3-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (85)
Figure imgf000020_0001
85
The synthesis of l,3-bis(4-(l-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (86)
Figure imgf000020_0002
The synthesis of l,3-bis(4-(l-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (87)
Figure imgf000020_0003
87
The synthesis of l,3-bis(4-(l-(4-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (88)
Figure imgf000021_0001
The synthesis of l,3-bis(4-(l-(3-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (89)
Figure imgf000021_0002
The synthesis of l,3-bis-(2-(l-(4-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (90)
Figure imgf000021_0003
The synthesis of l,3-bis-(2-(l-(3-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (91)
22 + 79
Figure imgf000021_0004
Targets 101-106
Figure imgf000022_0001
The synthesis of l,3-bis(3-(4-(3-(dimethylamino)propanamido)phenylcarbamoyl) phenyl)urea (101)
Figure imgf000022_0002
The synthesis of l,3-bis(3-(4-(3-(piperidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (102)
Figure imgf000022_0003
The synthesis of l,3-bis(3-(4-(3-(morpholino)propanamido)phenylcarbamoyl) phenyl)urea (103)
Figure imgf000023_0001
The synthesis of l,3-bis(3-(l-(4-(3-(dimethylamino)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl)urea (104)
Figure imgf000023_0002
The synthesis of l,3-bis(3-(l-(4-(3-(piperidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (105)
Figure imgf000023_0003
The synthesis of l,3-bis(3-(l-(4-(3-(morpholino)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (106)
Figure imgf000024_0001
Hence, the present invention also provides a method of making compounds according to formula (I).
Another aspect of the present invention is 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 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.
In particular, 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.
In particular, 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. For use in medicine, 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) 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)ORa, wherein Ra is Ci_6 alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, or one of the following:
Figure imgf000026_0001
Amidated acid groups include groups of the formula -CONRbRc, wherein Rb is H, Ci-5 alkyl, phenyl, substituted phenyl, benzyl, or substituted benzyl, and Rc is -OH or one of the groups just recited for Rb.
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.
Thus, in the methods of treatment of the present invention, the term "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.
Pharmaceutically acceptable 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.
It is anticipated that the compounds of the invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical administration, and inhalation.
For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension. Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose. Corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatine. The lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Capsules for oral use include hard gelatine capsules in which the active ingredient is mixed with a solid diluent and soft gelatine capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
For intramuscular, intraperitoneal, subcutaneous and intravenous use, 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. Expressed as dosage per unit body weight, a typical dose will be expected to be between 0.01 μg/kg and 50 mg/kg, especially between 10 μg/kg and 10 mg/kg, between 100 μg/kg and 2 mg/kg. Where reference is made to di(hydrocarbyl) or di(heterocarbyl) groups, for example, dialkyl groups [e.g. N(Ci_6 alkyl)2], it is understood that the two alkyl groups may be the same or different.
In the interests of simplicity, terms which are normally used to refer to monovalent groups (such as "alkyl" or "phenyl") are also used herein to refer to divalent bridging groups which are formed from the corresponding monovalent group by the loss of one hydrogen atom. Whether such a term refers to a monovalent group or to a divalent group will be clear from the context.
Where a divalent bridging group is formed from a cyclic moiety, the linking bonds may be on any suitable ring atom, subject to the normal rules of valency.
Where any particular moiety is substituted, for example a pyrrolyl group comprising a substituent on the heteroaryl ring, unless specified otherwise, the term "substituted on the backbone" contemplates all possible isomeric forms. For example, pyrrolyl substituted on the backbone includes all of the following permutations:
Figure imgf000028_0001
where one of the bonds shown is to the rest of the molecule or another moiety, and the other bond is to the defined substituent. This applies correspondingly to groups having a plurality of substituents.
The term "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.
The terms "comprising" and "comprises" 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 word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.
"Optional" or "optionally" means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. "May" means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
Where the compounds according to this invention have at least one chiral center, they 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.
The term "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.
As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, 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. For purposes of this invention, 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.
Reference to the "backbone" preferably means the carbon backbone of the group being referred to. However, the term "backbone" includes the possibility for substitution on a heteroatom, such as nitrogen, which is located in the carbon backbone. As used herein, the term "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, C2-20 aralkyl, the carbon atom number indicates the total number of carbon atoms in the group.
As used herein, the term "heteroatom" includes N, O, S, P, Si and halogen (including F, Cl, Br and I).
As used herein, the term "alkyl" refers to a straight or branched saturated monovalent hydrocarbon radical, having the number of carbon atoms as indicated. By way of non-limiting example, suitable alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, dodecyl and eicosyl. As used herein, the term "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. By way of non-limiting example, 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.
As used herein, the term "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. By way of non-limiting example, 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.
As used herein, the term "cycloalkyl" refers to a cyclic saturated monovalent hydrocarbon radical, having the number of carbon atoms as indicated. By way of non-limiting example, suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclohexyl, dimethylcyclohexyl, trimethylcyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclododecyl, spiroundecyl, bicyclooctyl and adamantyl.
As used herein, the term "(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. By way of non-limiting example, suitable (cycloalkyl)alkyl groups include cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylethyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, methylcyclohexylmethyl, dimethylcyclohexylmethyl, trimethylcyclohexylmethyl, cycloheptylmethyl, cycloheptylethyl, cycloheptylpropyl, cycloheptylbutyl and adamantylmethyl. Alkoxy refers to the group "alkyl-O-", where alkyl is as defined above. By way of non-limiting example, suitable alkoxy groups include methoxy, ethoxy, propoxy, butoxy, pentoxy and hexoxy.
Aryloxy refers to the group "aryl-O-", where aryl is as defined herein. By way of non-limiting example, suitable aryloxy groups include phenoxy, tolyloxy and xylyloxy.
As used herein, the term "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. By way of non-limiting example, suitable alkoxyalkyl groups include methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, methoxypropyl and ethoxypropyl.
As used herein, the term "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. By way of non-limiting example, suitable alkoxyaryl groups include methoxyphenyl, ethoxyphenyl, dimethoxyphenyl and trimethoxyphenyl . As used herein, the term "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. Preferably, the term "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. By way of non-limiting example, 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, chloromethylphenyl, tolylphenyl, xylylphenyl, chloroethylphenyl, trichloromethylphenyl, dihydroindenyl, benzocycloheptyl and trifluoromethylphenyl.
The term "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. Preferably, "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. By way of non-limiting example, 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, chromenyl, chromanyl, isochromanyl, carbolinyl, thiazolyl, isoxazolyl, isoxazolonyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, benzodioxepinyl and pyridazyl. The term "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 atoms; a saturated or partially unsaturated ring having eight 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 bicyclic 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 a saturated or partially unsaturated bicyclic ring having ten 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. Preferably, heterocycles comprising peroxide groups are excluded from the definition of hetercyclyl. By way of non- limiting example, suitable heterocyclyl groups include pyrrolinyl, pyrrolidinyl, dioxolanyl, tetrahydrofuranyl, morpholinyl, imidazolinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrothiopyranyl and piperazinyl.
The term "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. By way of non-limiting example, suitable heterocyclylalkyl groups include methyl, ethyl, propyl, butyl, pentyl and hexyl substituted with one or more of the heterocyclyl groups indicated immediately above.
As used herein, the term "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. Particularly preferred examples of alkaryl include tolyl, xylyl, butylphenyl, mesityl, ethyltolyl, methylindanyl, methylnaphthyl, methyltetrahydronaphthyl, ethylnaphthyl, dimethylnaphthyl, propylnaphthyl, butylnaphthyl, methylfluoryl and methylchrysyl.
As used herein, the term "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. Particularly preferred examples of 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, propylbenzyl, butylbenzyl, dimethylethylbenzyl, phenylpentyl, tetramethylbenzyl, phenylhexyl, dipropylbenzyl, triethylbenzyl, cyclohexylbenzyl, naphthylmethyl, diphenylethyl, triphenylmethyl and hexamethylbenzyl.
The term "heteroaralkyl" 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. By way of non-limiting example, suitable heteroaralkyl groups include methyl, ethyl, propyl, butyl, pentyl and hexyl substituted with one or more of the specific heteroaryl groups indicated above. The term "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. By way of non-limiting example, suitable arylamino groups include phenylamino, biphenylamino, methylphenylamino, methoxyphenylamino, tolylamino and chlorophenylamino .
The term "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. With regard to one or more 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.
Other 'compound' group definitions will be readily understandable by the skilled person based on the previous definitions of their constituent parts, and the usual conventions of nomenclature.
Reference to cyclic systems, e.g., cycloalkyl, aryl, heteroaryl, etc., contemplates monocyclic and polycyclic systems. Such systems comprise fused, non-fused and spiro conformations, such as bicyclooctyl, adamantyl, biphenyl and benzofuran. Experimental
Reagent, Solvent and Apparatus Preparation
All reagents were reagent grade and purchased from Sigma-Aldrich, Alfa Aesar, Avocado Organics and Lancaster Synthesis and were used as supplied without further purification. Palladium catalyst was 10% wt. loading on activated carbon. Ammonia in Methanol (NH3/MeOH) (ca. 7 N) was purchaced from Sigma-Aldrich and diluted in MeOH as required. Solvents were supplied by BDH and Fisher Scientific. Anhydrous solvents where specified were purchased from Sigma- Aldrich. HPLC grade solvents were purchased from Fisher Scientific. 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
1H NMR and 13C 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; CDCl3 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 (J) 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. High resolution accurate mass spectra (HRMS) 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. Chromatographic Techniques Analytical-TLC was performed upon Merck silica gel 60 F254 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 μm; BDH) eluting with the specified solvent. Analytical HPLC (HPLC) were conducted upon a Gilson Chromatograph with a YMC C18 5μ (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
18 minutes;
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 5μ (100 x 20 mm) column. Spectra were manipulated in the Unipoint 5.11 software. Several HPLC solvent systems, gradients and methods of sample isolation were employed as specified: Method G: 0.1% TFA in MeOH and 0.1% aqueous TFA, 25% - 75% organic over
25 minutes, injected from 25% MeOH in 0.1% aqueous TFA (3 ml). Compound isolation by reduction of fraction volumes (5 ml), precipitation with 5% NH3 (aq.) and filtration;
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). Compound isolation by reduction of fraction volumes (5 ml), precipitation with 5% NH3 (aq.) and filtration;
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% NH3 (aq.) and filtration;
Method J: 0.1% FA in Acetonitrile and 0.1% aqueous FA, 5% - 50% organic over
28 minutes, injected from 5% Acetonitrile in 0.1% aqueous FA (3 ml). Compound isolation by reduction of fraction volumes (5 ml), precipitation with 5% NH3 (aq.), centrifugation to concentrate the precipitate and freeze drying upon a Savant A 160 SpeedVac Concentrator co-evaporating with EtOH (1 ml). Chemistry Experimental
Figure imgf000037_0001
The synthesis of 2-chloro-N-(4-nitrophenyl)acetamide (1)
4-nitroaniline (5.66 g, 40.98 mmol) was dissolved in THF (150 ml) and cooled to 0 0C prior to TEA (11.42 ml, 81.95 mmol, 2 equiv.) being added and the drop wise addition of 2-chloroacetyl chloride (4.89 ml, 61.464 mmol, 1.5 equiv.). The mixture was stirred overnight at room temperature under N2. The precipitate was filtered, the THF removed in vacuo and the resulting oil suspended in sat. NaHCO3 (aq.) (200 ml). The solid product was filtered and oven dried to a brown solid (6.42 g, 30.05 mmol, 73%). Rf 0.35 [DCM]. Mp. 177 - 180 0C. 1H NMR (400 MHz, dtf-DMSO) δ 10.94 (IH, s, NH), 8.25 (2H, d, J=9.3 Hz, 2ArH), 7.85 (2H, d, J=9.1 Hz, 2ArH), 4.35 (2H, s, CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 165.6 (C=O), 144.6 (ArC), 142.6 (ArC), 125.0 (2ArCH), 119.1 (2ArCH), 43.5 (CH2) ppm. IR (neat) υmax 3274, 3227, 3162, 3103, 2941, 1683, 1565, 1494, 1332, 849 cm"1. MS m/z calc. C8H7ClN2O3 [M] 214.0, found [M-I] 212.9. The synthesis of N-(4-nitrophenyl)-2-(pyrrolidin-l-yl)acetamide (2)
Figure imgf000037_0002
2-chloro-N-(4-nitrophenyl)acetamide (1) (2.00 g, 9.319 mmol) was dissolved in MeOH (100 ml) and to this was added pyrrolidine (1.56 ml, 18.634 mmol, 2 equiv.) and the mixture heated at reflux for 4 hours and stirred at room temperature overnight. The MeOH was removed in vacuo and the resulting oil suspended in 5% NH3 (aq.) (50 ml), and the observed precipitate was isolated by filtration as a brown solid (2.00 g, 8.014 mmol, 86%). Rf 0.58 [MeOH]. Mp. 78 - 81 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.39 (IH, s, NH), 8.26 (2H, d, J=9.6 Hz, 2ArH), 7.98 (2H, d, J= 9.4 Hz, 2ArH), 3.37 (2H, s, CH2), 2.64 (4H, m, 2CH2), 1.80 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.8 (C=O), 144.9 (ArC), 142.2 (ArC), 124.8 (2ArCH), 119.1 (2ArCH), 59.5 (CH2), 53.6 (2CH2), 23.4 (2CH2) ppm. IR (neat) υmax 3237, 2970, 2868, 2820, 2798, 1738, 1697, 1600, 1503, 1307, 1109, 851 cm"1. MS m/z calc. Ci2H15N3O3 [M] 249.1, found [M-I] 248.0. The synthesis of N-(4-aminophenyl)-2-(pyrrolidin-l-yl)acetamide (3)
Figure imgf000038_0001
3
N-(4-nitrophenyl)-2-(pyrrolidin-l-yl)acetamide (2) (1.50 g, 6.018 mmol) was dissolved in MeOH (30 ml) and stirred under N2. 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. The mixture was filtered through celite, MeOH removed in vacuo and the residue suspended in chloroform (50 ml) and washed with 5% NH3 (aq.) (2 x 20 ml), brine (20 ml) and dried over K2CO3 to yield a brown oil (820.8 mg, 3.743 mmol, 62%). Rf 0.33 [MeOH]. 1H NMR (400 MHz, CDCl3) δ 8.88 (IH, s, NH), 7.54 (2H, d, J=8.6 Hz, 2ArH), 6.85 (2H, d, J=8.8 Hz, 2ArH), 3.78 (2H, br s, NH2), 3.45 (2H, s, CH2), 2.88 (4H, m, 2CH2), 2.04 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 168.6 (C=O), 143.0 (ArC), 129.3 (ArC), 121.4 (2ArCH), 115.4 (2ArCH), 59.7 (CH2), 54.6 (2CH2), 24.1 (2CH2) ppm. IR (neat) υmax 3431, 3308, 3190, 3033, 2969, 2879, 2819, 1738, 1671, 1508, 1422, 1231, 998 cm"1. HRMS m/z calc. Ci2H17NO [M+ 1] 220.1444, found [M+l] 220.1447.
The synthesis of 2-chloro-N-(3-nitrophenyl)acetamide (4)
Figure imgf000038_0002
3-nitroaniline (6.37 g, 46.12 mmol) was dissolved in THF (150 ml) and cooled to 0 0C 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 N2. The precipitate was filtered, THF removed in vacuo and the resulting oil suspended in sat. NaHCO3 (aq.) (200 ml) and the solid product filtered and oven dried as a brown solid (7.68 g, 35.952 mmol, 78%). Rf 0.46 [DCM]. Mp. 113 - 115 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.83 (IH, s, NH), 8.62 (IH, t, 7=2.6» Hz, ArH), 7.97 (IH, ddd, J=8.2, 2.3, 0.9 Hz, ArH), 7.93 (IH, ddd, J=8.2, 2.2, 1.0 Hz, ArH), 7.65 (IH, t, J=8.2 Hz, ArH), 4.33 (2H, s, CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 165.4 (C=O), 148.0 (ArC), 139.6 (ArC), 130.3 (ArCH), 125.3 (ArCH), 118.4 (ArCH), 113.5 (ArCH), 43.4 (CH2) ppm. IR (neat) υmax 3269, 3142, 3070, 3022, 2970, 1738, 1672, 1521, 1345, 886 cm4. MS m/z calc. C8H7ClN2O3 [M] 214.0, found [M-I] 212.9. The synthesis of N-(3-nitrophenyl)-2-(pyrrolidin-l-yl)acetamide (5)
Figure imgf000039_0001
2-chloro-N-(3-nitrophenyl)acetamide (4) (2.07 g, 9.659 mmol) was dissolved in MeOH (100 ml) and to this was added pyrrolidine (1.61 ml, 19.319 mmol, 2 equiv.) and the mixture heated at reflux for 4 hours and stirred at room temperature overnight. The MeOH was removed in vacuo and the resulting oil suspended in 5% NH3 (aq.) (50 ml), extracted into CHCl3 (2 x 50 ml) washing with brine (20 ml) and dried over MgSO4 to yield a brown oil (2.390 g, 9.587 mmol, 99%). Rf 0.58 [MeOH]. 1H NMR (400 MHz, d6- DMSO) δ 10.23 (IH, s, NH), 8.71 (IH, t, J=2.2 Hz, ArH), 8.04 (IH, ddd, J=8.1, 2.2, 0.9 Hz, ArH), 7.91 (IH, ddd, J=8.1, 2.2, 0.9 Hz, ArH), 7.60 (IH, t, J=8.2 Hz, ArH), 3.30 (2H, s, CH2), 2.60 (4H, m, 2CH2), 1.76 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6- DMSO) δ 169.7 (C=O), 147.9 (ArC), 139.9 (ArC), 130.0 (ArCH), 125.5 (ArCH), 117.8 (ArCH), 113.6 (ArCH), 59.5 (CH2), 53.6 (2CH2), 23.5 (2CH2) ppm. IR (neat) υmax 3273, 2969, 1738, 1531, 1352, 1217 cm"1. HRMS m/z calc. Ci2Hi5N3O3 [M+l] 250.1186, found [M+l] 250.1181.
The synthesis of N-(3-aminophenyl)-2-(pyrrolidin-l-yl)acetamide (6)
Figure imgf000039_0002
N-(3-nitrophenyl)-2-(pyrrolidin-l-yl)acetamide (5) (1.5 g, 6.018 mmol) was dissolved in MeOH (30 ml) and stirred under N2. 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. The mixture was filtered through celite, MeOH removed in vacuo and the residue suspended in chloroform (50 ml), washed with a 5% NH3 (aq.) (2 x 20 ml), brine (20 ml) and dried over K2CO3 to yield a yellow oil (1.24 g, 5.655 mmol, 94%). Rf 0.37 [MeOH]. 1H NMR (400 MHz, CDCl3) δ 8.99 (IH, s, NH), 7.22 (IH, t, J=2.2 Hz, ArH), 7.08 (IH, t, J=7.9 Hz, ArH), 7.73 (IH, ddd, J= 7.9, 2.0, 1.0 Hz, ArH), 6.42 (IH, ddd, J = 7.9, 2.3, 1.0 Hz, ArH), 3.71 (2H, br s, NH2), 3.25 (2H, s, CH2), 2.68 (4H, m, 2CH2), 1.84 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ
169.1 (C=O), 147.3 (ArC), 138.8 (ArC), 129.7 (ArCH), 110.9 (ArCH), 109.5 (ArCH),
106.2 (ArCH), 59.8 (CH2), 54.6 (2CH2), 24.1 (2CH2) ppm. IR (neat) υmax 3331, 2965, 2813, 1676, 1608, 1530 cm"1. HRMS m/z calc. Ci2H17NO [M+l] 220.1444, found [M+l]
220.1438.
The synthesis of 3-chloro-N-(4-nitrophenyl)propanamide (7)
Figure imgf000040_0001
7 To 3-chloropropanoyl chloride (45 ml, 0.471 mol) at room temperature was added in portions 4-nitroaniline (15.0 g, 0.109 mol) with heavy stirring, followed by stirring at 50 0C overnight. The mixture was cooled to 0 0C and the precipitate isolated by filtration and washed with ether (50 ml) to yield a pale yellow solid (24.9207 g, 0.109 mol, quant.). Rf 0.26 [DCM]. Mp. 167 - 169 0C. 1H NMR (400 MHz, J6-DMSO) δ 10.69 (IH, s, NH), 8.22 (2H, d, J=9.2 Hz, 2ArH), 7.85 (2H, d, J=9.2 Hz, 2ArH), 3.89 (2H, t, J=6.2 Hz, CH2), 2.91 (2H, t, J=6.2 Hz, CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 168.9 (C=O), 144.9 (ArC), 142.1 (ArC), 124.9 (2ArCH), 118.7 (2ArCH), 40.3 (CH2), 39.3 (CH2) ppm. IR (neat) υmax 3338, 1705, 1485, 1304, 1240, 1097, 850 cm"1. MS m/z calc. C9H9ClN2O3 [M] 228.0, found [M+l] 228.9. The synthesis of N-(4-nitrophenyl)-3-(pyrrolidin-l-yl)propanamide (8)
Figure imgf000040_0002
3-chloro-N-(4-nitrophenyl)propanamide (7) (20.90 g, 0.091 mol) was dissolved in pyrrolidine (20 ml, 0.330 mol) prior to the addition of THF (100 ml) and the mixture stirred at 30 0C overnight. The THF and pyrrolidine was removed in vacuo and the resulting solid suspended in 5% NH3 (aq.) (200 ml) with stirring for 1 hour prior to isolation by filtration, washing with distilled water (10 ml) and oven drying to yield a yellow solid (23.3803 g, 0.888 mol, 98%). Rf 0.30 [MeOH]. Mp. 112 - 116 0C. 1H NMR (400 MHz, dtf-DMSO) δ 10.74 (IH, s, NH), 8.27 (2H, d, J=9.3 Hz, 2ArH), 7.88 (2H, d, J=9.3 Hz, 2ArH), 2.78 (2H, t, J=6.9 Hz, CH2), 2.61 (2H, t, J =6.9 Hz, CH2), 2.52 (4H, m, 2CH2), 1.74 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.2 (C=O), 145.4 (ArC), 142.0 (ArC), 125.0 (2ArCH), 118.6 (2ArCH), 53.4 (2CH2), 51.2 (CH2), 36.2 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 2970, 2807, 1738, 1549, 1321, 1217, 1108, 849 cm"1. MS m/z calc. C13H17N3O3 [M] 263.1, found [M-I] found 262.0. The synthesis of N-(4-aminophenyl)-3-(pyrrolidin-l-yl)propanamide (9)
Figure imgf000041_0001
N-(4-nitrophenyl)-3-(pyrrolidin-l-yl)propanamide (8) (5.00 g, 18.990 mmol) was dissolved in MeOH (100 ml) and stirred under N2. 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. The mixture was filtered through celite, the MeOH removed in vacuo and the residue suspended in chloroform (100 ml), washed with a 5% NH3 (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4 to yield a purple oil which solidified on standing (3.723 g, 15.957 mmol, 84%). Rf 0.29 [MeOH]. Mp. 92 - 95 0C. 1H NMR (400 MHz, CDCl3) δ 10.71 (IH, s, NH), 7.28 (2H, d, J=8.8 Hz, 2ArH), 6.64 (2H, d, J=8.8 Hz, 2ArH), 3.55 (2H, br s, NH2), 2.86 (2H, t, J=5.8 Hz, CH2), 2.69 (4H, m, 2CH2), 2.53 (2H, t, J=5.9 Hz, CH2), 1.88 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.4 (C=O), 142.6 (ArC), 130.5 (ArC), 121.3 (2ArCH), 115.5 (2ArCH), 53.2 (2CH2), 51.5 (CH2), 34.6 (CH2), 23.7 (2CH2) ppm. IR (neat) υmax 3436, 3332, 3184, 3015, 2969, 2807, 1738, 1651, 1507, 1360, 1217 cm"1. MS m/z calc. Ci3H19N3O3 [M] 233.2, found [M+l] found 233.9. The synthesis of N-(4-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (10)
Figure imgf000041_0002
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 0C prior to the addition of cone. HCl (12 M, 3.34 ml, 40.08 mmol, 5.5 equiv.) followed by 1BuONO (2.16 ml, 18.218 mmol, 2.5 equiv.) and the mixture stirred at 0 0C for 1 hour. After this period NaN3 (1.42 g, 21.861 mmol, 3 equiv.) was added followed by CAUTIOUS addition of distilled water (10 ml) and the mixture allowed to warm to room temperature and stirred overnight. The reaction was quenched with sat. NaHCO3 (aq.) (8.4 ml), the THF removed in vacuo and to the resulting aqueous was added sat. NaHCO3 (aq.) (50 ml) and the product extracted into EtOAc (3 x 100 ml) prior to drying over MgSO4 and evaporation to yield a pale brown solid (1.4888 g, 5.741 mmol, 79%). Rf 0.33 [10% MeOH in DCM]. Mp. 67 - 69 0C. 1H NMR (400 MHz, CDCl3) δ 11.19 (IH, s, NH), 7.50 (2H, d, J=8.9 Hz, 2ArH), 6.96 (2H, d, J=8.9 Hz, 2ArH), 2.88 (2H, t, J= 6.0 Hz, CH2), 2.71 (4H, m, 2CH2), 2.56 (2H, t, J=6.0 Hz, CH2), 1.91 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.5 (C=O), 135.9 (ArC), 134.8 (ArC), 120.9 (2ArCH), 119.3 (2ArCH), 53.1 (2CH2), 51.2 (CH2), 34.4 (CH2), 23.6 (2CH2) ppm. IR (neat) υmax 3279, 2948, 2822, 2106, 1656, 1607, 1498, 1277 cm"1. HRMS m/z calc. Ci3H17N5O [M+l] 260.1511, found [M+l] 260.1516. The synthesis 3-chloro-N-(3-nitrophenyl)propanamide (11)
Figure imgf000042_0001
11
To 3-chloropropanoyl chloride (45 ml, 0.471 mol) at room temperature was added in portions 3-nitroaniline (15.0 g, 0.109 mol) with heavy stirring, followed by stirring at 50 0C overnight. The mixture was cooled to 0 0C and ether (50 ml) was added inducing precipitation which was isolated by filtration and washed with ether (10 ml) to yield a white crystaline solid (21.7173 g, 0.095 mol, 87%). Rf 0.21 [DCM]. Mp. 100 - 102 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.57 (IH, s, NH), 8.64 (IH, t, J=2.0 Hz, NH), 7.91 (2H, d, J=8.2 Hz, ArH), 7.61 (IH, t, J=8.2 Hz, ArH), 3.90 (2H, t, J=6.2 Hz, CH2), 2.88 (2H, t, J=6.2 Hz, CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 168.7 (C=O), 147.9 (ArC), 139.9 (ArC), 130.1 (ArCH), 124.9 (ArCH), 117.7 (ArCH), 113.0 (ArCH), 40.4 (CH2), 39.2 (CH2) ppm. IR (neat) υmax 3105, 1691, 1524, 1326, 1210 cm"1. MS mJz calc. C9H9ClN2O3 [M] 228.03, found [M+l] 228.98. The synthesis of N-(3-nitrophenyl)-3-(pyrrolidin-l-yl)propanamide (12)
Figure imgf000042_0002
3-chloro-N-(3-nitrophenyl)propanamide (11) (20.60 g, 0.0901 mol) was dissolved in pyrrolidine (20 ml, 0.330 mol) prior to the addition of THF (50 ml) and the mixture stirred at 30 0C overnight. The THF and pyrrolidine was removed in vacuo and the resulting oil suspended in 5% NH3 (aq.) (200 ml) and extracted into chloroform (300 ml), washed with brine (100 ml) and dried over MgSO4 prior to evaporation to yield a brown oil (23.7224 g, 0.0901 mol, quant.). Rf 0.30 [MeOH]. 1H NMR (400 MHz, d6-OMSO) δ 10.59 (IH, s, NH), 8.70 (IH, t, J=2.2 Hz, ArH), 7.95 (2H, dt, J=8.2, 2.4 Hz, 2ArH), 7.65 (IH, t, J=8.2 Hz, ArH), 2.78 (2H, t, J=7.1 Hz, CH2), 2.58 (2H, t, J=6.9 Hz, CH2), 2.52 (4H, m, 2CH2), 1.74 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.9 (C=O), 147.9 (ArC), 140.3 (ArC), 130.1 (ArCH), 124.9 (ArCH), 117.5 (ArCH), 113.0 (ArCH), 53.4 (2CH2), 51.3 (CH2), 36.2 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 2969, 2806, 1738, 1527, 1351, 1217 cm"1. MS m/z calc. Ci3H17N3O3 [M] 263.1, found [M-I] found 262.0. The synthesis of N-(3-aminophenyl)-3-(pyrrolidin-l-yl)propanamide (13)
Figure imgf000043_0001
N-(3-nitrophenyl)-3-(pyrrolidin-l-yl)propanamide (12) (5.06 g, 19.218 mmol) was dissolved in MeOH (100 ml) and stirred under N2. 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. The mixture was filtered through celite, the MeOH removed in vacuo and the residue suspended in chloroform (100 ml), washed with a 5% NH3 (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4 to yield a yellow oil which solidified on standing (4.4838 g, 19.218 mmol, quant.). Rf 0.18 [MeOH]. Mp. 100 - 102 0C. 1H NMR (400 MHz, CDCl3) δ 10.99 (IH, s, NH), 7.19 (IH, t, /=2.6» Hz, ArH), 7.05 (IH, t, J= 7.9 Hz, ArH), 6.61 (IH, dd, J=7.9, 1.9 Hz, ArH), 6.39 (IH, dd, 7=8.0, 2.3 Hz, ArH), 3.68 (2H, s, NH2), 2.85 (2H, t, 7=5.9 Hz, CH2), 2.68 (4H, m, 2CH2), 2.53 (2H, t, 7=5.9 Hz, CH2), 1.89 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.8 (C=O), 147.2 (ArC), 139.9 (ArC), 129.6 (ArCH), 110.4 (ArCH), 109.7 (ArCH), 106.5 (ArCH), 53.1 (2CH2), 51.4 (CH2), 34.8 (CH2), 23.7 (2CH2) ppm. IR (neat) υmax 3322, 2964, 2807, 1664, 1612, 1559, 1496, 1458 cm"1. MS m/z calc. Ci3H19N3O3 [M] 233.2, found [M+l] found 233.9. The synthesis of N-(3-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (14)
Figure imgf000043_0002
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 0C prior to the addition of cone. HCl (12 M, 3.34 ml, 40.08 mmol, 5.5 equiv.) followed by 1BuONO (2.16 ml, 18.218 mmol, 2.5 equiv.) and the mixture stirred at 0 0C for 1 hour. After this period NaN3 (1.42 g, 21.861 mmol, 3 equiv.) was added followed by CAUTIOUS addition of distilled water (10 ml) and the mixture allowed to warm to room temperature and stirred overnight. The reaction was quenched with sat. NaHCO3 (aq.) (8.4 ml), the THF removed in vacuo and to the resulting aqueous was added sat. NaHCO3 (aq.) (50 ml) and the product extracted into EtOAc (3 x 100 ml) prior to drying over MgSO4 and evaporation to yield an orange oil which was used without further purification (1.7478 g, 6.740 mmol, 93%). Rf 0.28 [10% MeOH in DCM]. 1H NMR (400 MHz, CDCl3) δ 11.35 (IH, s, NH), 7.35 (IH, t, J=2.1 Hz, ArH), 7.25 (IH, t, J=8.3 Hz, ArH), 7.14 (IH, ddd, J=8.1, 1.9, 0.9 Hz, ArH), 6.73 (IH, ddd, J=8.0, 2.2, 0.9 Hz, ArH), 2.86 (2H, t, J=5.9 Hz, CH2), 2.69 (4H, m, 2CH2), 2.53 (2H, t, J=5.9 Hz, CH2), 1.90 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.9 (C=O), 140.6 (ArC), 140.3 (ArC), 129.9 (ArCH), 115.9 (ArCH), 113.9 (ArCH), 110.2 (ArCH), 53.0 (2CH2), 51.2 (CH2), 34.5 (CH2), 23.6 (2CH2) ppm. IR (neat) υmax 3296, 2951, 2802, 2106), 1667, 1597, 1545, 1290 cm"1. HRMS m/z calc. Ci3H17N5O [M+l] 260.1511, found [M+l] 260.1512.
The synthesis of 4-chloro-N-(4-nitrophenyl)butanamide (15)
Figure imgf000044_0001
15
To 4-chlorobutanoyl chloride (45 ml, 0.401 mol) at room temperature was added in portions 4-nitroaniline (15.0 g, 0.109 mol) with heavy stirring, prior to the addition of a further portion of 4-chlorobutanoyl chloride (15 ml, 0.134 mol) followed by stirring at 50 0C overnight. The mixture was cooled to 0 0C and the precipitate isolated by filtration washing with ether (50 ml) to yield a pale yellow crystalline solid (20.7253 g, 0.085 mol, 78%). Rf 0.68 [DCM]. Mp. 78 - 80 0C. 1H NMR (400 MHz, CDCl3) δ 8.22 (2H, d, /= 9.2 Hz, 2ArH), 7.77 (IH, s, NH), 7.71 (2H, d, /= 9.2 Hz, 2ArH), 3.68 (2H, t, J=6.0 Hz, CH2), 2.64 (2H, t, 7=7.6» Hz, CH2), 2.23 (2H, m, CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.5 (C=O), 143.6 (ArC), 143.5 (ArC), 125.1 (2ArCH), 119.1 (2ArCH), 44.2 (CH2), 34.2 (CH2), 27.5 (CH2) ppm. IR (neat) υmax 3276, 3216, 3088, 3016, 2970, 1738, 1668, 1506, 1328, 853 cm"1. HRMS m/z calc. Ci0H11ClN2O3 [M+l] 243.0531, found [M+l] 243.0536. The synthesis of N-(4-nitrophenyl)-4-(pyrrolidin-l-yl)butanamide (16)
Figure imgf000045_0001
16
4-chloro-N-(4-nitrophenyl)butanamide (15) (20.40 g, 0.084 mol) was dissolved in pyrrolidine (20 ml, 0.330 mol) and the mixture stirred at 30 0C overnight. The pyrrolidine was removed in vacuo and the resulting oil suspended in 5% NH3 (aq.) (200 ml) and extracted into chloroform (500 ml), washed with brine (100 ml) and dried over MgSO4 prior to evaporation to yield a brown oil (21.6884 g). The product was purified by flash chromatography [dry load; 1 - EtOAc; 2 - 10% MeOH in EtOAc; 3 - 5% DEA and 10% MeOH in EtOAc] to yield a brown solid (13.9505 g, 0.050 mol, 60%). Rf 0.51 [5% DEA and 10% MeOH in EtOAc]. Mp. 159 - 161 0C. 1H NMR (400 MHz, CDCl3) δ 10.93 (IH, s, NH), 8.91 (2H, d, J=9.3 Hz, 2ArH), 7.66 (2H, d, J=9.3 Hz, 2ArH), 2.67 (2H, t, J=6.7 Hz, CH2), 2.66 (4H, m, 2CH2), 2.59 (2H, t, J=6.5 Hz, CH2), 1.89 (6H, m, 3CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 172.3 (C=O), 145.0 (ArC), 143.0 (ArC), 125.0 (2ArCH), 118.7 (2ArCH), 56.2 (CH2), 54.0 (2CH2), 37.8 (CH2), 23.7 (2CH2), 23.5 (CH2) ppm. IR (neat) υmax 3292, 2970, 2952, 2871, 1738, 1591, 1540, 1439, 1298, 1104, 833, 750 cm"1. HRMS m/z calc. C14H19N3O3 [M+ 1] 278.1505, found [M+l] 278.1500. The synthesis of N-(4-aminophenyl)-4-(pyrrolidin-l-yl)butanamide (17)
Figure imgf000045_0002
17
N-(4-nitrophenyl)-4-(pyrrolidin- 1 -yl)butanamide (16) (5.0 g, 20.215 mmol) was dissolved in MeOH (100 ml) and stirred under N2. 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. The mixture was filtered through celite, the MeOH removed in vacuo and the residue suspended in chloroform (100 ml), washed with a 5% NH3 (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4 to yield a brown oil (1.9921 g, 8.054 mmol, 40%). Rf 0.19 [MeOH]. 1H NMR (400 MHz, CDCl3) δ 9.14 (IH, s, NH), 7.25 (2H, d, J=8.7 Hz, 2ArH), 6.61 (2H, d, J=8.7 Hz, 2ArH), 3.57 (2H, br s, NH2), 2.56 (2H, t, J=6.5 Hz, CH2), 2.55 (4H, m, 2CH2), 2.43 (2H, t, J=6.8 Hz, CH2), 1.86 (2H, m, CH2), 1.79 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 171.1 (C=O), 142.8 (ArC), 130.1 (ArC), 121.5 (2ArCH), 115.3 (2ArCH), 55.7 (CH2), 53.9 (2CH2), 36.5 (CH2), 24.1 (CH2), 23.5 (2CH2) ppm. IR (neat) υmax 3235, 2959, 2803, 1654, 1605, 1544, 1514, 1429, 1254, 1138, 830, 752 cm"1. HRMS m/z calc. C14H21N3O [M+l] 248.1763, found [M+l] 248.1752. The synthesis of N-(4-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (18)
Figure imgf000046_0001
18
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 0C prior to the addition of cone. HCl (12 M, 3.50 ml, 42.03 mmol, 5.5 equiv.) followed by 1BuONO (2.27 ml, 19.103 mmol, 2.5 equiv.) and the mixture stirred at 0 0C for 1 hour. After this period NaN3 (1.49 g, 22.923 mmol, 3 equiv.) was added followed by CAUTIOUS addition of distilled water (10 ml) and the mixture allowed to warm to room temperature and stirred overnight. The reaction was quenched with sat. NaHCO3 (aq.) (10 ml), the THF removed in vacuo and to the resulting aqueous was added sat. NaHCO3 (aq.) (50 ml) and the product extracted into EtOAc (3 x 100 ml) prior to drying over MgSO4 and evaporation to yield a brown oil which solidified overnight (1.4405 g, 5.270 mmol, 69%). Rf 0.20 [10% MeOH in DCM]. Mp 53 - 55 0C. 1H NMR (400 MHz, CDCl3) δ 9.90 (IH, s, NH), 7.52 (2H, d, J=8.8 Hz, ArH), 6.95 (2H, d, J=8.8 Hz, ArH), 2.65 (6H, m, 3CH2), 2.52 (2H, t, J=6.6 Hz, CH2), 1.91 (2H, m, CH2), 1.86 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 171.4 (C=O), 135.9 (ArC), 135.0 (ArC), 121.0 (2ArCH), 119.3 (2ArCH), 55.8 (CH2), 53.9 (2CH2), 36.8 (CH2), 23.7 (CH2), 23.5 (2CH2) ppm. IR (neat) υmax 3277, 3044, 2945, 2775, 2110, 1646, 1519, 1284, 1127 cm"1. HRMS m/z calc. C14H19N5O [M+l] 274.1668, found [M+l] 274.1671. The synthesis of 4-chloro-N-(3-nitrophenyl)butanamide (19)
Figure imgf000046_0002
19
To 4-chlorobutanoyl chloride (45 ml, 0.401 mol) at room temperature was added in portions 3-nitroaniline (15.0 g, 0.109 mol) with heavy stirring prior to stirring at 50 0C overnight. The mixture was cooled to 0 0C and the precipitate isolated by filtration washing with ether (50 ml) to yield a white solid (16.4459 g, 0.068 mol, 62%). Rf 0.83 [DCM]. Mp. 65 - 67 0C. 1H NMR (400 MHz, CDCl3) δ 8.40 (IH, t, J=2.1 Hz, ArH), 7.97 (IH, ddd, J=8.2, 2.1, 0.8 Hz, ArH), 7.92 (IH, dd, J=8.1, 1.4 Hz, ArH), 7.53 (IH, s, NH), 7.50 (IH, t, J=8.2 Hz, ArH), 3.68 (2H, t, J =6.1 Hz, CH2), 2.63 (2H, t, 7=7.6» Hz, CH2), 2.23 (2H, m, CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.3 (C=O), 148.6 (ArC), 138.8 (ArC), 129.9 (ArCH), 125.4 (ArCH), 119.0 (ArCH), 114.6 (ArCH), 44.3 (CH2), 34.1 (CH2), 27.7 (CH2) ppm. IR (neat) υmax 3313, 3123, 3088, 2970, 2866, 1738, 1662, 1532, 1343, 737 cm"1. HRMS m/z calc. Ci0H11ClN2O3 [M+l] 243.0531, found [M+l] 243.0541. The synthesis of N-(3-nitrophenyl)-4-(pyrrolidin-l-yl)butanamide (20)
Figure imgf000047_0001
20
4-chloro-N-(3-nitrophenyl)butanamide (19) (16.25 g, 0.067 mol) was dissolved in pyrrolidine (20 ml, 0.330 mol) and the mixture stirred at 30 0C overnight. The pyrrolidine was removed in vacuo and the resulting oil suspended in 5% NH3 (aq.) (200 ml) and extracted into chloroform (500 ml), washed with brine (100 ml) and dried over MgSO4 prior to evaporation to yield a brown oil (17.4581 g). The product was purified by flash chromatography [dry load; 1 - EtOAc; 2 - 10% MeOH in EtOAc; 3 - 5% DEA and 10% MeOH in EtOAc] to yield a brown oil (16.3007 g, 0.059 mmol, 88%). Rf 0.27 [5% DEA and 10% MeOH in EtOAc]. 1H NMR (400 MHz, CDCl3) δ 11.14 (IH, s, NH), 8.19 (IH, t, J=2.2 Hz, ArH), 8.07 (IH, ddd, J=8.1, 2.1, 0.9 Hz, ArH), 7.89 (IH, ddd, J=8.3, 2.2, 0.9 Hz, ArH), 7.45 (IH, t, J=8.2 Hz, ArH), 2.67 (6H, m, 3CH2), 2.59 (2H, m, CH2), 1.91 (6H, m, 3CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 172.2 (C=O), 148.5 (ArC), 140.3 (ArC), 129.8 (ArCH), 125.4 (ArCH), 118.0 (ArCH), 114.0 (ArCH), 56.7 (CH2), 54.1 (2CH2), 38.1 (CH2), 23.7 (2CH2), 23.5 (CH2) ppm. IR (neat) υmax 3435, 2969, 2879, 2805, 1738, 1678, 1615, 1558, 1522, 1349, 1217 cm4. HRMS m/z calc. C14H19N3O3 [M+l] 278.1505, found [M+l] 278.1507. The synthesis of N-(3-aminophenyl)-4-(pyrrolidin-l-yl)butanamide (21)
Figure imgf000047_0002
21 N-(3-nitrophenyl)-4-(pyrrolidin-l-yl)butanamide (20) (5.0 g, 20.215 mmol) was dissolved in MeOH (100 ml) and stirred under N2. 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. The mixture was filtered through celite, the MeOH removed in vacuo and the residue suspended in chloroform (100 ml), washed with a 5% NH3 (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4 to yield a brown oil (2.0943 g, 8.467 mmol, 42%). Rf 0.39 [5% TEA and 5% MeOH in DCM]. 1H NMR (400 MHz, CDCl3) δ 9.30 (IH, s, NH), 7.19 (IH, s, ArH), 7.03 (IH, t, J=8.0 Hz, ArH), 6.63 (IH, dd, 7=8.0, 1.1 Hz, ArH), 6.38 (IH, dd, 7=7.9, 1.7 Hz, ArH), 3.63 (2H, br s, NH2), 2.57 (6H, m, 3CH2), 2.45 (2H, t, 7=6.7 Hz, CH2), 1.87 (2H, m, CH2), 1.82 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 171.5 (C=O), 147.2 (ArC), 139.6 (ArC), 129.5 (ArCH), 110.5 (ArCH), 109.4 (ArCH), 106.4 (ArCH), 55.5 (CH2), 53.9 (2CH2), 36.7 (CH2), 24.0 (CH2), 23.5 (2CH2) ppm. IR (neat) υmax 3329, 2966, 2807, 1670, 1616, 1558, 1497, 1458, 1351 , 1217, 1164, 757 cm"1. HRMS m/z calc. Ci4H21N3O [M+l] 248.1763, found [M+l] 248.1755.
The synthesis of N-(3-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (22)
Figure imgf000048_0001
22
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 0C prior to the addition of cone. HCl (12 M, 3.80 ml, 45.64 mmol, 5.5 equiv.) followed by 1BuONO (2.46 ml, 20.745 mmol, 2.5 equiv.) and the mixture stirred at 0 0C for 1 hour. After this period NaN3 (1.618 g, 24.894 mmol, 3 equiv.) was added followed by CAUTIOUS addition of distilled water (12 ml) and the mixture allowed to warm to room temperature and stirred overnight. The reaction was quenched with sat. NaHCO3 (aq.) (12 ml), the THF removed in vacuo and to the resulting aqueous was added sat. NaHCO3 (aq.) (50 ml) and the product extracted into EtOAc (3 x 100 ml) prior to drying over MgSO4 and evaporation to yield a yellow oil (1.8039 g, 6.600 mmol, 80%). Rf 0.15 [10% MeOH in DCM]. 1H NMR (400 MHz, CDCl3) δ 10.08 (IH, s, NH), 7.37 (IH, s, ArH), 7.31 (IH, t, 7=8.0 Hz, ArH), 7.28 (IH, d, J=8.1 Hz, ArH), 6.73 (IH, d, 7=7.3 Hz, ArH), 2.64 (6H, m, 3CH2), 2.52 (2H, t, J =6.6 Hz, CH2), 1.90 (2H, m, CH2), 1.86 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 171.8 (C=O), 140.7 (ArC), 140.4 (ArC), 130.0 (ArCH), 115.9 (ArCH), 114.2 (ArCH), 110.2 (ArCH), 56.0 (CH2), 54.0 (2CH2), 37.1 (CH2), 23.8 (CH2), 23.6 (2CH2) ppm. IR (neat) υmax 3280, 2949, 2791, 2104, 1667, 1597, 1541, 1433, 1291, 1236 cm4. HRMS m/z calc. C14H19N5O [M+l] 274.1668, found [M+l] 274.1661. The synthesis of tert-butyl 3-nitrobenzoate (23)
Figure imgf000049_0001
23 2-nitrobenzoic acid (5.0 g, 29.919 mmol) was dissolved in anhydrous DCM (200 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 0C 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 3 hours. The precipitate was filtered from the reaction and the DCM washed with 1 M HCl (aq.) (2 x 50 ml) and brine (50 ml) prior to drying over MgSO4. The mixture was evaporated to a yellow oil which was purified by flash chromatography [10% - 20% EtOAc in Hexane] to yield a colourless oil (5.2496 g, 23.517 mmol, 79%). Rf 0.64 [14% EtOAc in Hexane]. 1H NMR (400 MHz, CDCl3) δ 8.72 (IH, t, J=1.8 Hz, ArH), 8.31 (IH, ddd, J=8.2, 2.2, 1.0 Hz, ArH), 8.24 (IH, dt, J=7.8, 1.1 Hz, ArH), 7.55 (IH, t, J=8.0 Hz, ArH), 1.56 (9H, s, C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3) δ 163.5 (C=O), 148.3 (ArC), 135.1 (ArCH), 133.8 (ArC), 129.4 (ArCH), 126.9 (ArCH), 124.4 (ArCH), 82.6 (C(CH3)3), 28.1 (C(CH3)3) ppm. IR (neat) υmax 3094, 3003, 2981, 2933, 1716, 1525, 1367, 1347, 1300, 1131 cm"1. MS not detected in ES+ or ES-. The Synthesis of tert-butyl 3-aminobenzoate (24)
Figure imgf000049_0002
24
Tert-butyl 3-nitrobenzoate (23) (5.20 g, 23.294 mmol) was dissolved in MeOH
(100 ml) and added with CAUTION was Pd/C (520 mg, 0.1 equiv. w/w) and the flask evacuated prior to purging with N2, re-evacuation and purging with H2. The mixture was stirred under H2 for 3.5 hours until complete. The mixture was filtered through celite and evaporated to a white solid (4.1848 g, 21.656 mmol, 93%). Rf 0.33 [20% EtOAc in Hexane]. Mp. 80 - 82 0C. 1H NMR (400 MHz, CDCl3) δ 7.38 (IH, dt, J=7.7, 1.2 Hz, ArH), 7.30 (IH, t, 7=2.6» Hz, ArH), 7.19 (IH, t, J=7.8 Hz, ArH), 6.82 (IH, ddd, J=7.9, 2.4, 1.0 Hz, ArH), 3.75 (2H, br s, NH2), 1.58 (9H, s, C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3) δ 165.9 (C=O), 146.3 (ArC), 133.1 (ArC), 129.0 (ArCH), 119.6 (ArCH), 118.9 (ArCH), 115.7 (ArCH), 80.8 (C(CH3)3), 28.2 (C(CH3)3) ppm. IR (neat) υmax 3401, 3323, 3223, 2976, 2931, 1690, 1605, 1366, 1292, 1242, 1158, 1105 cm"1. HRMS m/z calc. CnH15NO2 [M+l] 194.1176, found [M+l] 194.1172. The synthesis of 3,3'-ureylene-di-(tert-butyl benzoate) (25)
Figure imgf000050_0001
Tert-butyl 3-aminobenzoate (24) (4.0085 g, 20.744 mmol) was dissolved in anhydrous THF (50 ml, c = 0.42 M) and CDI (2.018 g, 12.446 mmol, 0.6 equiv.) was added prior to the mixture being heated at reflux under N2 overnight. The THF was evaporated and the crude residue dissolved in EtOAc (500 ml) and washed with 1 M HCl (aq.) (2 x 100 ml), brine (50 ml) and dried over MgSO4. The solvent was evaporated to yield a white fluffy solid (3.9727 g, 9.585 mmol, 92%). Rf 0.24 [17% Hexane in EtOAc]. Mp. 296 0C dec. 1H NMR (400 MHz, dtf-DMSO) δ 8.97 (2H, s, 2NH), 8.09 (2H, t, J=1.8 Hz, 2ArH), 7.75 (2H, ddd, 7=8.6», 2.1, 1.0 Hz, 2ArH), 7.58 (2H, dt, 7=7.8, 1.2 Hz, 2ArH), 7.46 (2H, t, 7=7.9 Hz, 2ArH), 1.61 (18H, s, 2(C(CH3)3)) ppm. 13C NMR (100 MHz, d6- DMSO) δ 164.8 (2C=O; ester), 152.5 (C=O; urea), 139.8 (2ArC), 131.9 (2ArC), 129.0 (2ArCH), 122.5 (2ArCH), 122.5 (2ArCH), 118.7 (2ArCH), 80.7 (2(C(CH3)3)), 27.8 (2(C(CH3)3)) ppm. IR (neat) υmax 3308, 2977, 1713, 1645, 1590, 1557, 1431, 1366, 1301, 1229, 1155 cm"1. HRMS m/z calc. C23H28N2O5 [M+l] calc. 413.2071, found [M+l] 413.2075. The synthesis of 3,3'-ureylene-di-benzoic acid (26)
Figure imgf000050_0002
26 3,3'-ureylene-di-(tert-butyl benzoate) (25) (3.0115 g, 7.266 mmol) was suspended in TFA (30 ml) and stirred vigorously at room temperature for 1 hour. The precipitate was isolated by filtration and washed with DCM (10 ml) and oven dried as a white fluffy solid (2.182 g, 7.267 mmol, quant.). Mp. 321 - 323 0C. 1H NMR (400 MHz, d6-OMSO) δ 12.84 (2H, br s, 2COOH), 8.97 (2H, s, 2NH), 8.15 (2H, t, J=IJ Hz, 2ArH), 7.66 (2H, ddd, J=8.1, 2.0, 0.9 Hz, 2ArH), 7.57 (2H, dt, J=7.8, 1.0 Hz, 2ArH), 7.41 (2H, t, J=7.9 Hz, 2ArH) ppm. 13C NMR (100 MHz, d6-OMSO) δ 167.2 (2COOH), 152.5 (C=O; urea), 139.8 (2ArC), 131.3 (2ArC), 129.0 (2ArCH), 122.8 (2ArCH), 122.5, (2ArCH) 119.0 (2ArCH) ppm. IR (neat) υmax 3304, 2970, 2823, 2552, 1684, 1647, 1591, 1561, 1450, 1307, 1217, 755 cm 1. HRMS m/z calc. C15H12N2O5 [M+l] calc. 301.0819, found [M+l] 301.0807 The synthesis of tert-butyl 4-nitrobenzoate (27)
Figure imgf000051_0001
27
4-nitrobenzoic acid (5.0 g, 29.919 mmol) was dissolved in anhydrous DCM (150 ml) and anhydrous DMF (17 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 0C 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 3 hours until complete. The precipitate was filtered from the reaction and the DCM washed with 1 M HCl (aq.) (2 x 50 ml) and brine (50 ml) prior to drying over MgSO4. The mixture was evaporated to a pale yellow oil with considerable precipitate, which was purified by flash chromatography [10%
EtOAc in Hexane], and concentrated in vacuo to a white crystalline solid (4.82 g, 21.592 mmol, 72%). Rf 0.83 [20% EtOAc in Hexane]. Mp. 114 - 116 0C. 1H NMR (400 MHz,
CDCl3) δ 8.26 (2H, dt, 7=9.6», 2.1 Hz, 2ArH), 8.14 (2H, dt, 7=9.6», 2.1 Hz, 2ArH), 1.62 (9H, s, C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3) δ 163.7 (C=O), 150.3 (ArC), 137.4,
(ArC) 130.5 (2ArCH), 123.4 (2ArCH), 82.6 (C(CH3)3), 28.1 (C(CH3)3) ppm. IR (neat) υmax 3115, 2979, 1711, 1519, 1364, 1344, 1295, 1251, 1101 cm 1. MS not detected in ES+ or ES-. The Synthesis of tert-butyl 4-aminobenzoate (28)
Figure imgf000052_0001
Tert-butyl 4-nitrobenzoate (27) (4.75 g, 21.279 mmol) was dissolved in MeOH (200 ml) and added with CAUTION was Pd/C (475 mg, 0.1 equiv. w/w) and the flask evacuated prior to purging with N2, re-evacuation and purging with H2. The mixture was stirred under H2 for 5 hours until complete. The mixture was filtered through celite and evaporated to a white solid (4.11 g, 21.269 mmol, quant.). Rf 0.29 [20% EtOAc in Hexane]. Mp. I l l - 113 0C. 1H NMR (400 MHz, CDCl3) δ 7.79 (2H, dt, J=8.6, 2.3 Hz, 2ArH), 6.61 (2H, dt, J=8.6, 2.3 Hz, 2ArH), 4.00 (2H, br s, NH2), 1.56 (9H, s, C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3) δ 165.9 (C=O), 150.4 (ArC), 131.3 (ArCH), 121.7 (ArC), 113.7 (ArCH), 80.0 (C(CH3)3), 28.3 (C(CH3)3) ppm. IR (neat) υmax 3415, 3345, 3235, 2972, 1682, 1598, 1367, 1287, 1154, 1115 cm 1. HRMS mJz calc. C11H15NO2 [M+l] 194.1176, found [M+l] 194.1179. The synthesis of 4,4'-ureylene-di-(tert-butyl benzoate) (29)
Figure imgf000052_0002
29
Tert-butyl 4-aminobenzoate (28) (3.7105 g, 19.202 mmol) was dissolved in anhydrous THF (40 ml, c = 0.48 M) under N2 and CDI (1.868 g, 11.521 mmol, 0.6 equiv.) was added prior to the mixture being heated at reflux overnight, at which point TLC showed it to be incomplete and a further portion of CDI (1.868 g, 11.521 mmol, 0.6 equiv.) was added and reflux continued over a second night. The THF was removed in vacuo and the residue dissolved in EtOAc (200 ml) and washed with 1 M HCl (aq.) (2 x 100 ml), brine (50 ml), dried over MgSO4 and evaporated to a white solid (3.9372 g, 9.552 mmol, 99%) of crude 4,4'-ureylene-di-(tert-butyl benzoate) (29) which was unable to be purified further and hence used crude. Rf 0.13 [20% EtOAc in Hexane]. 1H NMR (400 MHz, CDCl3) δ 9.23 (2H, s, 2NH), 7.89 (4H, d, J=8.8 Hz, 4ArH), 7.63 (4H, d, J=8.8 Hz, 4ArH), 1.59 (18H, s, 2(C(CHj)3)) ppm. IR (neat) υmax 3377, 1668, 1587, 1366 cm"1. HRMS m/z calc. C23H28N2O5 [M+l] 413.2071, found [M+l] 413.2092. The synthesis of 4,4'-ureylene-di-benzoic acid (30)
Figure imgf000053_0001
30
4,4'-ureylene-di-(tert-butyl benzoate) (29) (3.1154 g, 7.558 mmol) was suspended in TFA (30 ml) and stirred vigorously at room temperature for 1 hour at which point the precipitate was isolated by filtration, washed with DCM (50 ml) and oven dried to a fine white powder (2.1534 g, 7.1715 mmol, 95%). Mp. > 350 0C. 1H NMR (400 MHz, DMSO) δ 12.62 (2H, br s, 2COOH), 9.21 (2H, s, 2NH), 7.88 (4H, d, J=8.7 Hz, 4ArH), 7.58 (4H, d, J=8.7 Hz, 4ArH) ppm. 13C NMR (100 MHz, DMSO) δ 166.9 (2COOH), 151.9 (C=O; urea), 143.6 (2ArC), 130.5 (2ArCH), 123.9 (2ArC), 117.3 (2ArCH) ppm. IR (neat) υmax 3314, 2823, 2666, 2548, 1651, 1589, 1533, 1423, 1293, 1218, 1175, 860 cm"1. HRMS m/z calc. Ci5H12N2O5 [M+l] 301.0819, found [M+l] 301.0817. The synthesis of tert-butyl 2-nitrobenzoate (31)
Figure imgf000053_0002
31
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 0C 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. The precipitate was filtered and the DCM washed with 1 M HCl (aq.) (2 x 50 ml) and brine (50 ml) prior to drying over MgSO4. The mixture was evaporated to a yellow oil which was purified by flash chromatography [10% - 20% EtOAc in Hexane] to yield a pale yellow oil (5.7272 g, 25.6560 mmol, 86%). Rf 0.60 [10% EtOAc in Hexane]. 1H NMR (400 MHz, CDCl3) δ 7.86 (IH, dd, 7=8.0, 1.2 Hz, ArH), 7.75 (IH, dd, 7=7.5, 1.6 Hz, ArH), 7.66 (IH, td, 7=7.5, 1.4 Hz, ArH), 7.60 (IH, td, 7=7.8, 1.6 Hz, ArH), 1.59 (9H, s, C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3) δ 164.2 (C=O), 148.4 (ArC), 132.6 (ArCH), 131.2 (ArCH), 129.9 (ArCH), 129.0 (ArC), 123.6 (ArCH), 83.7 (C(CH3)3), 27.7 (C(CHs)3) ppm. IR (neat) υmax 2979, 1722, 1532, 1367, 1299, 1126 cm"1. MS not detected in ES+ or ES-. The Synthesis of tert-butyl 2-aminobenzoate (32)
Figure imgf000054_0001
32 Tert-butyl 2-nitrobenzoate (31) (4.83 g, 21.637 mmol) was dissolved in MeOH
(100 ml) and added with CAUTION was Pd/C (480 mg, 0.1 equiv. w/w) and the flask evacuated prior to purging with N2, re-evacuation and purging with H2. The mixture was stirred under H2 for 3.5 hours until complete. The mixture was filtered through celite and evaporated to a pale yellow oil (3.5046 g, 18.136 mmol, 84%). Rf 0.63 [17% EtOAc in Hexane]. 1H NMR (400 MHz, CDCl3) δ 7.81 (IH, m, ArH), 7.22 (IH, m ArH), 6.63 (IH, d, J=7.6 Hz, ArH), 6.61 (IH, dd, J=8.6, 1.2 Hz, ArH), 5.68 (2H, br s, NH2), 1.58 (9H, s, C(CH3)3) ppm. 13C NMR (100 MHz, CDCl3) δ 167.7 (C=O), 150.3 (ArC), 133.5 (ArCH), 131.5 (ArCH), 116.7 (ArCH), 116.1 (ArCH), 112.7 (ArC), 80.6 (C(CH3)3), 28.4 (C(CHs)3) ppm. IR (neat) υmax 3480, 3369, 2975, 1682, 1614, 1585, 1367, 1297, 1103 cm" \ HRMS m/z calc. CnH15NO2 [M+l] 194.1176, found [M+l] 194.1177. The synthesis of 2,2'-ureylene-di-(tert-butyl benzoate) (33)
Figure imgf000054_0002
33
Tert-butyl 2-aminobenzoate (32) (3.3793 g, 17.488 mmol) was dissolved in anhydrous THF (44 ml, c = 0.4 M) and CDI (2.836 g, 17.488 mmol, 1.0 equliv.) was added and the mixture heated at reflux for 24 hours under N2. TLC showed the reaction to be incomplete so a further portion of CDI (1.7016 g, 10.4928 mmol, 0.6 equiv.) was added and reflux continued for a further 24 hours at which point LCMS confirmed the success of the reaction. The THF was removed in vacuo and the residue dissolved in EtOAc (200 ml) and washed with 1 M HCl (aq.) (2 x 100 ml), brine (50 ml) and dried over MgSO4. The solvent was removed in vacuo to a yellow semi-solid oil of crude 2,2'- ureylene-di-(tert-butyl benzoate) (33) (3.5949 g, 8.721 mmol, quant.) which was unable to be purified further and hence used crude. 1H NMR (400 MHz, CDCl3) 10.69 (2H, s, 2NH), 8.46 (2H, dd, J=8.5, 0.9 Hz, 2ArH), 7.96 (2H, dd, 7=8.0, 1.6 Hz, 2ArH), 7.49 (2H, ddd, J= 8.7, 7.3, 1.7 Rz, 2ArH), 6.99 (2H, ddd, 7=8.2, 7.3, 1.1 Hz, 2ArH), 1.64 (18H, s, 2(C(CHs)3)) ppm. 13C NMR (100 MHz, CDCl3) 167.8 (2C=O; ester), 152.4 (C=O; urea), 142.4 (2ArC), 133.8 (2ArCH), 131.0 (2ArCH), 121.1 (2ArCH), 120.0 (2ArCH), 116.4 (2ArC), 82.3 (2C(CH3)3), 28.3 (2C(CH3)3) ppm. IR (neat) υmax 3250, 1677, 1582, 1365 cm"1. HRMS m/z calc. C23H28N2O5 [M+l] 413.2071, found [M+l] 413.2065. The synthesis of 2,2'-ureylene-di-benzoic acid (34)
Figure imgf000055_0001
34
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 0C 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 0C. 1H NMR (400 MHz, d6- DMSO) 13.48 (2H, br s, 2COOH), 10.73 (2H, s, 2NH), 8.29 (2H, d, 7=8.3 Hz, 2ArH), 7.95 (2H, dd, 7=7.9, 1.2 Hz, 2ArH), 7.56 (2H, dd, 7=7.6, 1.2 Hz, 2ArH), 7.09 (2H, t, J=7.6 Hz, 2ArH) ppm. 13C NMR (100 MHz, d6-OMSO) 169.7 (2COOH), 151.7 (C=O; urea), 141.6 (2ArC), 133.8 (2ArCH), 131.0 (2ArCH), 121.5 (2ArCH), 119.7 (2ArCH), 116.4 (2ArC) ppm. IR (neat) υmax 3324, 2970, 2810, 2560, 1669, 1583, 1534, 1451, 1260, 749 cm 1. HRMS m/z calc. C15H12N2O5 [M+l] 301.0819, found [M+l] 301.0814. The synthesis of l,3-bis(3-(4-(2-(pyrrolidin-l-yl)acetamido)phenylcarbamoyl)phenyl) urea (35)
Figure imgf000056_0001
35
3,3'-ureylene-di-benzoic acid (26) (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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication, and the precipitant isolated by filtration. The solid residue was suspended in NH3MeOH (1 M, 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a pale brown solid (333.0 mg, 0.474 mmol, 95%). Semi-Prep HPLC (method G) was conducted (60 mg) to yield a white solid (17.4 mg). HPLC (method A) 97%, RT 17.69 minutes. Mp. 286 - 288 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.20 (2H, s, 2NH; amide), 9.73 (2H, s, 2NH; amide), 8.98 (2H, s, 2NH; urea), 7.98 (2H, s, 2ArH), 7.70 (6H, d, J=8.9 Hz, 6ArH), 7.61 (4H, d, J=8.9 Hz, 4ArH), 7.56 (2H, d, J=7.8 Hz, 2ArH), 7.44 (2H, t, J=7.9 Hz, 2ArH), 3.32 (4H, s, 2CH2), 2.67 (8H, m, 4CH2), 1.77 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 167.9 (2C=O; amide), 165.2 (2C=O; amide), 152.4 (C=O; urea), 139.7 (2ArC), 135.7 (2ArC), 134.6 (2ArC), 134.3 (2ArC), 128.6 (2ArCH), 121.1 (2ArCH), 120.8 (2ArCH), 120.6 (4ArCH), 119.6 (4ArCH), 117.6 (2ArCH), 59.0 (2CH2), 53.7 (4CH2), 23.3 (4CH2) ppm. HRMS m/z calc. C39H42N8O5 [M+l] 703.3351, found [M+ 1] 703.3386. The synthesis of l,3-bis(3-(4-(3-(pyrrolidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (36)
Figure imgf000057_0001
36
3,3'-ureylene-di-benzoic acid (26) (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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication. The organic solvent was decanted and the oily residue suspended in NH3MeOH (1 M, 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a white solid (346.0 mg, 0.473 mmol, 95%). HPLC (method A) 98%, RT 19.00 minutes. Mp. 3300C dec. 1H NMR (400 MHz, d6-OMSO) δ 10.18 (2H, s, 2NH; amide), 10.05 (2H, s, 2NH; amide), 8.95 (2H, s, 2NH; urea), 7.97 (2H, s, 2ArH), 7.70 (6H, m, 6ArH), 7.55 (6H, m, 6ArH), 7.44 (2H, t, J=7.9 Hz, 2ArH), 2.75 (4H, t, J=7.1 Hz, 2CH2), 2.50 (12H, m, 6CH2), 1.70 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, J5-DMSO) δ 169.6 (2C=O; amide), 165.2 (2C=O; amide), 152.4 (C=O; urea), 139.6 (2ArC), 135.7 (2ArC), 135.0 (2ArC), 134.3 (2ArC), 128.7 (2ArCH), 121.1 (2ArCH), 120.8 (2ArCH), 120.7 (4ArCH), 119.1 (4ArCH), 117.6 (2ArCH), 53.3 (4CH2), 51.4 (2CH2), 35.7 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C4IH46N8O5 [M+l] 731.3664, found [M+l] 731.3690. Anal. CHN calc. C4IH46N8O5 C 67.4%, H 6.3%, N 15.3%, found C 67.2%, H, 6.3%, N 15.2%. The synthesis of l,3-bis(3-(4-(4-(pyrrolidin-l-yl)butanamido)phenylcarbamoyl) phenyl)urea (37)
Figure imgf000058_0001
37
3,3'-ureylene-di-benzoic acid (26) (150.0 mg, 0.500 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(4-aminophenyl)-4-(pyrrolidin-l- 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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication. The organic solvent was decanted and the oily residue suspended in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2θ (2 x 5 ml) and oven dried to yield a white solid (344.8 mg, 0.454 mmol, 91%). HPLC (method A) 96%, RT 19.86 minutes. Mp. 239 - 241 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.20 (2H, s, 2NH; amide) , 9.89 (2H, s, 2NH; amide), 9.00 (2H, s, 2NH; urea), 7.99 (2H, s, 2ArH), 7.70 (6H, m, 6ArH), 7.57 (6H, m, 6ArH), 7.44 (2H, t, J=7.9 Hz, 2ArH), 2.50 (12H, m, 6CH2), 2.35 (4H, t, J=7.4 Hz, 2CH2), 1.77 (4H, m, 2CH2), 1.70 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.7 (2C=O; amide), 165.2 (2C=O; amide), 152.4 (C=O; urea), 139.7 (2ArC), 135.7 (2ArC), 135.1 (2ArC), 134.2 (2ArC), 128.6 (2ArCH), 121.1 (2ArCH), 120.8 (2ArCH), 120.6 (4ArCH), 119.1 (4ArCH), 117.6 (2ArCH), 54.9 (2CH2), 53.4 (4CH2), 45.7 (2CH2), 34.1 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C43H50N8O5 [M+l] 759.3977, found [M+l] 759.3940. Anal. CHN calc. C43H50N8O5-H2O C 66.5%, H 6.8%, N 14.4%, found C 66.2%, H 6.5%, N 14.6%. The synthesis of l,3-bis(3-(3-(2-(pyrrolidin-l-yl)acetamido)phenylcarbamoyl)phenyl) urea (38)
Figure imgf000059_0001
3,3'-ureylene-di-benzoic acid (26) (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) (438.12 mg, 1.998 mmol, 4 equiv.) and PyBOP (779.9 mg, 1.499 mmol, 3 equiv.) and the mixture stirred at room temperature under nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication, and the precipitate isolated by filtration. The precipitate was dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2θ (2 x 5 ml) and oven dried to yield a pale yellow solid (310.1 mg, 0.441 mmol, 88%). HPLC (method A) 91%, RT 18.69 minutes. Mp. 154 - 157 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.27 (2H, s, 2NH; amide), 9.78 (2H, s, 2NH; amide), 8.96 (2H, s, 2NH; urea), 8.12 (2H, s, 2ArH), 8.79 (2H, s, 2ArH), 7.71 (2H, d, J=8.0 Hz, 2ArH), 7.57 (2H, d, J=7.7 Hz, 2ArH), 7.44 (4H, m. 4ArH), 7.38 (2H, d, J=8.3 Hz, 2ArH), 7.26 (2H, t, J=8.0 Hz, 2ArH), 3.31 (4H, s, 2CH2), 2.65 (8H, m, 4CH2), 1.76 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, J5-DMSO) δ 168.3 (2C=O; amide), 165.6 (2C=O; amide), 152.6 (C=O; urea), 139.7 (2ArC), 139.4 (2ArC), 138.8 (2ArC), 135.8 (2ArC), 128.8 (2ArCH), 128.7 (2ArCH), 121.3 (2ArCH), 121.0 (2ArCH), 117.8 (2ArCH), 115.7 (2ArCH), 115.0 (2ArCH), 111.8 (2ArCH), 59.2 (2CH2), 53.7 (4CH2), 23.4 (4CH2) ppm. HRMS m/z calc. C39H42N8O5 [M+l] 703.3351, found [M+l] 703.3344.
The synthesis of l,3-bis(3-(3-(3-(pyrrolidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (39)
Figure imgf000060_0001
3,3'-ureylene-di-benzoic acid (26) (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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication. The organic solvent was decanted and the oily residue dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a pale yellow solid (292.4 mg, 0.400 mmol, 80%). HPLC (method A) 93%, RT 19.39 minutes. Mp. 160 - 163 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.26 (2H, s, 2NH; amide), 10.13 (2H, s, 2NH; amide), 9.00 (2H, s, 2NH; urea), 8.10 (2H, s, 2ArH), 7.98 (2H, s, 2ArH), 7.72 (2H, d, J=7.9 Hz, 2ArH), 7.57 (2H, d, J=7.7 Hz, 2ArH), 7.45 (2H, t, J=7.9 Hz, 2ArH), 7.42 (2H, d, J=7.7 Hz, 2ArH), 7.38 (2H, d, J=7.9 Hz, 2ArH), 7.25 (2H, t, J=7.7 Hz, 2ArH), 2.72 (4H, t, J=6.9 Hz, 2CH2), 2.49 (12H, m, 6CH2), 1.70 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.0 (2C=O; amide), 165.5 (2C=O; amide), 152.6 (C=O; urea), 139.6 (2ArC), 139.3 (2ArC), 139.3 (2ArC), 135.7 (2ArC), 128.6 (2ArCH), 128.5 (2ArCH), 121.2 (2ArCH), 120.9 (2ArCH), 117.7 (2ArCH), 115.2 (2ArCH), 114.5 (2ArCH), 111.2 (2ArCH), 53.3 (4CH2), 51.4 (2CH2), 35.9 (2CH2), 23.1 (4CH2) ppm. HRMS m/z calc. C41H46N8O5 [M+l] 731.3664, found [M+l] 731.3651. Anal. CHN calc. C4iH46N8O5.2H2O C 64.2%, H 6.6%, N 14.6%, found C 64.5%, H 6.3%, N 14.3%. The synthesis of l,3-bis(3-(3-(4-(pyrrolidin-l-yl)butanamido)phenylcarbamoyl) phenyl)urea (40)
Figure imgf000061_0001
3,3'-ureylene-di-benzoic acid (26) (50.0 mg, 0.167 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(3-aminophenyl)-4-(pyrrolidin-l- yl)butanamide (21) (164.75 mg, 0.666 mmol, 4 equiv.) and PyBOP (260.72 mg, 0.500 mmol, 3 equiv.) and the mixture stirred at room temperature under nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication, and the organic layer decanted. The residue was dissolved in NH3/MeOH (1 M; 2 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a white solid (63.2 mg, 0.083 mmol, 50%). HPLC (method A) 96%, RT 19.56 minutes. Mp. 234 - 236 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.26 (2H, s, 2NH; amide), 9.94 (2H, s, 2NH; amide), 9.01 (2H, s, 2NH; urea), 8.11 (2H, s, 2ArH), 7.99 (2H, s, 2ArH), 7.72 (2H, d, J=8.0 Hz, 2ArH), 7.57 (2H, d, J=7.6 Hz, 2ArH), 7.43 (4H, m, 4ArH), 7.37 (2H, d, J=7.9 Hz, 2ArH), 7.25 (2H, t, J=8.1 Hz, 2ArH), 2.48 (12H, m, 6CH2), 2.37 (4H, t, J=7.3 Hz, 2CH2), 1.77 (4H, m, 2CH2), 1.69 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.0 (2C=O; amide), 165.5 (2C=O; amide), 152.5 (C=O; urea), 139.6 (2ArC), 139.4 (2ArC), 139.3 (2ArC), 135.7 (2ArC), 128.6 (2ArCH), 128.5 (2ArCH), 121.2 (2ArCH), 120.9 (2ArCH), 117.7 (2ArCH), 115.1 (2ArCH), 114.5 (2ArCH), 111.3 (2ArCH), 54.9 (2CH2), 53.4 (4CH2), 34.2 (2CH2), 24.1 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C43H50N8O5 [M+l] 759.3977, found [M+ 1] 759.3942.
The synthesis of l,3-bis(4-(4-(2-(pyrrolidin-l-yl)acetamido)phenylcarbamoyl)phenyl) urea (41)
Figure imgf000061_0002
4,4'-ureylene-di-benzoic acid (30) (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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication, and the precipitate isolated by filtration. The solid residue was suspended in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a dark brown solid (325.3 mg, 0.463 mmol, 93%). To aid solubility the acetate salt was formed by suspending the title compound (41) (100 mg) in acetic acid (1 ml) and adding water until dissolved. The salt was isolated by freeze drying. Semi-Prep HPLC (method G) was conducted (60 mg) to yield an off white solid (16.4 mg). HPLC (method A; 280nm) 98%, RT 19.23 minutes. Mp. 296 - 298 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.04 (2H, s, 2NH; amide), 9.65 (2H, s, 2NH; amide), 9.14 (2H, s, 2NH; urea), 7.94 (4H, d, J=8.8 Hz, 4ArH), 7.69 (4H, d, 7=9.6» Hz, 4ArH), 7.61 (4H, d, J=8.8 Hz, 4ArH), 7.60 (4H, d, 7=9.6» Hz, 4ArH), 3.25 (4H, s, 2CH2), 2.61 (8H, m, 4CH2), 1.76 (8H, m, 4CH2) ppm. HRMS m/z calc. C39H42N8O5 [M+l] 703.3351, found [M+l] 703.3353.
The synthesis of l,3-bis(4-(4-(3-(pyrrolidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (42)
Figure imgf000062_0001
4,4'-ureylene-di-benzoic acid (30) (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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication and the resulting pink precipitate isolated by filtration. The precipitate was suspended in NH3/MeOH (1 M; 5 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield an off white/pale pink solid (298.6 mg, 0.409 mmol, 82%). HPLC (method A; 280nm) 99%, RT 18.65 minutes. Mp. 299 - 301 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.04 (4H, s, 4NH; amide), 9.15 (2H, s, 2NH; urea), 7.94 (4H, d, J=8.6 Hz, 4ArH), 7.69 (4H, d, 7=8.8 Hz, 4ArH), 7.62 (4H, d, J=8.6 Hz, 4ArH), 7.55 (4H, d, J=8.8 Hz, 4ArH), 2.77 (4H, t, J=6.4 Hz, 2CH2), 2.50 (12H, m, 6CH2), 1.72 (8H, m, 4CH2) ppm. HRMS m/z calc. C4IH46N8O5 [M+l] 731.3664, found [M+l] 731.3653. Anal. CHN calc. C4IH46N8O5-H2O C 65.8%, H 6.5%, N 15.0%, found C 66.1%, H 6.3%, N 15.2%. To aid solubility the acetate salt was formed by suspending the title compound (42) (41.7 mg) in acetic acid (1 ml) and adding water until dissolved. The salt was isolated after freeze drying and found to not significantly aid solubility.
The synthesis of l,3-bis(4-(4-(4-(pyrrolidin-l-yl)butanamido)phenylcarbamoyl) phenyl)urea (43)
Figure imgf000063_0001
4,4'-ureylene-di-benzoic acid (30) (150.0 mg, 0.500 mmol) was dissolved in anhydrous DMF (8 ml) and to this was added N-(4-aminophenyl)-4-(pyrrolidin-l- 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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication and the resulting pale pink solid isolated by filtration. The solid was suspended in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a brown crystalline solid (332.3 mg, 0.438 mmol, 88%). HPLC (method A; 280nm) 91%, RT 20.01 minutes. Mp. 289 - 291 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.02 (2H, s, 2NH; amide), 9.85 (2H, s, 2NH; amide), 9.16 (2H, s, 2NH; urea), 7.93 (4H, d, J=8.7 Rz, 4ArH), 7.67 (4H, d, J=8.9 Hz, 4ArH), 7.61 (4H, d, J=8.7 Hz, 4ArH), 7.55 (4H, d, J=8.9 Hz, 4ArH), 2.5 (12H, m, 6CH2), 2.35 (4H, t, J=7.3 Hz, 2CH2), 1.77 (4H, m, 2CH2), 1.69 (8H, m, 4CH2) ppm. HRMS m/z calc. C43H50N8O5 [M+l] 759.3977, found [M+l] 759.3992. Anal. CHN calc. C43H50N8O5.1.5 H2O C 65.7%, H 6.8%, N 14.3%, found C 65.7%, H 6.8%, N 14.6%. To aid solubility the acetate salt was formed by suspending the title compound (43) (100 mg) in acetic acid (1 ml) and adding water until dissolved. The salt was isolated by freeze drying and found to not significantly aid solubility. The synthesis of l,3-bis(4-(3-(2-(pyrrolidin-l-yl)acetamido)phenylcarbamoyl)phenyl) urea (44)
Figure imgf000064_0001
4,4'-ureylene-di-benzoic acid (30) (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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCI3 (20 ml) with sonication, and the precipitant isolated by filtration. The residue was suspended in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2θ (2 x 5 ml) and oven dried to yield a pale yellow solid (265.0 mg, 0.377 mmol, 75%). Semi-Prep HPLC (method G) was conducted (60mg) to yield a white solid (32.5 mg). HPLC (method A) 99%, RT 18.35 minutes. Mp. 232 - 234 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.10 (2H, s, 2NH; amide), 9.71 (2H, s, 2NH; amide), 9.15 (2H, s, 2NH; urea), 8.11 (2H, s, 2ArH), 7.95 (4H, d, J=8.8 Hz, 4ArH), 7.62 (4H, d, J=8.8 Hz, 4ArH), 7.46 (2H, d, J=8.2 Hz, 2ArH), 7.36 (2H, d, J=8.5 Hz, 2ArH), 7.25 (2H, t, J=8.1 Hz, 2ArH), 3.28 (4H, s, 2CH2), 2.63 (8H, m, 4CH2), 1.76 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 168.5 (2C=O; amide), 164.8 (2C=O; amide), 152.1 (C=O; urea), 142.6 (2ArC), 139.5 (2ArC), 138.7 (2ArC), 128.8 (4ArCH), 128.6 (2ArC), 127.9 (2ArCH), 117.2 (4ArCH), 115.6 (2ArCH), 114.7 (2ArCH), 111.7 (2ArCH), 59.3 (2CH2), 53.6 (4CH2), 23.4 (4CH2) ppm. HRMS m/z calc. C39H42N8O5 [M+ 1] 703.3351, found [M+l] 703.3375.
The synthesis of l,3-bis(4-(3-(3-(pyrrolidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (45)
Figure imgf000064_0002
4,4'-ureylene-di-benzoic acid (30) (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 nitrogen 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. The residue was dissolved in NH3/MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2θ (2 x 5 ml) and oven dried to yield a pale yellow solid (233.1 mg, 0.319 mmol, 64%). Semi-Prep HPLC (method G) was conducted (60 mg) to yield a white solid (18.0 mg). HPLC (method A) 99%, RT 19.01 minutes. Mp. 231 - 233 0C. 1H NMR (400 MHz, dtf-DMSO) δ 10.11 (2H, s, 2NH; amide), 10.10 (2H, s, 2NH; amide), 9.17 (2H, s, 2NH; urea), 8.11 (2H, s, 2ArH), 7.96 (4H, d, J=8.8 Hz, 4ArH), 7.62 (4H, d, J=8.8 Hz, 4ArH), 7.42 (2H, d, J= 8.3 Hz, 2ArH), 7.35 (2H, d, J=8.2 Hz, 2ArH), 7.25 (2H, t, J=8.1 Hz, 2ArH), 2.76 (4H, t, 7=7.6» Hz, 2CH2), 2.50 (12H, m, 6CH2), 1.71 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.9 (2C=O; amide), 164.8 (2C=O; amide), 152.0 (C=O; urea), 142.5 (2ArC), 139.5 (2ArC), 139.3 (2ArC), 128.7 (4ArCH), 128.5 (2ArC), 127.9 (2ArCH), 117.2 (4ArCH), 115.2 (2ArCH), 114.3 (2ArCH), 111.3 (2ArCH), 53.3 (4CH2), 51.4 (2CH2), 35.8 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C4IH46N8O5 [M+l] 731.3664, found [M+l] 731.3668. Anal. CHN calc. C4IH46N8O5-LS H2O C 65.0%, H 6.5%, N 14.8%, found C 65.3%, H 6.3%, N 15.0%. The synthesis of l,3-bis(4-(3-(4-(pyrrolidin-l-yl)butanamido)phenylcarbamoyl) phenyl)urea (46)
Figure imgf000065_0001
4,4'-ureylene-di-benzoic acid (30) (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 nitrogen for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication and the resulting solid isolated by decanting the organic solvent. The solid was dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a white solid (173.4 mg, 0.228 mmol, 46%). Semi-Prep HPLC (method H) was conducted (90 mg) to yield a brown solid (31.0 mg). HPLC (method A) 97%, RT 20.40 minutes. Mp. 249 - 252 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.09 (2H, s, 2NH; amide), 9.91 (2H, s, 2NH; amide), 9.31 (2H, s, 2NH; urea), 8.1 1 (2H, s, 2ArH), 7.95 (4H, d, J=8.5 Hz, 4ArH), 7.63 (4H, d, J=8.5 Hz, 4ArH), 7.41 (2H, d, J=8.1 Hz, 2ArH), 7.34 (2H, d, J=7.9 Hz, 2ArH), 7.23 (2H, t, J=8.0 Hz, 2ArH), 2.52 (12H, m, 6CH2), 2.37 (4H, t, J=7.2 Hz, 2CH2), 1.78 (4H, m, 2CH2), 1.70 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.9 (2C=O; amide), 164.7 (2C=O; amide), 152.1 (C=O; urea), 142.6 (2ArC), 139.4 (2ArC), 139.4 (2ArC), 128.7 (4ArCH), 128.4 (2ArC), 127.9 (2ArCH), 117.2 (4ArCH), 115.2 (2ArCH), 114.3 (2ArCH), 111.4 (2ArCH), 54.9 (2CH2), 53.4 (4CH2), 34.1 (2CH2), 23.9 (2CH2), 22.9 (4CH2) ppm. HRMS m/z calc. C43H50N8O5 [M+l] 759.3977, found [M+ 1] 759.3975.
The synthesis of N-(4-(2-(pyrrolidin-l-yl)acetamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3 (4H)-yl)benzamide (47)
Figure imgf000066_0001
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 N2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication, and the organic solvent decanted from the oily residue. The residue was dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a pale brown solid (154.8 mg, 0.320 mmol, 64%). Semi-Prep HPLC (method I) was conducted (40 mg) to yield an off white solid (22.0 mg). HPLC (method B) 99%, RT 8.40 minutes. Mp. 238 - 239 0C. 1H NMR (400 MHz, d6-OMSO) δ 11.49 (IH, s, NH; urea), 10.31 (IH, s, NH; amide), 9.69 (IH, s, NH; amide), 7.91 (IH, dd, J=8.1, 1.4 Hz, ArH), 7.80 (IH, d, J=7.6 Hz, ArH), 7.69 (IH, m, ArH), 7.65 (IH, td, J=7.7, 1.5 Hz, ArH), 7.58 (IH, td, J=7.5, 1.1 Hz, ArH), 7.52 (4H, m, 4ArH), 7.45 (IH, dd, J=7.6, 1.2 Hz, ArH), 7.21 (2H, m, ArH), 3.31 (2H, s, CH2), 2.66 (4H, m, 2CH2), 1.76 (4H, m, 2CH2,) ppm. 13C NMR (100 MHz, d6-OMSO) δ 167.8 (C=O; amide), 164.5 (C=O; amide), 162.0 (C=O), 150.0 (C=O; urea), 139.8 (ArC), 135.2 (ArCH), 134.8 (ArC), 134.3 (ArC), 134.3 (ArC), 134.0 (ArC), 130.9 (ArCH), 130.7 (ArCH), 128.6 (ArCH), 128.3 (ArCH), 127.6 (ArCH), 122.5 (ArCH), 120.2 (2ArCH), 119.7 (2ArCH), 115.2 (ArCH), 114.2 (ArC), 59.0 (CH2), 53.8 (2CH2), 23.4 (2CH2) ppm. IR (neat) υmax 3365, 1656, 1513, 1397, 751 cm"1. HRMS m/z calc. C27H25N5O4 [M+H]+ 484.1979, found [M+H]+ 484.1968. Anal. CHN C27H25N5O4.0.5 H2O calc. C 65.8%, H 5.3%, N 14.2%, found C 65.5%, H 5.0%, 14.0%. The synthesis of N-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3(4H)-yl)benzamide (48)
Figure imgf000067_0001
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 N2 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. The residue was dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a grey solid (164.8 mg, 0.331 mmol, 66%). Semi-Prep HPLC (method I) was conducted (40 mg) to yield a white solid (10.8 mg). HPLC (method B) 100%, RT 9.29 minutes. Mp. 193 - 195 0C. 1H NMR (400 MHz, d6-OMSO) δ 11.49 (IH, s, NH; urea), 10.29 (IH, s, NH; amide), 9.98 (IH, s, NH; amide), 7.92 (IH, dd, J=8.1, 1.4 Hz, ArH), 7.81 (IH, d, J=7.4 Hz, ArH), 7.68 (2H, m, 2ArH), 7.58 (IH, td, J=7.5, 1.0 Hz, ArH), 7.52 (2H, d, J=9.1 Hz, 2ArH), 7.46 (2H, d, J=9.1 Hz, 2ArH), 7.45 (IH, dd, J=7.7, 1.2 Hz, ArH), 7.22 (2H, m, ArH), 2.69 (2H, t, J=7.1 Hz, CH2), 2.46 (4H, m, 2CH2), 2.44 (2H, t, J=7.1 Hz, CH2), 1.68 (4H, m, CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.7 (C=O; amide), 164.3 (C=O; amide), 161.9 (C=O), 149.9 (C=O; urea), 139.7 (ArC), 135.1 (ArCH), 134.9 (ArC), 134.3 (ArC), 134.2 (ArC), 133.9 (ArC), 130.8 (ArCH), 130.6 (ArCH), 128.5 (ArCH), 128.2 (ArCH), 127.5 (ArCH), 122.4 (ArCH), 120.1 (2ArCH), 119.0 (2ArCH), 115.1 (ArCH), 114.0 (ArC), 53.3 (2CH2), 51.4 (CH2), 35.9 (CH2), 23.0 (2CH2) ppm. IR (neat) υmax 3293, 1718, 1647, 1513, 1394, 756 cm"1. HRMS m/z calc. C28H27N5O4 [M+H]+ 498.2136, found [M+Hf 498.2139. The synthesis of N-(4-(4-(pyrrolidin-l-yl)butanamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3 (4H)-yl)benzamide (49)
Figure imgf000068_0001
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 N2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication, and the organic solvent decanted from the oily residue. The residue was dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a brown solid (246.4 mg, 0.482 mmol, 96%). Semi-Prep HPLC (method I) was conducted (40 mg) to yield a white solid (17.0 mg). HPLC (method B) 100%, RT 10.38 minutes. Mp. 188 - 190 0C. 1H NMR (400 MHz, d6-OMSO) δ 11.48 (IH, s, NH; urea), 10.28 (IH, s, NH; amide), 9.80 (IH, s, NH; amide), 7.91 (IH, dd, J=8.1, 1.4 Hz, ArH), 7.80 (IH, dd, J=7.4, 0.8 Hz, ArH), 7.69 (IH, dd, J=7.4, 1.5 Hz, ArH), 7.65 (IH, td, J=7.6, 1.5 Hz, ArH), 7.58 (IH, td, J=7.5, 1.1 Hz, ArH), 7.50 (2H, d, J=9.1 Hz, 2ArH), 7.46 (2H, d, J=9.1 Hz, 2ArH), 7.45 (IH, dd, J=7.7, 1.3 Hz, ArH), 7.21 (2H, m, 2ArH), 2.44 (6H, m, 3CH2), 2.31 (2H, t, J=7.4 Hz, CH2), 1.73 (2H, m, CH2), 1.67 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.8 (C=O; amide), 164.4 (C=O; amide), 162.0 (C=O), 150.0 (C=O; urea), 139.8 (ArC), 135.2 (ArCH), 135.1 (ArC), 134.3 (ArC), 134.3 (ArC), 134.0 (ArC), 130.9 (ArCH), 130.7 (ArCH), 128.6 (ArCH), 128.3 (ArCH), 127.6 (ArCH), 122.5 (ArCH), 120.2 (2ArCH), 119.7 (2ArCH), 1 15.2 (ArCH), 1 14.2 (ArC), 55.1 (CH2), 53.5 (2CH2), 34.3 (CH2), 24.2 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 3273, 1653, 1511, 1395, 755 cm 1. HRMS m/z calc. C29H29N5O4 [M+H]+ 512.2292, found [M+H]+ 512.2266. The synthesis of N-(3-(2-(pyrrolidin-l-yl)acetamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3 (4H)-yl)benzamide (50)
Figure imgf000069_0001
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 N2 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. The residue was dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a pale yellow powder (109.8 mg, 0.227 mmol, 45%). Semi-Prep HPLC (method I) was conducted (40 mg) to yield a white solid (13.5 mg). HPLC (method B) 100%, RT 9.92 minutes. Mp. 169 - 171 0C. 1H NMR (400 MHz, d6-OMSO) δ 11.50 (IH, s, NH; urea), 10.41 (IH, s, NH; amide), 9.71 (IH, s, NH; amide), 7.96 (IH, t, J=1.8 Hz, ArH), 7.92 (IH, dd, J=8.1, 1.4 Hz, ArH), 7.81 (IH, dd, J=7.6, 1.3 Hz, ArH), 7.70 (IH, dd, J=7.3, 1.5 Hz, ArH), 7.66 (IH, td, J=7.6, 1.6 Hz, ArH), 7.59 (IH, td, J=7.6, 1.3 Hz, ArH), 7.49 (IH, dd, J=7.7, 1.2 Hz, ArH), 7.38 (IH, m, ArH), 7.21 (4H, m, 4ArH), 3.27 (2H, s, CH2), 2.62 (4H, m, 2CH2), 1.74 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 168.1 (C=O; amide), 164.6 (C=O; amide), 161.9 (C=O), 149.9 (C=O; urea), 139.7 (ArC), 139.3 (ArC), 138.7 (ArC), 135.1 (ArCH), 134.2 (ArC), 133.8 (ArC), 130.9 (ArCH), 130.6 (ArCH), 128.6 (ArCH), 128.5 (ArCH), 128.2 (ArCH), 127.5 (ArCH), 122.4 (ArCH), 115.1 (ArCH), 115.0 (ArCH), 114.7 (ArCH), 114.0 (ArC), 111.1 (ArCH), 59.0 (CH2), 53.5 (2CH2), 23.3 (2CH2) ppm. IR (neat) υmax 3267, 1662, 1604, 758 cm 1. HRMS m/z calc. C27H25N5O4 [M+Hf 484.1979, found [M+H]+ 484.1979. Anal. CHN calc. C27H25N5O4 C 67.1%, H 5.2%, N 14.5%, found C 67.3%, H 4.9%, N 14.3%. The synthesis of N-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3(4H)-yl)benzamide (51)
Figure imgf000070_0001
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 N2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication, and the organic solvent decanted from the oily residue. The residue was dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a sticky brown solid (127.0 mg, 0.2556 mmol, 51%). Semi-Prep HPLC (method I) was conducted (40 mg) to yield a white solid (15.3 mg). HPLC (method B) 99%, RT 10.39 minutes. Mp. 156 - 158 0C. 1H NMR (400 MHz, d6-OMSO) δ 11.49 (IH, s, NH; urea), 10.39 (IH, s, NH; amide), 10.04 (IH, s, NH; amide), 7.92 (2H, m, 2ArH), 7.80 (IH, dd, J=7.6, 1.3 Hz, ArH), 7.69 (IH, m, ArH), 7.66 (IH, td, J=7.6, 1.5 Hz, ArH), 7.58 (IH, td, J=7.5, 1.2 Hz, ArH), 7.45 (IH, dd, J=7.7, 1.2 Hz, ArH), 7.39 (IH, d, J=7.8 Hz, ArH), 7.19 (4H, m, 4ArH), 2.70 (2H, t, J=6.9 Hz, CH2), 2.47 (4H, m, 2CH2), 2.43 (2H, t, 7=7.6» Hz, CH2), 1.66 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, dtf-DMSO) δ 169.9 (C=O; amide), 164.6 (C=O; amide), 161.9 (C=O), 149.9 (C=O; urea), 139.7 (ArC), 139.4 (ArC), 139.3 (ArC), 135.1 (ArCH), 134.2 (ArC), 133.8 (ArC), 130.9 (ArCH), 130.6 (ArCH), 128.6 (ArCH), 128.5 (ArCH), 128.2 (ArCH), 127.5 (ArCH), 122.4 (ArCH), 115.1 (ArCH), 114.6 (ArCH), 114.2 (ArCH), 114.1 (ArC), 110.5 (ArCH), 53.2 (2CH2), 51.3 (CH2), 35.7 (CH2), 23.0 (2CH2) ppm. IR (neat) υmax 3260, 1718, 1654, 1603, 756 cm"1. HRMS m/z calc. C28H27N5O4 [M+H]+ 498.2136, found [M+H]+ 498.2141. The synthesis of N-(3-(4-(pyrrolidin-l-yl)butanamido)phenyl)-3-(l,2-dihydro-2,4- dioxoquinazolin-3 (4H)-yl)benzamide (52)
Figure imgf000071_0001
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 N2 for 22 hours. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication, and the organic solvent decanted from the oily residue. The residue was dissolved in NH3MeOH (1 M; 3 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a pale brown solid (136.9 mg, 0.268 mmol, 54%). Semi-Prep HPLC (method I) was conducted (40 mg) to yield a brown solid (14.3 mg). HPLC (method B) 96%, RT 11.33 minutes. Mp. 161 - 163 0C 1H NMR (400 MHz, d6-OMSO) δ 11.49 (IH, s, NH; urea), 10.38 (IH, s, NH; amide), 9.82 (IH, s, NH; amide), 7.92 (2H, m, 2ArH), 7.80 (IH ,dd, J=7.6, 1.3 Hz, ArH), 7.69 (IH, dd, J=7.4, 1.5 Hz, ArH), 7.65 (IH, td, J=7.6, 1.5 Hz, ArH), 7.58 (IH, td, J=7.6, 1.3 Hz, ArH), 7.45 (IH, dd, J=7.8, 1.2 Hz, ArH), 7.38 (IH, d, J=7.8 Hz, ArH), 7.17 (4H, m, 4ArH), 2.42 (4H, m, 2CH2), 2.39 (2H, t, J=7.3 Hz, CH2), 2.29 (2H, t, J=7.4 Hz, CH2), 1.71 (2H, m, CH2), 1.65 (4H, m, CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.0 (C=O; amide), 164.5 (C=O; amide), 161.9 (C=O), 149.9 (C=O; urea), 139.7 (ArC), 139.4 (ArC), 139.3 (ArC), 135.1 (ArCH), 134.2 (ArC), 133.8 (ArC), 130.9 (ArCH), 130.6 (ArCH), 128.6 (ArCH), 128.4 (ArCH), 128.2 (ArCH), 127.5 (ArCH), 122.4 (ArCH), 115.1 (ArCH), 114.6 (ArCH), 114.3 (ArCH), 114.1 (ArC), 110.6 (ArCH), 55.0 (CH2), 53.4 (2CH2), 34.2 (CH2), 24.1 (CH2), 23.0 (2CH2) ppm. IR (neat) υmax 3260, 1652, 1603, 755 cm 1. HRMS m/z calc. C29H29N5O4 [M+H]+ 512.2292, found [M+Hf 512.2281. Anal. CHN C29H29N5O4-LSH2O calc. C 64.7%, H 6.0%, N 13.0%, found C 64.7%, H 6.0%, N 13.0%. The synthesis of 3-amino-5-nitro-N-phenylbenzamide (53)
Figure imgf000072_0001
53
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 N2 at room temperature for 24 hours. The precipitate was filtered from the reaction, the DMF removed in vacuo, and to the residue was added 5% NH3 (aq.) (100 ml) and the product extracted into EtOAc (3 x 100 ml), dried over K2CO3 and the solvent removed in vacuo. The desired product was obtained by flash chromatography [50% EtOAc in Hexane] as a pale orange powder (1.2911 g, 5.019 mmol, 91%). Rf 0.47 [50% EtOAc in Hexane]. Mp. 202 - 204 0C. 1H NMR (400 MHz, d6-OMSO) δlθ.40 (IH, s, NH), 7.90 (IH, t, J=I.8 Hz, ArH), 7.77 (2H, d, J=7.6 Hz, 2ArH), 7.57 (IH, t, J=2.2 Hz, ArH), 7.51 (IH, t, J=1.8 Hz, ArH), 7.37 (2H, t, J=7.9 Hz, 2ArH), 7.12 (IH, t, J= 7.4 Hz, ArH), 6.09 (2H, s, NH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 164.2 (C=O), 150.2 (ArC), 148.7 (ArC), 138.8 (ArC), 136.9 (ArC), 128.6 (2ArCH), 123.9 (ArCH), 120.5 (2ArCH), 118.8 (ArCH), 109.5 (ArCH), 108.6 (ArCH) ppm. IR (neat) υmax 3442, 3318, 2927, 2849, 1650, 1523, 1327, 1246 cm"1. HRMS m/z calc. Ci3H11N3O3 [M+l] 258.0873, found [M+l] 258.0867. The synthesis of 3-(2-chloroacetamido)-5-nitro-N-phenylbenzamide (54)
Figure imgf000072_0002
54 3-amino-5-nitro-N-phenylbenzamide (53) (150 mg, 0.584 mmol) was dissolved in anhydrous THF (5 ml) and TEA (0.16 ml, 1.168 mmol, 2 equiv.) was added and the mixture cooled to 0 0C prior to the drop wise addition of 2-chloroacetyl chloride (0.10 ml, 1.168 mmol, 2 equiv.), and the mixture was stirred overnight at room temperature. The solid precipitate was filtered from the reaction and the THF removed in vacuo. The residue was suspended in ether which upon filtration yielded a pink solid (127.8 mg, 0.383 mmol, 66%). Rf 0.52 [50% EtOAc in Hexane]. Mp. 187 0C dec. 1H NMR (400 MHz, d6-OMSO) δ 11.03 (IH, s, NH), 10.65 (IH, s, NH), 8.82 (IH, t, 7=2.6» Hz, ArH), 8.59 (IH, t, 7=2.6» Hz, ArH), 8.46 (IH, t, 7=2.6» Hz, ArH), 7.78 (2H, dd, J=8.6, 1.3 Hz, 2ArH), 7.40 (2H, dd, J=8.6, 7.4 Hz, 2ArH), 7.15 (IH, tt, 7=7.3, 1.3 Hz, ArH), 4.37 (2H, s, CH2) ppm. 13C NMR (100 MHz, dtf-DMSO) δ 165.6 (C=O), 163.1 (C=O), 148.0 (ArC), 139.7 (ArC), 138.6 (ArC), 136.8 (ArC), 128.7 (2ArCH), 124.6 (ArCH), 124.2 (ArCH), 120.6 (2ArCH), 117.1 (ArCH), 116.0 (ArCH), 43.4 (CH2) ppm. IR (neat) υmax 3327, 3258, 3087, 2935, 1655, 1530, 1324, 899 cm"1. HRMS m/z calc. Ci5H12ClN3O4 [M+l] 334.0589, found [M+l] 334.0582.
The synthesis of 3-(2-(pyrrolidin-l-yl)acetamido)-5-nitro-N-phenylbenzamide (55)
Figure imgf000073_0001
55
3-(2-chloroacetamido)-5-nitro-N-phenylbenzamide (54) (102.2 mg, 0.306 mmol) was dissolved in THF (5 ml) prior to the addition of pyrrolidine (0.05 ml, 0.612 mmol, 2 equiv.) and the mixture stirred under N2 at room temperature overnight. The THF was removed in vacuo and 5% NH3 (aq.) (50 ml) was added and the solid isolated by filtration washing with ether to yield a beige solid (102.9 mg, 0.2793 mmol, 91%). Rf 0.64 [MeOH]. Mp. 181 - 184 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.57 (IH, s, NH), 10.41 (IH, s, NH), 8.92 (IH, t, 7=2.6» Hz, ArH), 8.58 (IH, t, 7=i.7 Hz, ArH), 8.51 (IH, t, J=I.8 Hz, ArH), 7.77 (2H, dd, J=LO, 8.6 Hz, 2ArH), 7.39 (2H, t, 7=7.9 Hz, 2ArH), 7.15 (IH, tt, J=LO, 7.3 Hz, ArH), 3.33 (2H, s, CH2), 2.57 (4H, m, 2CH2), 1.72 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.0 (C=O), 163.4 (C=O), 147.9 (ArC), 140.1 (ArC), 138.7 (ArC), 136.7 (ArC), 128.7 (2ArCH), 124.9 (ArCH), 124.1 (ArCH), 120.6 (2ArCH), 116.5 (ArCH), 116.2 (ArCH), 59.5 (CH2), 53.7 (2CH2), 23.5 (2CH2) ppm. IR (neat) υmax 3315, 3235, 3088, 2961, 2770, 1664, 1600, 1533, 1330, 1244, 897 cm"1. HRMS m/z calc.
Ci9H20N4O4 [M+l] 369.1557, found [M+l] 369.1539.
The synthesis of 3-(2-(pyrrolidin-l-yl)acetamido)-5-amino-N-phenylbenzamide (56)
Figure imgf000074_0001
56 3-(2-(pyrrolidin-l-yl)acetamido)-5-nitro-N-phenylbenzamide (55) (300 mg, 0.814 mmol) was suspended in absolute EtOH (4 ml) and ammonium formate (205.32 mg, 3.256 mmol, 4 equiv.) and Pd/C (30 mg, 0.1 equiv. w/w) was added prior to the mixture being heated by microwave irradiation at 120 0C for 10 minutes under N2 (pressure ~ 7 bar). The resulting solution was filtered through celite, solvent removed in vacuo, and the residue dissolved in EtOAc (100 ml) and washed with 5% NH3 (aq.) (2 x 25 ml), brine (25 ml) and dried over K2CO3. Evaporation yielded a brown solid (254.1 mg, 0.751 mmol, 92%). Rf 0.33 [EtOH]. Mp. 88 - 91 0C. 1H NMR (400 MHz, J5-DMSO) δlθ.16 (IH, s, NH), 9.61 (IH, s, NH), 7.80 (2H, d, /=7.7 Hz, 2ArH), 7.39 (2H, t, J=7.8 Hz, 2ArH), 7.29 (IH, s, ArH), 7.20 (IH, s, ArH), 7.13 (IH, t, J=7.4, ArH), 6.82 (IH, s, ArH), 5.43 (2H, br s, NH2), 3.28 (2H, s, CH2) 2.65 (4H, m, 2CH2), 1.81 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 168.6 (C=O), 166.4 (C=O), 149.0 (ArC), 139.4 (ArC), 139.1 (ArC), 136.5 (ArC), 128.5 (2ArCH), 123.3 (ArCH), 120.1 (2ArCH), 108.4 (ArCH), 107.5 (ArCH), 106.7 (ArCH), 59.4 (CH2), 53.7 (2CH2), 23.5 (2CH2) ppm. IR (neat) υmax 3246, 2970, 2808, 1737, 1655, 1593, 1522, 1352, 1217, 864, 753 cm"1. HRMS m/z calc. C19H22N4O2 [M+l] 339.1815, found [M+l] 339.1797.
The Synthesis of l,3-bis(3-(phenylcarbamoyl)-5-(2-(pyrrolidin-l-yl)acetamido) phenyl)urea (57)
Figure imgf000074_0002
57 3-(2-(pyrrolidin-l-yl)acetamido)-5-amino-N-phenylbenzamide (56) (500 mg, 1.478 mmol) was dissolved in anhydrous THF (3.7 ml, c = 0.4 M) and CDI (239.7 mg, 1.478 mmol, 1 equiv.) was added and the mixture heated by microwave irradiation at 75 0C for 20 minutes under N2 (pressure ~ 0 bar). Precipitation was observed prior to the mixture being cooled to room temperature and flooded with EtOAc (10 ml). The title compound was isolated by filtration, washed with EtOAc (2 ml), Et2O (2 ml) and oven dried to yield a white solid (231.0 mg, 0.329 mmol, 44%). Semi-Prep HPLC (method G) was conducted (40 mg) to yield a white solid (15.8 mg). HPLC (method A) 96%, RT 21.02 minutes. Mp. 179 - 181 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.28 (2H, s, 2NH; amide), 9.92 (2H, s, 2NH; amide), 8.94 (2H, s, 2NH; urea), 8.14 (2H, t, J=U Hz, 2ArH), 7.77 (4H, d, J=7.8 Hz, 4ArH), 7.72 (2H, t, J=U Hz, 2ArH), 7.70 (2H, t, J=U Hz, 2ArH), 7.36 (4H, t, J=7.9 Hz, 4ArH), 7.11 (2H, t, J=7.4 Hz, 2ArH), 3.33 (4H, s, 2CH2), 2.66 (8H, m, 4CH2), 1.78 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 168.7 (2C=O; amide), 165.7 (2C=O; amide), 152.2 (C=O; urea), 139.8 (2ArC), 139.1 (2ArC), 139.0 (2ArC), 136.4 (2ArC), 128.5 (4ArCH), 123.5 (2ArCH), 120.1 (4ArCH), 112.5 (2ArCH), 112.5 (2ArCH), 111.9 (2ArCH), 59.2 (2CH2), 53.6 (4CH2), 23.4 (4CH2) ppm. HRMS m/z calc. C39H42N8O5 [M+l] 703.3351, found [M+l] 703.3369. The synthesis of 3-(acrylamido)-5-nitro-N-phenylbenzamide (58)
Figure imgf000075_0001
58 3-amino-5-nitro-N-phenylbenzamide (53) (1.5 g, 5.831 mmol) was dissolved in anhydrous THF (50 ml) and TEA (1.63 ml, 11.66 mmol, 2 equiv.) was added and the mixture cooled to 0 0C prior to the drop wise addition of 3-chloropropanoyl chloride (1.12 ml, 11.66 mmol, 2 equiv.). The mixture was stirred overnight at room temperature. The solid precipitate was filtered from the reaction, the THF removed in vacuo, and the residue suspended in ether which upon filtration yielded a cream solid (1.223 g, 3.928 mmol, 67%). Rf 0.21 [DCM]. Mp. 197 - 199 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.67 (IH, s, NH), 10.61 (IH, s NH), 8.93 (IH, t, J=2.1 Hz, ArH), 8.55 (IH, t, J=1.8 Hz, ArH), 8.51 (IH, t, J=I.8 Hz, ArH), 7.76 (2H, dd, J=8.6, 1.0 Hz, 2ArH), 7.39 (2H, t, J=8.0 Hz, 2ArH), 7.15 (IH, tt, J=7.4, 1.0 Hz, ArH), 6.48 (IH, dd, 7=77.6», 9.9 Hz, CH=CH2), 6.36 (IH, dd, 7=77.6», 2.6» Hz, CH=CH2), 5.88 (IH, dd, 7=9.9, 2.6» Hz, CH=CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 163.8 (C=O), 163.2 (C=O), 147.9 (ArC), 140.2 (ArC), 138.5 (ArC), 136.7 (ArC), 131.0 (CH=CH2), 128.6 (2ArCH), 128.4 (CH=CH2), 124.5 (ArCH), 124.1 (ArCH), 120.5 (2ArCH), 116.6 (ArCH), 115.9 (ArCH) ppm. IR (neat) υmax 3246, 3084, 2970, 1738, 1650, 1526, 1334, 897 cm"1. HRMS m/z calc. Ci6H13N3O4 [M+l] 312.0976, found [M+l] 312.0965.
The synthesis of 3-(3-(pyrrolidin-l-yl)propanamido)-5-nitro-N-phenylbenzamide (59)
Figure imgf000076_0001
59
3-(acrylamido)-5-nitro-N-phenylbenzamide (58) (1 g, 3.212 mmol) was dissolved in THF (20 ml) under N2 and pyrrolidine (0.54 ml, 6.425 mmol, 2 equiv.) was added and the mixture stirred at room temperature overnight. The solvent and excess pyrrolidine was removed in vacuo and the residue dissolved in EtOAc (50 ml) and washed with 5% NH3 (aq.) (2 x 25 ml), brine (25 ml), and dried over MgSO4. Removal of the solvent yielded a cream solid which was suspended in Et2O (15 ml) and collected by filtration (947.8 mg, 2.478 mmol, 77%). Rf 0.13 [DCM]. Mp. 151- 153 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.69 (IH, s, NH), 10.59 (IH, s, NH), 8.85 (IH, t, J=1.8 Hz, ArH), 8.51 (IH, s, ArH), 8.41 (IH, s, ArH), 7.77 (2H, d, 7=8.6» Hz, 2ArH), 7.38 (2H, t, 7=7.8 Hz, 2ArH), 7.14 (IH, t, 7=7.4 Hz, ArH), 2.75 (2H, t, J=6.9 Hz, CH2), 2.55 (2H, t, J=6.9 Hz, CH2), 2.48 (4H, m, 2CH2), 1.68 (4H, m, 2CH2) ppm.13C NMR (100 MHz, d6-OMSO) δ 171.0 (C=O), 163.2 (C=O), 147.8 (ArC), 140.4 (ArC), 138.6 (ArC), 136.6 (ArC), 128.6 (2ArCH), 124.1 (ArCH), 124.0 (ArCH), 120.5 (2ArCH), 116.1 (ArCH), 115.5 (ArCH), 53.3 (2CH2), 51.2 (CH2), 36.0 (CH2), 23.0 (2CH2) ppm. IR (neat) υmax 3260, 2970, 2803, 1738, 1655, 1597, 1532, 1441, 1342, 1235, 896 cm"1. HRMS m/z calc. C20H22N4O4 [M+l] 383.1714, found [M+l] 383.1727. The synthesis of 3-(3-(pyrrolidin-l-yl)propanamido)-5-amino-N-phenylbenzamide (60)
Figure imgf000077_0001
3-(3-(pyrrolidin-l-yl)acetamido)-5-nitro-N-phenylbenzamide (59) (300 mg, 0.784 mmol) was suspended in absolute EtOH (4 ml) and ammonium formate (197.9 mg, 3.138 mmol, 4 equiv.) and Pd/C (30 mg, 0.1 equiv. w/w) were added and the mixture heated under microwave irradiation at 120 0C for 10 minutes under N2 (pressure ~ 7 bar). The resulting solution was filtered through celite, solvent removed in vacuo and the residue dissolved in EtOAc (100 ml) and washed with 5% NH3 (aq.) (2 x 25 ml), brine (25 ml), dried over K2CO3 and evaporated to a white solid (225.7 mg, 0.640 mmol, 82%). Rf 0.05 [EtOH]. Mp. 196 - 198 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.12 (IH, s, NH), 9.97 (IH, s, NH), 7.74 (2H, d, J=7.7 Hz, 2ArH), 7.32 (2H, t, J=7.9 Hz, 2ArH), 7.16 (IH, s, ArH), 7.10 (IH, s, ArH), 7.06(1H, t, J=7.4 Hz, ArH), 6.72 (IH, s, ArH), 5.35 (2H, br s, NH2), 2.64 (2H, t, 7=7.6» Hz, CH2), 2.45 (6H, m, 3CH2), 1.68 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.0 (C=O), 166.5 (C=O), 148.9 (ArC), 139.7 (ArC), 139.3 (ArC), 136.5 (ArC), 128.4 (2ArCH), 123.2 (ArCH), 119.9 (2ArCH), 107.9 (ArCH), 107.1 (ArCH), 106.4 (ArCH), 53.3 (2CH2), 51.5 (CH2), 36.0 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 3446, 3328, 3280, 3220, 3016, 2970, 2807, 1738, 1574, 1533, 1436, 1365, 1217, 878, 750 cm"1. HRMS m/z calc. C20H24N4O2 [M+l] 353.1972, found [M+ 1] 353.1955.
The synthesis of l,3-bis(3-(phenylcarbamoyl)-5-(3-(pyrrolidin-l-yl)propanamido) phenyl)urea (61)
Figure imgf000078_0001
3-(3-(pyrrolidin-l-yl)propanamido)-5-amino-N-phenylbenzamide (60) (500 mg,
1.419 mmol) was suspended in anhydrous THF (3.55 ml, c = 0.4 M) and CDI (230.1 mg, 1.419 mmol, 1 equiv.) was added and the mixture heated under microwave irradiation at 75 0C for 20 minutes under N2 (pressure ~ 0 bar). Precipitation was observed prior to the mixture being cooled to room temperature and flooded with EtOAc (10 ml). The title compound was isolated by filtration and washed with EtOAc (2 ml), Et2O (2 ml) and oven dried to yield a white solid (245.8 mg, 0.336 mmol, 47%). Semi-Prep HPLC (method G) was conducted (60 mg) to yield a white crystalline solid (21.4 mg). HPLC (method A) 99%, RT 21.01 minutes. Rf 0.21 [5% TEA in MeOH]. Mp. 167 - 169 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.27 (2H, s, 2NH; amide), 10.22 (2H, s, 2NH; amide), 8.90 (2H, s, 2NH; urea), 8.08 (2H, s, 2ArH), 7.74 (4H, d, J=8.6 Hz, 4ArH), 7.65 (2H, s, 2ArH), 7.62 (2H, s, 2ArH), 7.33 (4H, t, J=7.8 Hz, 4ArH), 7.07 (2H, t, J=7.4 Hz, 2ArH), 2.75 (4H, t, J=6.9 Hz, 2CH2), 2.50 (12H, m, 6CH2), 1.68 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.2 (2C=O; amide), 165.8 (2C=O; amide), 152.2 (C=O; urea), 139.8 (2ArC), 139.6 (2ArC), 139.1 (2ArC), 136.5 (2ArC), 128.5 (4ArCH), 123.4 (2ArCH), 120.1 (4ArCH), 112.1 (2ArCH), 112.1 (2ArCH), 111.4 (2ArCH), 53.3 (4CH2), 51.3 (2CH2), 35.8 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C4IH46N8O5 [M+ 1] 731.3664, found [M+l] 731.3674. Anal. CHN calc. C4IH46N8O5-SH2O C, 62.7%, H 6.7%, N 14.3%, found C 62.8%, H 6.4%, N 14.4%. The synthesis of 3-(4-chlorobutanamido)-5-nitro-N-phenylbenzamide (62)
Figure imgf000079_0001
62
3-amino-5-nitro-N-phenylbenzamide (53) (1.5 g, 5.831 mmol) was dissolved in anhydrous THF (50 ml) and TEA (1.63 ml, 11.66 mmol, 2 equiv.) was added and the mixture cooled to 0 0C prior to the drop wise addition of 4-chlorobutanoyl chloride (1.30 ml, 11.66 mmol, 2 equiv.) and stirring of the mixture overnight at room temperature. The solid precipitate was filtered from the reaction, the THF removed in vacuo and the residue suspended in ether (15 ml) which upon filtration yielded a cream solid (1.9392 g, 5.360 mmol, 92%). Rf 0.34 [DCM]. Mp. 213 - 215°C. 1H NMR (400 MHz, d6-OMSO) 10.69 (IH, s, NH), 10.60 (IH, s, NH), 8.85 (IH, t, J=2.1 Hz, ArH), 8.52 (IH, t, J=1.8 Hz, ArH), 8.44 (IH, t, J=1.8 Hz, ArH), 7.78 (2H, dd, J=8.6, 1.0 Hz, 2ArH), 7.39 (2H, t, J=8.0 Hz, 2ArH), 7.15 (IH, tt, J=7.4, 1.1 Hz, ArH), 3.74 (2H, t, J=6.5 Hz, CH2), 2.58 (2H, t, J=7.3 Hz, CH2), 2.09 (2H, m, CH2) ppm. 13C NMR (100 MHz, d6-OMSO) 171.2 (C=O), 163.3 (C=O), 147.9 (ArC), 140.4 (ArC), 138.6 (ArC), 136.7 (ArC), 128.7 (2ArCH), 124.3 (ArCH), 124.1 (ArCH), 120.6 (2ArCH), 116.3 (ArCH), 115.7 (ArCH), 44.9 (CH2), 33.4 (CH2), 27.6 (CH2) ppm. IR (neat) υmax 3247, 3084, 3029, 2970, 1738, 1647, 1522, 1350, 1230, 1216, 895 cm 1. HRMS m/z calc. Ci7H16ClN3O4 [M+l] 362.0908, found [M+l] 362.0900. The synthesis of 3-(4-(pyrrolidin-l-yl)butanamido)-5-nitro-N-phenylbenzamide (63)
Figure imgf000079_0002
3-(4-chlorobutanamido)-5-nitro-N-phenylbenzamide (62) (1.0 g, 2.764 mmol) was dissolved in THF (20 ml) under N2 and pyrrolidine (0.46 ml, 5.528 mmol, 2 equiv.) was added and the mixture stirred at room temperature overnight. TLC showed the reaction was proceeding so a further portion of pyrrolidine (0.23 ml, 2.764 mmol, 1 equiv.) was added and the mixture heated at reflux for 6 hours and stirred overnight at room temperature. The solvent and excess pyrrolidine was removed in vacuo and the residue dissolved in EtOAc (50 ml) and washed with 5% NH3 (aq.) (2 x 25 ml), brine (25 ml), and dried over MgSO4. Removal of the solvent yielded a pale yellow solid which was suspended in Et2O (10 ml) and isolated by filtration (861.1 mg, 2.172 mmol, 79%). Rf 0.17 [DCM]. Mp. 157 - 161 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.58 (2H, s, 2NH), 8.84 (IH, t, 7=2.6» Hz, ArH), 8.50 (IH, t, J=I.8 Hz, ArH), 8.42 (IH, t, J=U Hz, ArH), 7.77 (2H, d, J=8.6 Hz, 2ArH), 7.38 (2H, t Hz, 2ArH), 7.14 (IH, tt, J=7.7, 1.1 Hz, ArH), 2.43 (8H, m, 4CH2), 1.79 (2H, m, CH2), 1.66 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 172.1 (C=O), 163.2 (C=O), 147.8 (ArC), 140.5 (ArC), 138.5 (ArC), 136.6 (ArC), 128.6 (2ArCH), 124.1 (ArCH), 124.0 (ArCH), 120.5 (2ArCH), 116.0 (ArCH), 115.5 (ArCH), 54.9 (CH2), 53.4 (2CH2), 34.4 (CH2), 24.0 (CH2), 23.0 (2CH2) ppm. IR (neat) υmax 3280, 3087, 2959, 2869, 2788, 1738, 1653, 1530, 1442, 1331, 1217, 1017, 895 cm"1. HRMS m/z calc. C2IH24N4O4 [M+l] 397.1876, found [M+l] 397.1860.
The synthesis of 3-(4-(pyrrolidin-l-yl)butanamido)-5-amino-N-phenylbenzamide (64)
Figure imgf000080_0001
64
3-(4-(pyrrolidin-l-yl)butanamido)-5-nitro-N-phenylbenzamide (63) (299.7 mg, 0.756 mmol) was dissolved in EtOH (4 ml), ammonium formate (190.7 mg, 3.024 mmol, 4 equiv.) and Pd/C (30 mg, 0.1 equiv. w/w) was added and the mixture heated under microwave irradiation at 120 0C for 10 minutes (pressure ~ 8 bar). This procedure was repeated as required (801.0 mg, 2.021 mmol). The reactions were combined prior to the solvent being evaporated and the residue dissolved in EtOAc (100 ml) and washed with 5% NH3 (aq.) (2 x 25 ml), brine (25 ml) and dried over MgSO4. The solvent was removed in vacuo affording a white solid (646.9 mg, 1.765 mmol, 87%). Rf 0.12 [MeOH]. Mp. 159 - 161 0C. 1H NMR (400 MHz, J6-DMSO) δ 10.07 (IH, s, NH), 9.75 (IH, s, NH), 7.74 (2H, dd, J=SA, 0.8 Hz, 2ArH), 7.32 (2H, t, J=8.0 Hz, 2ArH), 7.16 (IH, t, J=1.8 Hz, ArH), 7.12 (IH, t, J= 1.6 Hz, ArH), 7.06 (IH, tt, J=7A, 1.0 Hz, ArH), 6.72 (IH, t, J=IJ Hz, ArH), 5.30 (2H, br s, NH2), 2.42 (4H, m, 2CH2), 2.40 (2H, t, J=7.2 Hz, CH2), 2.30 (2H, t, J=7.4 Hz, CH2), 1.74 (2H, m, CH2), 1.67 (4H, m, 2CH) ppm. 13C NMR (100 MHz, J6-DMSO) δ 171.0 (C=O), 166.6 (C=O), 148.8 (ArC), 139.8 (ArC), 139.3 (ArC), 136.5 (ArC), 128.4 (2ArCH), 123.2 (ArCH), 119.9 (2ArCH), 107.9 (ArCH), 107.2 (ArCH), 106.4 (ArCH), 55.0 (CH2), 53.4 (2CH2), 34.4 (CH2), 24.3 (CH2), 23.0 (2CH2) ppm. IR (neat) υmax 3310, 3026, 2970, 2807, 1738, 1648, 1594, 1526, 1437, 1365, 1217, 864 cm"1. HRMS m/z calc. C2iH26N4O2 [M+ 1] 367.2134, found [M+l] 367.2088. The synthesis of l,3-bis(3-(phenylcarbamoyl)-5-(4-(pyrrolidin-l-yl)butanamido) phenyl)urea (65)
Figure imgf000081_0001
3-(4-(pyrrolidin-l-yl)butanamido)-5-amino-N-phenylbenzamide (64) (50.0 mg,
1.364 mmol) was suspended in anhydrous THF (3.40 ml, c = 0.4 M) and CDI (221.2 mg, 1.364 mmol, 1 equiv.) was added and the mixture heated under microwave irradiation at 75 0C for 20 minutes (pressure ~ 0 bar). The peach coloured mixture was cooled to room temperature and flooded with EtOAc (10 ml). The title compound was filtered and washed with EtOAc (2 ml), Et2O (2 ml) and oven dried to yield a white solid (288.9 mg, 0.381 mmol, 56%). Semi-Prep HPLC (method G) was conducted (60 mg) to yield a white crystalline solid (15.7 mg). HPLC (method A) 97%, RT 21.89 minutes. Mp. 176 - 178 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.28 (2H, s, 2NH; amide), 10.09 (2H, s, 2NH; amide), 8.91 (2H, s, 2NH; urea), 8.10 (2H, s, 2ArH), 7.76 (4H, d, J=7.7 Hz, 4ArH), 7.69 (2H, s, 2ArH), 7.65 (2H, s, 2ArH), 7.36 (4H, t, J=7.4 Hz, 4ArH), 7.10 (2H, t, J=7.4 Hz, 2ArH), 2.46 (12H, m, 6CH2), 2.39 (4H, t, J=7.4 Hz, 2CH2), 1.78 (4H, m, 2CH2), 1.69 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.3 (2C=O; amide), 165.9 (2C=O; amide), 152.2 (C=O; urea), 139.7 (2ArC), 139.7 (2ArC), 139.1 (2ArC), 136.4 (2ArC), 128.5 (4ArCH), 123.4 (2ArCH), 120.1 (4ArCH), 112.1 (2ArCH), 112.0 (2ArCH), 111.5 (2ArCH), 55.0 (2CH2), 53.4 (4CH2), 34.3 (2CH2), 24.1 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C43H50N8O5 [M+l] 759.3977, found [M+l] 759.4016. The synthesis of l-(3-amino-5-nitrophenyl)-3-phenylurea (66)
Figure imgf000082_0001
66
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 N2 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. NaHCO3 (aq.) (50 ml), brine (25 ml) prior to drying over MgSO4, and loading to silica for flash chromatography [50% EtOAc in Hexane]. Evaporation of solvent yielded a solid which was collected by filtration from Et2O (20 ml) to yield a bright yellow solid (3.14 g, 12.526 mmol, 65%). Rf 0.50 [50% EtOAc in Hexane]. Mp. 206 - 209 0C. 1H NMR (400 MHz, d6-OMSO) δ 8.87 (IH, s, NH), 8.64 (IH, s, NH), 7.61 (IH, t, 7=2.6» Hz, ArH), 7.45 (2H, dd, J=8.6, 1.0 Hz, 2ArH), 7.29 (2H, tt, J=7.5, 1.8 Hz, 2ArH), 7.03 (IH, t, J=2.1 Hz, ArH), 6.98 (2H, m, ArH), 5.78 (2H, br s, NH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 152.3 (C=O), 150.2 (ArC), 149.0 (ArC), 141.2 (ArC), 139.4 (ArC), 128.8 (2ArCH), 122.1 (ArCH), 118.4 (2ArCH), 108.3 (ArCH), 101.5 (ArCH), 100.0 (ArCH) ppm. IR (neat) υmax 3467, 3282, 3087, 1625, 1516, 1452, 1339, 1225 cm"1. HRMS m/z calc. Ci3H12N4O3 [M+l] 273.0982, found [M+l] 273.0994. The synthesis of l-(3-(acrylamido)-5-nitrophenyl)-3-phenylurea (67)
Figure imgf000082_0002
67 l-(3-amino-5-nitrophenyl)-3-phenylurea (66) (350.0 mg, 1.286 mmol) was dissolved in THF (30 ml) and cooled to 0 0C prior to the addition of TEA (0.36 ml, 2.571 mmol, 2 equiv.) and 3-chloropropanoyl chloride (0.25 ml, 2.571 mmol, 2 equiv.) and the mixture stirred at room temperature overnight. The precipitate was filtered and the solvent removed in vacuo. The resulting oil was dissolved in EtOAc (50 ml), washed with 5% NH3 (aq.) (50 ml) and the solid precipitant filtered prior to washing again with 5% NH3 (aq.) (50 ml), brine (50 ml) and drying over MgSO4. All solvent was removed in vacuo and co-evaporated with Et2O until a solid formed on Et2O addition (5 ml), which was isolated by filtration as a yellow solid (214.8 mg, 0.658 mmol, 51%). Rf 0.29 [50% EtOAc in Hexane]. Mp. 156 - 159 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.59 (IH, s, NH; amide), 9.29 (IH, s, NH; urea), 8.75 (IH, s, NH; urea), 8.28 (IH, t, 7=2.6» Hz, ArH), 8.21 (IH, t, 7=2.6» Hz, ArH), 8.14 (IH, t, J=1.9 Hz, ArH), 7.48 (2H, dd, J=8.4, 0.8 Hz, 2ArH), 7.31 (2H, t, 7=7.9 Hz, 2ArH), 7.01 (IH, t, 7=7.4 Hz, ArH), 6.45 (IH, dd, 7=i7.6», i6>.6> Hz, CH=CH2), 6.33 (IH, dd, 7=77.6», 2.6» Hz, CH=CH2), 5.84 (IH, dd, 7=i6>.6>, 2.6» Hz, CH=CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 163.7 (C=O; amide), 152.3 (C=O; urea), 148.3 (ArC), 141.2 (ArC), 140.2 (ArC), 139.2 (ArC), 131.3 (CH=CH2), 128.8 (2ArCH), 128.0 (CH=CH2), 122.3 (ArCH), 118.5 (2ArCH), 113.8 (ArCH), 107.2 (ArCH), 106.9 (ArCH) ppm. IR (neat) υmax 3285, 1658, 1603, 1528, 1426, 1336, 1209 cm 1. HRMS m/z calc. Ci6H14N4O4 [M+l] 327.1088, found [M+l] 327.1099. The synthesis of l-(3-(3-(pyrrolidin-l-yl)propanamido)-5-nitrophenyl)-3-phenylurea (68)
Figure imgf000083_0001
68 l-(3-(acrylamido)-5-nitrophenyl)-3-phenylurea (67) (200.0 mg, 0.613 mmol) was dissolved in THF (5 ml) and pyrrolidine (0.11 ml, 1.226 mmol, 2 equiv.) added and the mixture stirred at room temperature overnight. The solvent was removed in vacuo and the residue dissolved in EtOAc (30 ml) and washed with 5% NH3 (aq.) (2 x 30 ml), brine (30 ml) and dried over MgSO4. The solvent was removed in vacuo and the resulting solid suspended in Et2O (5 ml) and isolated by filtration to yield a pale yellow solid (175.5 mg, 0.442 mmol, 72%). Rf 0.43 [5% MeOH and 5% TEA in DCM]. Mp. 169 - 172 0C. 1H NMR (400 MHz, d6-OMS0) δ 10.49 (IH, s, NH; amide), 9.26 (IH, s, NH; urea), 8.74 (IH, s, NH; urea), 8.18 (2H, m, 2ArH), 8.03 (IH, t, J=1.8 Hz, ArH), 7.47 (2H, d, J=7.6 Hz, 2ArH), 7.30 (2H, t, J=7.9 Hz, 2ArH), 7.00 (IH, t, J= 7.4 Hz, ArH), 2.74 (2H, t, J=6.9 Hz, CH2), 2.52 (2H, t, J=6.9 Hz, CH2), 2.49 (4H, m, 2CH2), 1.69 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMS0) δ 170.9 (C=O; amide), 152.3 (C=O; urea), 148.3 (ArC), 141.2 (ArC), 140.4 (ArC), 139.2 (ArC), 128.8 (2ArCH), 122.3 (ArCH), 118.5 (2ArCH), 113.5 (ArCH), 106.8 (ArCH), 106.6 (ArCH), 53.4 (2CH2), 51.3 (CH2), 36.2 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 3343, 2955, 2806, 1648, 1529, 1330, 1199 cm"1. HRMS m/z calc. C20H23N5O4 [M+ 1] 398.1823, found [M+l] 398.1838.
The synthesis of l-(3-(3-(pyrrolidin-l-yl)propanamido)-5-aminophenyl)-3- phenylurea (69)
Figure imgf000084_0001
69 l-(3-(3-(pyrrolidin-l-yl)propanamido)-5-nitrophenyl)-3-phenylurea (68) (440.9 mg, 1.110 mmol) was suspended in EtOH (4 ml) prior to the addition of ammonium formate (700 mg, 11.10 mmol, 10 equiv.) and Pd/C (44 mg, 0.1 equiv. w/w) and in two portions the mixture was heated by microwave irradiation at 120 0C for 10 minutes under nitrogen (pressure ~ 8 bar). The portions were combined and filtered through celite, prior to evaporation of the solvent. The residue was dissolved in EtOAc (30 ml) and washed with 5% NH3 (aq.) (3 x 30 ml), brine (30 ml) and dried over MgSO4. Removal of the solvent in vacuo afforded a cream crystalline solid (301.9 mg, 0.822 mmol, 74%). Rf 0.23
[5% MeOH and 5% TEA in DCM]. Mp. 124 - 126 0C. 1H NMR (400 MHz, d6-OMSO) δ
9.77 (IH, s, NH; amide), 8.47 (IH, s, NH; urea), 8.36 (IH, s, NH; urea), 7.42 (2H, d,
J=7.6 Hz, 2ArH), 7.26 (2H, t, J=7.9 Hz, 2ArH), 6.94 (IH, t, J=7.3 Hz, ArH), 6.86 (IH, s, ArH), 6.55 (IH, s, ArH), 6.50 (IH, t, J=1.9 Hz, ArH), 5.03 (2H, s, NH2), 2.68 (2H, t,
7=7.6» Hz, CH2), 2.46 (4H, m, 2CH2), 2.42 (2H, t, 7=7.6» Hz, CH2), 1.69 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.8 (C=O; amide), 152.3 (C=O; urea), 149.2 (ArC), 140.2 (ArC), 140.0 (ArC), 139.8 (ArC), 128.7 (2ArCH), 121.6 (ArCH), 117.9 (2ArCH), 99.2 (ArCH), 99.1 (ArCH), 97.6 (ArCH), 53.3 (2CH2), 51.6 (CH2), 36.1 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 3311, 2954, 2804, 1605, 1538, 1435, 1305, 1203 cm 1. HRMS m/z calc. C20H25N5O2 [M+l] 368.2081, found [M+l] 368.2081. The synthesis of l,3-bis(l-(3-phenylureido)-5-(3-(pyrrolidin-l-yl)propanamido) phenyl)urea (70)
Figure imgf000085_0001
70 l-(3-(3-(pyrrolidin-l-yl)propanamido)-5-aminophenyl)-3-phenylurea (69) (281.5 mg, 0.766 mmol) was dissolved in anhydrous THF (3.26 ml, c = 0.4 M) and CDI (211.6 mg, 1.305 mmol, 1 equiv.) was added under N2 and the mixture heated by microwave irradiation at 75 0C for 20 minutes (pressure ~ 0 bar). A thick white precipitate formed which was suspended in EtOAc (5 ml) and isolated by filtration, washed with EtOAc (2 ml) and Et2O (2 ml) and oven dried to yield a white solid (162.4 mg, 0.213 mmol, 56%). Semi-Prep HPLC (method J) was conducted (60 mg) to yield a white solid (17.8 mg). HPLC (method D) 96%, RT 20.11 minutes. Mp. 178 - 180 0C. 1H NMR (400 MHz, d6- DMSO) δ 10.05 (2H, s, 2NH; amide), 8.80 (2H, s, 2NH; urea), 8.64 (2H, s, 2NH; urea), 8.61 (2H, s, 2NH; urea), 7.47 (8H, m, 8ArH), 7.40 (2H, s, 2ArH), 7.29 (4H, t, J=7.9 Hz, 4ArCH), 6.97 (2H, t, J=7.3 Hz, 2ArCH), 2.75 (4H, t, 7=7.6» Hz, 2CH2), 2.52 (8H, m, 4CH2), 2.49 (4H, t, J=7.1 Hz, 2CH2), 1.71 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6- DMSO) δ 170.1 (2C=O; amide), 152.3 (2C=O; urea), 152.1 (C=O; urea), 140.2 (2ArC), 140.2 (2ArC), 139.9 (2ArC), 139.7 (2ArC), 128.8 (4ArCH), 121.7 (2ArCH), 118.0 (4ArCH), 118.0 (2ArCH), 102.6 (2ArCH), 102.5 (2ArCH), 53.4 (4CH2), 51.5 (2CH2), 36.0 (2CH2), 23.1 (4CH2) ppm. HRMS m/z calc. C4IH48Ni0O5 [M+l] 761.3887, found [M+l] 761.3952. The synthesis of l-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3- (3-nitrophenyl)urea (71)
Figure imgf000086_0001
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 N2 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 CHCI3 (2 ml) and toluene (2 ml) and added to the reaction prior to heating the reaction at 115 0C for 16 hours. The brown solution was cooled to room temperature and quenched with sat. NaHCO3 (aq.) (20 ml) and the reaction mixture extracted into CHCl3 (30 ml), washing with sat. NaHCCh (aq.) (3 x 20 ml), brine (20 ml), dried over MgSO4 and the solvent removed in vacuo to yield a yellow oil which was purified by flash chromatography [5% MeOH and 5% TEA in DCM] to yield a bright yellow solid (412.4 mg, 1.038 mmol, 33%). Rf 0.37 [5% MeOH and 5% TEA in DCM]. Mp. 207 - 209 0C. 1H NMR (400 MHz, d6-OMSO) δ 9.97 (IH, s, NH; amide), 9.16 (IH, s, NH; urea), 8.74 (IH, s, NH; urea), 8.55 (IH, t, J=2.1 Hz, ArH), 7.81 (IH, dd, J=I.6, 8.1 Hz, ArH), 7.70 (IH, dd, J=IJ, 8.1 Hz, ArH), 7.55 (IH, t, J=8.2 Hz, ArH), 7.51 (2H, d, J=8.9 Hz, 2ArH), 7.39 (2H, d, J=8.9 Hz, 2ArH), 2.70 (2H, t, J=7.1 Hz, CH2), 2.47 (4H, m, 2CH2), 2.45 (2H, t, J=7.2 Hz, CH2), 1.68 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.7 (C=O; amide), 152.4 (C=O; urea), 148.1 (ArC), 141.2 (ArC), 134.4 (ArC), 134.1 (ArC), 130.0 (ArCH), 124.2 (ArCH), 119.6 (2ArCH), 119.0 (2ArCH), 116.1 (ArCH), 112.0 (ArCH), 53.4 (2CH2), 51.6 (CH2), 36.0 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 3328, 2808, 1707, 1559, 1511, 1408, 1312, 1201, 1126 Cm HRMS mZz CaIc C20H23N5O4 [M+l] 398.1828, found [M+l] 398.1821. The synthesis of l-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)- 3-(3-aminophenyl)urea (72)
Figure imgf000087_0001
72
In two portions l-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(3- nitrophenyl)urea (71) (313.8 mg, 0.790 mmol) was suspended in absolute EtOH (10 ml) and ammonium formate (497.9 mg, 7.895 mmol, 10 equiv.) and Pd/C (40 mg, 0.1 equiv. w/w) added and the mixture heated by microwave irradiation at 120 0C for 10 minutes under N2 (pressure ~ 7 bar). The resulting solutions were combined and filtered through celite, solvent removed in vacuo and the residue dissolved in EtOAc (50 ml) and washed with 5% NH3 (aq.) (3 x 10 ml), brine (10 ml), dried over MgSO4 and evaporated to a white solid (226.5 mg, 0.616 mmol, 78%). Rf 0.22 [5% MeOH and 5% TEA in DCM]. Mp. 237 - 239 0C. 1H NMR (400 MHz, d6-OMSO) δ 9.95 (IH, s, NH; amide), 8.46 (IH, s, NH; urea), 8.30 (IH, s, NH; urea), 7.47 (2H, d, 7=9.6» Hz, 2ArH), 7.34 (2H, d, 7=9.6» Hz, 2ArH), 6.87 (IH, t, 7=8.6» Hz, ArH), 6.75 (IH, t, 7=2.6» Hz, ArH), 6.55 (IH, dd, 7=8.6», 1.1 Hz, ArH), 6.17 (IH, ddd, 7=7.9, 2.6», 0.6 Hz, ArH), 5.02 (2H, s, NH2), 2.69 (2H, t, J= 7.1 Hz, CH2), 2.46 (4H, m, 2CH2), 2.44 (2H, t, 7=7.2 Hz, CH2), 1.68 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.9 (C=O; amide), 152.4 (C=O; urea), 149.1 (ArC), 140.3 (ArC), 135.1 (ArC), 133.5 (ArC), 129.0 (ArCH), 119.6 (2ArCH), 118.3 (2ArCH), 108.0 (ArCH), 106.0 (ArCH), 103.6 (ArCH), 53.4 (2CH2), 51.6 (CH2), 36.0 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 3279, 3046, 2944, 2797, 1650, 1539, 1393, 1300, 1218 cm"1. HRMS m/z calc. C20H25N5O2 [M+ 1] 368.2086, found [M+l] 368.2086. The synthesis of 5,5'-ureylene-di-(l-(4-(3-(pyrrolidin-l-yl)propanamido)-phenyl)-3- phenylurea) (73)
Figure imgf000088_0001
73 l-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(3-aminophenyl)urea (72) (208.6 mg, 0.568 mmol) was suspended in anhydrous THF (1.42 ml, c = 0.4 M) and CDI (92.1 mg, 0.568 mmol, 1 equiv.) was added under N2 and the mixture heated by microwave irradiation at 75 0C for 20 minutes (pressure ~ 0 bar). A thick white precipitate formed which was suspended in EtOAc (5 ml), isolated by filtration, washed with EtOAc (2 ml) and Et2O (2 ml) and oven dried to yield a white solid (172.7 mg, 0.227 mmol, 80%). Semi- Prep HPLC (method J) was conducted (60 mg) to yield a white solid (27.0 mg). HPLC (method D) 96%, RT 17.81 minutes. Mp. 276 0C dec. 1H NMR (400 MHz, d6- DMSO) δ 9.96 (2H, s, 2NH; amide), 8.67 (2H, s, 2NH; urea), 8.64 (2H, s, 2NH; urea), 8.53 (2H, s, 2NH; urea), 7.70 (2H, s, 2ArH), 7.50 (4H, d, J=8.9 Hz, 4ArH), 7.38 (4H, d, J=8.9 Hz, 4ArH), 7.16 (2H, t, J=8.0 Hz, 2ArH) 7.06 (4H d, J=7.6 Hz, 4ArH), 2.73 (4H, t, 7=7.0 Hz, 2CH2), 2.50 (8H, m, 4CH2), 2.46 (4H, t, J=7.1 Hz, 2CH2), 1.70 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.6 (2C=O; amide), 152.5 (2C=O; urea), 152.3 (C=O; urea), 140.3 (2ArC), 140.1 (2ArC), 134.9 (2ArC), 133.7 (2ArC), 129.0 (2ArCH), 119.6 (4ArCH), 118.6 (4ArCH), 11 1.6 (2ArCH), 111.5 (2ArCH), 107.7 (2ArCH), 53.4 (4CH2), 51.6 (2CH2), 35.9 (2CH2), 23.1 (4CH2) ppm. HRMS m/z calc. C4IH48Ni0O5 [M+l] 761.3887, found [M+l] 761.3889. Anal. CHN C4IH48Ni0O5 calc. C 64.7%, H 6.4%, N 18.4%, found C 64.7%, H 6.2%, N 18.4%. The synthesis of l-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(3-nitrophenyl) urea (74)
Figure imgf000089_0001
74
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 N2 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 CHCI3 (2 ml) and added to the reaction prior to heating the reaction at 115 0C for 16 hours. The brown solution was cooled to room temperature and quenched with sat. NaHCO3 (aq.) (20 ml) and the reaction mixture extracted into CHCI3 (30 ml) which was washed with sat. NaHCO3 (aq.) (3 x 20 ml), brine (20 ml), dried over MgSO4. The solvent was removed in vacuo to yield a yellow oil which was purified by flash chromatography [5% MeOH and 5%TEA in DCM] to yield a bright yellow solid (303.7 mg, 0.764 mmol, 21%). Rf 0.53 [5% MeOH and 5%TEA in DCM]. Mp. 118 - 120 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.07 (IH, s, NH; amide), 9.22 (IH, s, NH; urea), 8.94 (IH, s, NH; urea), 8.60 (IH, t, J=2.2 Hz, ArH), 7.84 (IH, t, J=2.2 Hz, ArH), 7.82 (IH, ddd, J=8.2, 2.3, 0.8 Hz, ArH), 7.69 (IH ,ddd, J=8.1, 1.9, 0.6 Hz, ArH), 7.56 (IH, t, J=8.2 Hz, ArH), 7.26 (IH, dt, J=8.0, 1.4 Hz, ArH), 7.19 (IH, t, J=8.0 Hz, ArH), 7.13 (IH, dt, J=8.1, 1.5 Hz, ArH), 2.73 (2H, t, J=7.0 Hz, CH2), 2.50 (4H, m, CH2), 2.48 (2H, t, 7=7.6» Hz, CH2), 1.69 (4H, m, CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.1 (C=O; amide), 152.3 (C=O; urea), 148.1 (ArC), 141.1 (ArC), 139.7 (ArC), 139.5 (ArC), 130.0 (ArCH), 129.0 (ArCH), 124.2 (ArCH), 116.2 (ArCH), 113.1 (ArCH), 113.0 (ArCH), 112.0 (ArCH), 109.1 (ArCH), 53.4 (2CH2), 51.5 (CH2), 36.0 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 3341, 2955, 2796, 1710, 1645, 1525, 1325, 1201 cm"1. HRMS m/z calc. C20H23N5O4 [M+ 1] 398.1828, found [M+l] 398.1811. The synthesis of l-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(3-aminophenyl) urea (75)
Figure imgf000090_0001
75 l-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(3-nitrophenyl)urea (74) (227.7 mg, 0.573 mmol) was suspended in absolute EtOH (4 ml) and ammonium formate (361.3 mg, 5.730 mmol, 10 equiv.) and Pd/C (30 mg, 0.1 equiv. w/w) were added and the mixture heated by microwave irradiation at 120 0C for 10 minutes under N2 (pressure ~ 7 bar). The resulting solution was filtered through celite, solvent removed in vacuo and the residue dissolved in EtOAc (50 ml) and washed with 5% NH3 (aq.) (3 x 10 ml), brine (10 ml), dried over MgSO4 and evaporated to a white solid (155.3 mg, 0.423 mmol, 74%). Rf 0.23 [5% MeOH and 5% TEA in DCM]. Mp. 99 - 102 0C. 1H NMR (400 MHz, d6- DMSO) δ 10.05 (IH, s, NH; amide), 8.58 (IH, s, NH; urea), 8.29 (IH, s, NH; urea), 7.77 (IH, d, J=LO Hz, ArH), 7.15 (3H, m, 3ArH), 6.88 (IH, t, J=8.0 Hz, ArH), 6.75 (IH, t, J=2.0 Hz, ArH), 6.55 (IH, ddd, J=8.0, 1.8, 0.7 Rz, ArH), 6.18 (IH, ddd, J=8.0, 2.0, 0.7 Hz, ArH), 5.03 (2H, s, NH2), 2.70 (2H, t, 7=7.6» Hz, CH2), 2.46 (6H, m, 3CH2), 1.68 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.1 (C=O; amide), 152.2 (C=O; urea), 149.1 (ArC), 140.2 (ArC), 140.1 (ArC), 139.7 (ArC), 129.0 (ArCH), 128.9 (ArCH), 112.6 (ArCH), 112.4 (ArCH), 108.6 (ArCH), 108.0 (ArCH), 105.9 (ArCH), 103.6 (ArCH), 53.4 (2CH2), 51.6 (CH2), 36.1 (CH2), 23.1 (2CH2) ppm. IR (neat) υmax 3289, 2949, 2804, 1605, 1539, 1481, 1443, 1304, 1209 cm"1. HRMS m/z calc. C20H25N5O2 [M+ 1] 368.2086, found [M+l] 368.2056. The synthesis of 5,5'-ureylene-di-(l-(3-(3-(pyrrolidin-l-yl)propanamido)-phenyl)-3- phenylurea) (76)
Figure imgf000091_0001
l-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-3-(3-aminophenyl)urea (75) (147.6 mg, 0.402 mmol) was dissolved in anhydrous THF (1.0 ml, c = 0.4 M) and CDI (65.2 mg, 0.568 mmol, 1 equiv.) was added under N2 and the mixture heated by microwave irradiation at 75 0C for 20 minutes (pressure ~ 0 bar). A thick white precipitate formed which was suspended in EtOAc (2.5 ml), isolated by filtration, washed with EtOAc (2 ml) and Et2θ (2 ml) and oven dried to yield a white solid (109.3 mg, 0.144 mmol, 71%). Semi-Prep HPLC (method H) was conducted (60 mg) and to yield a white solid (37.0 mg). HPLC (method D) 98%, RT 18.62 minutes. Mp. 276 0C dec. 1H NMR (400 MHz, d6-OMSO) δ 10.06 (2H, s, 2NH; amide), 8.68 (6H, s, 6NH; urea), 7.82 (2H, s, 2ArH), 7.75 (2H, t, J=I.7 Hz, 2ArH), 7.18 (8H, m, 8ArH), 7.06 (4H, m, 4ArH), 2.74 (4H, t, 7=7.6» Hz, 2CH2), 2.50 (8H, m, 4CH2), 2.49 (4H, t, J=7.1 Hz, 2CH2), 1.70 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.1 (2C=O; amide), 152.3 (2C=O; urea), 152.3 (C=O; urea), 140.2 (2ArC), 140.2 (2ArC), 140.0 (2ArC), 139.7 (2ArC), 129.0 (2ArCH), 128.9 (2ArCH), 112.8 (2ArCH), 112.6 (2ArCH), 111.6 (2ArCH), 111.6 (2ArCH), 108.8 (2ArCH), 107.7 (2ArCH), 53.4 (4CH2), 51.5 (2CH2), 36.0 (2CH2), 23.1 (4CH2) ppm. HRMS m/z calc. C4IH48Ni0O5 [M+l] 761.3887, found [M+l] 761.3903. Anal. CHN calc. C 64.7%, H 6.4%, N 18.4%, found C 64.5%, H 6.2%, N 18.2%. The synthesis of l,3-bis(3-ethynylphenyl)urea (77)
Figure imgf000091_0002
To anhydrous THF (21.3 ml) under nitrogen was added 3-ethynylbenzenamine (1.0 g, 0.89 ml, 8.536 mmol, c = 0.4 M) followed by CDI (830.5 mg, 5.122 mmol, 0.6 equiv.) and the mixture heated at reflux for 4 hours. The THF was removed in vacuo and the solid residue dissolved in EtOAc (50 ml) and washed with 1 M HCl (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4. Evaporation of the solvent yielded a white solid (1.0362 g, 3.981 mmol, 93%). Rf 0.27 [20% EtOAc in Hexane]. Mp. 222 - 224 0C. 1H NMR (400 MHz, d6-OMSO) δ 8.82 (2H, s, 2NH), 7.67 (2H, s, 2ArH), 7.41 (2H, dd, J=8.3, 1.0 Hz, 2ArH), 7.29 (2H, t, J=7.9 Hz, 2ArH), 7.09 (2H, d, J=7.6 Hz, 2ArH), 4.14 (2H, s, 2C≡CH) ppm. 13C NMR (100 MHz, d6-OMSO) δ 152.3 (C=O), 139.7 (2ArC), 129.1 (2ArCH), 125.1 (2ArC), 122.0 (2ArCH), 121.0 (2ArCH), 118.9 (2ArCH), 83.4 (2C≡CH), 80.2 (2C≡CH) ppm. IR (neat) υmax 3284, 1547, 1476, 1219 cm"1. HRMS m/z calc. CnH12N2O [M+l] 261.1028, found [M+l] 261.1035. The synthesis of l,3-bis(4-ethynylphenyl)urea (78)
Figure imgf000092_0001
78
To anhydrous THF (10.7 ml) under nitrogen was added 4-ethynylbenzenamine
(500 mg, 4.268 mmol, c = 0.4 M) followed by CDI (415.2 mg, 2.561 mmol, 0.6 equiv.) and the mixture heated at reflux for 4 hours. The THF was removed in vacuo and the solid residue dissolved in EtOAc (75 ml) and washed with 1 M HCl (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4. Evaporation of the solvent yielded a pale yellow solid (556.1 mg, 2.136 mmol, quant.). Rf 0.19 [20% EtOAc in Hexane]. Mp. > 315 0C dec. 1H NMR (400 MHz, d6-OMSO) δ 8.92 (2H, s, 2NH), 7.47 (4H, d, J=8.7 Hz, 2ArH), 7.39 (4H, d, J=8.7 Hz, 2ArH), 4.30 (2H, s, 2C≡CH) ppm. 13C NMR (100 MHz, d6-OMSO) δ 151.9 (C=O), 140.0 (2ArC), 132.3 (2ArCH), 117.9 (2ArCH), 114.7 (2ArC), 83.6 (2C≡CH), 79.3 (2C≡CH) ppm. IR (neat) υmax 3270, 1584, 1539, 1215, 822 cm"1. HRMS m/z calc. Ci7H12N2O [M+l] 261.1022, found [M+l] 261.1026. The synthesis of l,3-bis(2-ethynylphenyl)urea (79)
Figure imgf000093_0001
To anhydrous THF (10.7 ml) under nitrogen was added 2-ethynylbenzenamine (531.1 mg, 4.520 mmol, 0.52 ml, c = 0.4 M) followed by CDI (415.2 mg, 2.561 mmol, 0.6 equiv.) and the mixture heated at reflux for 4 hours at which point the reaction was deemed incomplete by TLC and a further portion of CDI (276.8 mg, 0.4 equiv.) was added, and reflux continued overnight. The THF was removed in vacuo, the solid residue dissolved in EtOAc (75 ml) and washed with 1 M HCl (aq.) (3 x 50 ml), brine (50 ml) and dried over MgSO4. Evaporation of the solvent yielded a beige solid (588.3 mg, 2.260 mmol, quant.). Rf 0.41 [20% EtOAc in Hexane]. Mp. 188 - 190 0C. 1H NMR (400 MHz, d6-OMSO) δ 9.01 (2H, s, 2NH), 7.94 (2H, d, J=8.0 Hz, 2ArH), 7.45 (2H, dd, J=7.7, 1.5 Hz, 2ArH), 7.35 (2H, dd, J=7.1, 1.6 Hz, 2ArH), 7.03 (2H, dt, J=7.6, 1.0 Hz, 2ArH), 4.56 (2H, s, 2C≡CH) ppm. 13C NMR (100 MHz, d6-OMSO) δ 152.4 (C=O), 140.5 (2ArC), 132.6 (2ArCH), 129.3 (2ArCH), 122.5 (2ArCH), 121.0 (2ArCH), 112.0 (2ArC), 86.7 (2C≡CH), 79.9 (2C≡CH) ppm. IR (neat) υmax 3282, 3016, 1739, 1542, 1219, 756 cm"1. HRMS m/z calc. C17H12N2O [M+l] 261.1022, found [M+l] 261.1017. The synthesis of l,3-bis(3-(l-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (80) and l-(3-(l-(4-(3-(pyrrolidin-l- yl)propanamido)phenyl)-lH-l,2,3-triazol-4-yl)phenyl)-3-(3-(l-(4-(acrylamido)phenyl )-lH-l,2,3-triazol-4-yl)phenyl)urea (81)
Figure imgf000093_0002
N-(4-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (10) (149.5 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (2.5 ml) and dH2O (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated to 130 0C by microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a dark green solid (129.6 mg, 0.166 mmol, 87%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield;
(80) as a pale yellow solid (19.79 mg). HPLC (method D) 99%, RT 22.24 minutes. Mp. 228 - 230 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.38 (2H, s, 2NH; amide), 9.25 (2H, s, 2NH; urea), 9.20 (2H, s, 2C=CH), 8.20 (2H, s, 2ArH), 7.92 (4H, d, 7=9.6» Hz, 4ArH), 7.82 (4H, d, 7=9.6» Hz, 4ArH), 7.53 (2H, d, 7=7.4 Hz, 2ArH), 7.47 (2H, d, J=8.6 Hz, 2ArH), 7.40 (2H, t, 7=7.8 Hz, 2ArH), 2.80 (4H, t, 7=7.6» Hz, 2CH2), 2.55 (12H, m, 6CH2), 1.71 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.3 (2C=O; amide), 152.6 (C=O; urea), 147.2 (2C=CH), 140.4 (2ArC), 139.5 (2ArC), 131.7 (2ArC), 130.8 (2ArC), 129.4 (2ArCH), 120.7 (4ArCH), 119.7 (4ArCH), 119.4 (2C=CH), 119.2 (2ArCH), 118.1 (2ArCH), 115.0 (2ArCH), 53.4 (2CH2), 51.3 (CH2), 35.8 (CH2), 23.1 (2CH2) ppm. HRMS m/z calc. C43H46Ni2O3 [M+l] 779.3894, found [M+l] 779.3898.
(81) as a cream solid (6.21 mg). HPLC (method D) 95%, RT 25.91 minutes. Mp. > 259 0C dec. 1H NMR (400 MHz, d6-OMSO) δ 10.42 (IH, s, NH; amide), 10.35 (IH, s, NH; amide), 9.20 (IH, s, NH; urea), 9.18 (IH, s, NH; urea), 9.14 (2H, s, 2C=CH), 8.19 (2H, s, 2ArH), 7.95 (2H, d, 7=9.2 Hz, 2ArH), 7.91 (2H, d, 7=9.6» Hz, 2ArH), 7.91 (2H, d, J=9.1 Hz, 2ArH), 7.82 (2H, d, 7=9.0 Hz, 2ArH), 7.53 (2H, d, 7=7.5 Hz, 2ArH), 7.47 (2H, d, 7=8.6» Hz, 2ArH), 7.41 (2H, t, J=7.6 Hz, 2ArH), 6.48 (IH, dd, 7=77.6», 10.1 Hz, CH=CH2), 6.31 (IH, dd, J=17.0, 2.0 Hz, CH=CH2), 5.81 (IH, dd, 7=i6>.6>, 2.6» Hz, CH=CH2), 2.81 (2H, t, J =6.9 Hz, CH2), 2.56 (6H, m, 3CH2), 1.72 (4H, m, 2CH2) ppm. HRMS m/z calc. C39H37N11O3 [M+l] 708.3159, found [M+l] 708.3142.
The synthesis of l,3-bis(3-(l-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (82) and l-(3-(l-(3-(3-(pyrrolidin-l- yl)propanamido)phenyl)-lH-l,2,3-triazol-4-yl)phenyl)-3-(3-(l-(3-(acrylamido)phenyl )-lH-l,2,3-triazol-4-yl)phenyl)urea (83)
Figure imgf000094_0001
N-(3-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (14) (149.5 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated to 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5ml) and cooled on ice for 10 minutes. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a yellow powder (128.9 mg, 0.165 mmol, 86%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield; (82) as a cream powder (24.33 mg). HPLC (method D) 97%, RT 22.26 minutes.
Mp. 227 - 229 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.42 (2H, s, 2NH; amide), 9.27 (2H, s, 2NH; urea), 9.25 (2H, s, 2C=CH), 8.38 (2H, s, 2ArH), 8.24 (2H, s, 2ArH), 7.63 (4H, m, 4ArH), 7.55 (4H, m, 4ArH), 7.47 (2H, d, J=8.5 Hz, 2ArH), 7.40 (2H, t, J=7.8 Hz, 2ArH), 2.80 (4H, t, 7=7.6» Hz, 2CH2), 2.56 (12H, m, 6CH2), 1.70 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.5 (2C=O; amide), 152.7 (C=O; urea), 147.4 (2C=CH), 140.5 (2ArC), 140.4 (2ArC), 136.9 (2ArC), 130.7 (2ArC), 130.2 (2ArCH), 129.4 (2ArCH), 119.6 (2C=CH), 119.2 (2ArCH), 118.9 (2ArCH), 118.2 (2ArCH), 115.1 (2ArCH), 114.4 (2ArCH), 110.5 (2ArCH), 53.4 (4CH2), 51.3 (2CH2), 35.9 (2CH2), 23.1 (4CH2) ppm. HRMS m/z calc. C43H46Ni2O3 [M+l] 779.3894, found [M+l] 779.3875. (83) as a cream solid (6.36 mg). HPLC (method D) 87%, RT 26.17 minutes. Mp.
> 297 0C dec. 1H NMR (400 MHz, d6-OMSO) δ 10.49 (IH, s, NH; amide), 10.40 (IH, s, NH; amide), 9.24 (IH, s, NH; urea), 9.22 (IH, s, NH; urea), 9.13 (2H, s, 2C=CH), 8.43 (IH, t, J=1.9 Hz, ArH), 8.37 (IH, t, J=I.9 Hz, ArH), 8.22 (2H, m, 2ArH), 7.75 (IH, d, 7=8.6» Hz, ArH), 7.65 (2H, 2ArH), 7.60 (IH, s, ArH), 7.55 (4H, m, 4ArH), 7.45 (2H, d, 7=8.4 Hz, 2ArH), 7.41 (2H, t, 7=7.6 Hz, 2ArH), 6.48 (IH, dd, 7=77.6», 10.1 Hz, CH=CH2), 6.33 (IH, dd, 7=77.6», 1.9 Hz, CH=CH2), 5.82 (IH, dd, J=IOJ, 1.9 Hz, CH=CH2), 2.82 (2H, t, 7=7.6» Hz, CH2), 2.57 (6H, m, 3CH2), 1.71 (4H, m, 2CH2) ppm. HRMS m/z calc. C39H37NnO3 [M+l] 708.3159, found [M+l] 708.3160. The synthesis of l,3-bis(3-(l-(4-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (84)
Figure imgf000096_0001
N-(4-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (18) (157.4 mg, 0.576 mmol, 3 equiv.) was dissolved in 1BuOH (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated to 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a brown solid (142.0 mg, 0.176 mmol, 92%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield a cream powder (42.73 mg). HPLC (method D) 99%, RT 22.61 minutes. Mp. 234 - 236 0C. 1H NMR (400 MHz, d6- DMSO) δ 10.23 (2H, s, 2NH; amide), 9.42 (2H, s, 2NH; urea), 9.20 (2H, s, 2C=CH), 8.22 (2H, s, 2ArH), 7.92 (4H, d, 7=9.6» Hz, 4ArH), 7.83 (4H, d, 7=9.6» Hz, 4ArH), 7.53 (2H, d, 7=7.5 Hz, 2ArH), 7.48 (2H, d, J=8.6 Hz, 2ArH), 7.41 (2H, t, 7=7.8 Hz, 2ArH), 2.53 (12H, m, 6CH2), 2.42 (4H, t, 7=7.3 Hz, 2CH2), 1.81 (4H, m, 2CH2), 1.71 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.4 (2C=O; amide), 152.7 (C=O; urea), 147.3 (2C=CH), 140.5 (2ArC), 139.6 (2ArC), 131.6 (2ArC), 130.8 (2ArC), 129.3 (2ArCH), 120.6 (4ArCH), 119.7 (4ArCH), 1 19.3 (2C=CH), 119.1 (2ArCH), 118.1 (2ArCH), 115.1 (2ArCH), 54.9 (2CH2), 53.4 (4CH2), 34.3 (2CH2), 23.8 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C45H50Ni2O3 [M+l] 807.4207, found [M+l] 807.4179. The synthesis of l,3-bis(3-(l-(3-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (85)
Figure imgf000097_0001
85
N-(3-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (22) (157.4 mg, 0.576 mmol, 3 equiv.) was dissolved in 1BuOH (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated to 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a pale yellow solid (133.3 mg, 0.165 mmol, 86%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield a cream powder (53.0 mg). HPLC (method D) 94%, RT 22.83 minutes. Mp. 232 - 234 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.29 (2H, s, 2NH; amide), 9.45 (2H, s, 2NH; urea), 9.24 (2H, s, 2C=CH), 8.39 (2H, s, 2ArH), 8.25 (2H, s, 2ArH), 7.66 (2H, d, J=8.0 Hz, 2ArH), 7.62 (2H, d, J=8.9 Hz, 2ArH), 7.55 (4H, m, 4ArH), 7.48 (2H, d, J=7.5 Hz, 2ArH), 7.41 (2H, t, J=7.8 Hz, 2ArH), 2.54 (12H, m, 6CH2), 2.43 (4H, t, J=7.3 Hz, 2CH2), 1.81 (4H, m, 2CH2), 1.70 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.5 (2C=O; amide), 152.7 (C=O; urea), 147.4 (2C=CH), 140.6 (2ArC), 140.5 (2ArC), 136.9 (2ArC), 130.7 (2ArC), 130.1 (2ArCH), 129.3 (2ArCH), 1 19.5 (2C=CH), 119.1 (2ArCH), 118.9 (2ArCH), 118.2 (2ArCH), 115.1 (2ArCH), 114.3 (2ArCH), 110.5 (2ArCH), 54.8 (2CH2), 53.4 (4CH2), 34.3 (2CH2), 23.8 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C45H50Ni2O3 [M+l] 807.4207, found [M+l] 807.4209. Anal. CHN C45H50Ni2O3 C 67.0%, H 6.3%, N 20.8%, found C 66.7%, H 6.2%, N 20.5%. The synthesis of l,3-bis(4-(l-(4-(3-(pyrrolidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (86)
Figure imgf000098_0001
N-(4-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (10) (149.5 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (2.5 ml) and dH2O (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated at 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a beige solid (142.0 mg, 0.182 mmol, 85%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield a pale brown powder (28.4 mg) as the 1.84 formate salt. HPLC (method D) 94%, RT 21.41 minutes. Mp. > 315 0C dec. 1H NMR (400 MHz, d6-OMSO) δ 10.38 (2H, s, 2NH; amide), 9.60 (2H, s, 2NH; urea), 9.1 1 (2H, s, 2C=CH), 8.28 (1.84H, s, FA - HCOOH), 7.88 (4H, d, J=9.1 Hz, 4ArH), 7.85 (4H, d, 7=8.7 Hz, 4ArH), 7.82 (4H, d, J=9.1 Hz, 4ArH), 7.64 (4H, d, 7=8.7 Hz, 4ArH), 2.92 (4H, t, J=Zl Hz, 2CH2), 2.70 (8H, m, 4CH2), 2.62 (4H, t, 7=7.6» Hz, 2CH2), 1.76 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.8 (2C=O; amide), 164.3 (FA - HCOOH), 152.5 (C=O; urea), 147.2 (2C=CH), 140.0 (2ArC), 139.3 (2ArC), 131.7 (2ArC), 125.8 (4ArCH), 123.6 (2ArC), 120.4 (4ArCH), 119.7 (4ArCH), 118.3 (4ArCH), 118.3 (2C=CH), 53.2 (4CH2), 50.9 (2CH2), 35.0 (2CH2), 22.9 (4CH2) ppm. HRMS m/z calc. C43H46N12O3 [M+l] 779.3889, found [M+ 1] 779.3904. Anal. CHN C43H46N12O3 calc. C 66.3%, H 6.0%, N 21.6%, found C 66.1%, H 5.8%, N 21.4%. The synthesis of l,3-bis(4-(l-(3-(3-(pyrrolidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (87)
Figure imgf000099_0001
N-(3-azidophenyl)-3-(pyrrolidin-l-yl)propanamide (14) (149.5 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated at 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a yellow solid (97.4 mg, 0.125 mmol, 65%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield a cream solid (16.0 mg). HPLC (method D) 94%, RT 21.73 minutes. Mp. 235 - 237 0C. 1H NMR (400 MHz, d6- DMSO) δ 10.39 (2H, s, 2NH; amide), 9.17 (2H, s, 2NH; urea), 9.15 (2H, s, 2C=CH), 8.36 (2H, s, 2ArH), 7.89 (4H, d, J=8.5 Hz, 4ArH), 7.62 (6H, m, 6ArH), 7.56 (4H, m, 4ArH), 2.79 (4H, t, 7=7.6» Hz, 2CH2), 2.55 (12H, m, 6CH2), 1.71 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.4 (2C=O; amide), 152.4 (C=O; urea), 147.2 (2C=CH), 140.4 (2ArC), 139.8 (2ArC), 136.9 (2ArC), 130.1 (2ArCH), 125.9 (4ArCH), 123.7 (2ArC), 118.7 (2C=CH), 118.6 (2ArCH), 118.3 (4ArCH), 114.2 (2ArCH), 110.3 (2ArCH), 53.3 (4CH2), 51.2 (2CH2), 35.8 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C43H46Ni2O3 [M+l] 779.3889, found [M+l] 779.3892.
The synthesis of l,3-bis(4-(l-(4-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (88)
Figure imgf000100_0001
N-(4-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (18) (157.4 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated at 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a yellow solid (110.4 mg, 0.137 mmol, 71%). Semi-Prep HPLC (method J) was conducted (90.0 mg) to yield a pale yellow solid (29.2 mg) as the 1.60 formate salt. HPLC (method D) 91%, RT 22.03 minutes. Mp. > 315 0C dec. 1H NMR (400 MHz, d6-OMSO) δ 10.22 (2H, s, 2NH; amide), 9.83 (2H, s, 2NH; urea), 9.10 (2H, s, 2C=CH), 8.33 (1.60H, s, FA - HCOOH), 7.85 (12H, m, 12ArH), 7.65 (4H, d, J=8.7 Ηz, 4ArH), 2.72 (8H, m, 4CH2), 2.68 (4H, t, J=7.4 Hz, 2CH2), 2.43 (4H, t, J=7.3 Hz, 2CH2), 1.84 (4H, m, 2CH2), 1.76 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.1 (2C=O; amide), 165.1 (FA - HCOOH), 152.6 (C=O; urea), 147.2 (2C=CH), 140.0 (2ArC), 139.4 (2ArC), 131.6 (2ArC), 125.7 (4ArCH), 123.6 (2ArC), 120.4 (4ArCH), 1 19.6 (2C=CH), 119.6 (4ArCH), 118.3 (4ArCH), 54.5 (2CH2), 53.2 (4CH2), 34.0 (2CH2), 23.3 (2CH2), 22.9 (4CH2) ppm. HRMS m/z calc. C43H46Ni2O3 [M+l] 807.4202, found [M+ 1] 807.4217. The synthesis of l,3-bis(4-(l-(3-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (89)
Figure imgf000101_0001
N-(3-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (22) (157.4 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated at 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a brown solid (132.6 mg, 0.164 mmol, 86%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield a pale yellow solid (31.7 mg). HPLC (method D) 90%, RT 21.80 minutes. Mp. 240 - 242 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.24 (2H, s, 2NH; amide), 9.31 (2H, s, 2NH; urea), 9.15 (2H, s, 2C=CH), 8.36 (2H, s, 2ArH), 7.88 (4H, d, J=8.6 Hz, 4ArH), 7.63 (6H, m, 6ArH), 7.55 (4H, m, 4ArH), 2.50 (12H, m, 6ArH), 2.42 (4H, t, J=7.4 Hz, 2CH2), 1.80 (4H, m, 2CH2), 1.69 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.5 (2C=O; amide), 152.4 (C=O; urea), 147.3 (2C=CH), 140.5 (2ArC), 139.8 (2ArC), 136.8 (2ArC), 130.0 (2ArCH), 125.9 (4ArCH), 123.6 (2ArC), 118.7 (2ArCH), 118.5 (2C=CH), 118.3 (4ArCH), 114.1 (2ArCH), 110.3 (2ArCH), 54.8 (2CH2), 53.4 (4CH2), 34.3 (2CH2), 23.8 (2CH2), 23.0 (4CH2) ppm. HRMS m/z calc. C43H46Ni2O3 [M+l] 807.4202, found [M+l] 807.4211.
The synthesis of l,3-bis-(2-(l-(4-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (90)
Figure imgf000102_0001
N-(4-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (18) (157.4 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated at 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. The precipitate was isolated by centrifugation and washed with ice cold distilled water (2 ml) prior to freeze drying as a red solid (90.7 mg, 0.112 mmol, 59%). Semi-Prep HPLC (method J) was conducted (90.0 mg) to yield a red solid (28.0 mg). HPLC (method D) 97%, RT 23.05minutes. Mp. 130 - 132 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.24 (2H, s, 2NH; amide), 9.79 (2H, s, 2NH; urea), 9.14 (2H, s, 2C=CH), 8.02 (2H, d, J=8.2 Hz, 2ArH), 7.86 (4H, d, J=9.1 Hz, 4ArH), 7.84 (6H, m, 6ArH), 7.37 (2H, t, J=7.8 Hz, 2ArH), 7.19 (2H, t, J=7.5 Hz, 2ArH), 2.63 (8H, m, 4CH2), 2.60 (4H, t, J=7.2 Hz, 2CH2), 2.42 (4H, t, J=7.1 Hz, 2CH2), 1.82 (4H, m, 2CH2), 1.73 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.1 (2C=O; amide), 153.0 (C=O; urea), 145.8 (2C=CH), 139.7 (2ArC), 136.2 (2ArC), 131.2 (2ArC), 128.4 (2ArCH), 128.1 (2ArCH), 123.1 (2ArCH), 123.0 (2ArCH), 120.9 (2C=CH), 120.7 (4ArCH), 120.3 (2ArC), 119.6 (4ArCH), 54.5 (2CH2), 53.2 (4CH2), 34.0 (2CH2), 23.3 (2CH2), 22.9 (4CH2) ppm. HRMS m/z calc. C43H46Ni2O3 [M+l ] 807.4202, found [M+l] 807.4202 The synthesis of l,3-bis-(2-(l-(3-(4-(pyrrolidin-l-yl)butanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (91)
Figure imgf000103_0001
N-(3-azidophenyl)-4-(pyrrolidin-l-yl)propanamide (22) (157.4 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (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 CuSO4.5H2O (2.4 mg, 9.6 μmol, 5 mol %) and the mixture heated at 130 0C under microwave irradiation for 30 minutes. The reaction was cooled to 45 0C, flooded with distilled water (5 ml) and cooled on ice for 10 minutes. No precipitation was observed hence 5% NH3 (aq.) (10 ml) was added inducing precipitation which was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a brown solid (86.0 mg, 0.107 mmol, 56%). Semi-Prep HPLC (method J) was conducted (90.0 mg) to yield a brown solid (15.4 mg). HPLC (method D) 98%, RT 22.35 minutes. Mp. 128 - 130 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.29 (2H, s, 2NH; amide), 9.78 (2H, s, 2NH; urea), 9.16 (2H, s, 2C=CH), 8.37 (2H, s, 2ArH), 8.00 (2H, d, J=8.1 Hz, 2ArH), 7.85 (2H, dd, J=7.8, 1.1 Hz, 2ArH), 7.63 (2H, d, J=7.9 Hz, 2ArH), 7.54 (2H, d, J=8.3 Hz, 2ArH), 7.48 (2H, t, J=8.0 Hz, 2ArH), 7.37 (2H, dt, J=7.7, 1.3 Hz, 2ArH), 7.20 (2H, t, J=7.4 Hz, 2ArH), 2.60 (8H, m, 4CH2), 2.58 (4H, t, J=7.5 Hz, 2CH2), 2.41 (4H, t, J=7.3 Hz, 2CH2), 1.80 (4H, m, 2CH2), 1.71 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 171.3 (2C=O; amide), 153.1 (C=O; urea), 145.9 (2C=CH), 140.5 (2ArC), 136.6 (2ArC), 136.2 (2ArC), 130.0 (2ArCH), 128.5 (2ArCH), 128.2 (2ArCH), 123.2 (2ArCH), 123.2 (2ArCH), 121.1 (2C=CH), 120.4 (2ArC), 119.0 (2ArCH), 114.5 (2ArCH), 110.7 (2ArCH), 54.5 (2CH2), 53.2 (4CH2), 34.0 (2CH2), 23.3 (2CH2), 22.9 (4CH2) ppm. HRMS m/z calc. C43H46Ni2O3 [M+l] 807.4202, found [M+l] 807.4196. The synthesis of 3-(dimethylamino)-N-(4-nitrophenyl)propanamide (92)
Figure imgf000104_0001
3-chloro-N-(4-nitrophenyl)propanamide (7) (5.00 g, 21.869 mmol) was dissolved in dimethylamine (2.0 M in THF, 39.4 ml, 78.730 mmol) and the mixture stirred at 30 °C overnight. The precipitate was filtered from the reaction, and the THF and dimethylamine removed in vacuo. The resulting oil was suspended in 5% NH3 (aq.) (200 ml) and extracted into CHCl3 (3 x 100 ml), washing with brine and drying over MgSO4 to yield a pale yellow solid (5.0493 g, 21.282 mmol, 97%). Rf 0.19 [5% MeOH in CHCl3]. Mp. 96 - 98 0C. 1H NMR (400 MHz, CDCl3) δ 11.66 (IH, s, NH), 8.17 (2H, d, J=9.2 Hz, 2ArH), 7.65 (2H, d, J=9.2 Hz, 2ArH), 2.67 (2H, m, CH2), 2.53 (2H, m, CH2), 2.41 (6H, s, 2CH3) ppm. 13C NMR (100 MHz, CDCl3) δ 171.2 (C=O), 144.7 (ArC), 143.0 (ArC), 125.0 (2ArCH), 119.1 (2ArCH), 54.8 (CH2), 44.3 (2CH3), 33.3 (CH2) ppm. IR (neat) υmax 2946, 2860, 2814, 2769, 1699, 1549, 1504, 1323, 849, 749 cm"1. MS m/z calc. CnH15N3O3 [M+Hf 238.1186, found [M+Hf 238.1192. The synthesis of N-(4-aminophenyl)-3-(dimethylamino)propanamide (93)
Figure imgf000104_0002
3-(dimethylamino)-N-(4-nitrophenyl)propanamide (92) (4.07 g, 17.154 mmol) was dissolved in MeOH (100 ml) and stirred under N2. To this was added ammonium formate (10.82 g, 171.542 mmol, 10 equiv.) and Pd/C catalyst (400 mg, 0.1 equiv. w/w) and the mixture stirred at room temperature overnight. The mixture was filtered through celite, the MeOH removed in vacuo and the residue suspended in chloroform (100 ml), washed with 5% NH3 (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4 to yield a purple oil (3.4745 g, 16.763 mmol, 98%). Rf 0.29 [5% TEA in DCM]. 1H NMR (400 MHz, CDCl3) δ 10.51 (IH, s, NH), 7.28 (2H, d, J=8.7 Hz, 2ArH), 6.62 (2H, d, J=8.7 Hz, 2ArH), 3.54 (2H, br s, NH2), 2.60 (2H, m, CH2), 2.45 (2H, m, CH2), 2.32 (6H, s, 2CH3) ppm. 13C NMR (100 MHz, CDCl3) δ 170.2 (C=O), 142.6 (ArC), 130.2 (ArC), 121.5 (2ArCH), 115.3 (2ArCH), 55.2 (CH2), 44.4 (2CH3), 33.3 (CH2) ppm. IR (neat) υmax 3223, 3016, 2970, 2947, 1739, 1511, 1365, 1230, 1217, 826 cm"1. MS m/z calc. CnH17N3O
[M+Hf 208.1444, found [M+H]+ 208.1452.
The synthesis of N-(4-azidophenyl)-3-(dimethylamino)propanamide (94)
Figure imgf000105_0001
N-(4-aminophenyl)-3-(dimethylamino)propanamide (93) (1.5867 g, 7.6552 mmol) was dissolved in THF (100 ml) and cooled to 0 0C prior to the addition of cone. HCl (12 M, 3.51 ml, 42.1038 mmol, 5.5 equiv.) followed by 1BuONO (2.27 ml, 19.1380 mmol, 2.5 equiv.) and the mixture stirred at 0 0C for 1 hour. After this period NaN3 (1.49 g, 22.9656 mmol, 3 equiv.) was added followed by CAUTIOUS addition of distilled water (10 ml) and the mixture allowed to warm to room temperature and stirred overnight. The reaction was quenched with sat. NaHCO3 (aq.) (10 ml), the THF removed in vacuo and to the resulting aqueous was added sat. NaHCO3 (aq.) (50 ml) and the product extracted into EtOAc (3 x 100 ml) prior to drying over MgSO4 and evaporation to yield a brown oil (1.5206 g, 6.519 mmol, 85%). Rf 0.19 [MeOH]. 1H NMR (400 MHz, CDCl3) δ 10.92 (IH, s, NH), 7.50 (2H, d, J=8.9 Hz, 2ArH), 6.94 (2H, d, J=8.9 Hz, 2ArH), 2.65 (2H, m CH2), 2.49 (2H, m, CH2), 2.37 (6H, s, 2CH3) ppm. 13C NMR (100 MHz, CDCl3) δ 170.6 (C=O), 135.9 (ArC), 135.0 (ArC), 121.2 (2ArCH), 119.4 (2ArCH), 55.1 (CH2), 44.4 (2CH3), 33.4 (CH2) ppm. IR (neat) υmax 3455, 3016, 2970, 2947, 2107, 2071, 1734, 1379, 1219 cm"1. HRMS m/z calc. CnH15N5O [M+H]+ 234.1349, found
Figure imgf000105_0002
The synthesis of N-(4-nitrophenyl)-3-(piperidin-l-yl)propanamide (95)
Figure imgf000105_0003
3-chloro-N-(4-nitrophenyl)propanamide (7) (5.11 g, 22.351 mmol) was dissolved in piperidine (8.0 ml, 78.728 mmol) prior to the addition of THF (25 ml) and the mixture stirred at 30 0C overnight. The precipitate was filtered from the reaction, and the THF and piperidine removed in vacuo. The resulting solid was suspended in 5% NH3 (aq.) (200 ml) with stirring for 1 hour prior to isolation by filtration, washing with distilled water (10 ml) and oven drying to yield a yellow solid (5.5827 g, 20.130 mmol, 90%). Rf 0.36 [MeOH]. Mp. 99 - 101 0C. 1H NMR (400 MHz, CDCl3) δ 12.03 (IH, s, NH), 8.18 (2H, d, J=9.2 Hz, 2ArH), 7.70 (2H, d, J=9.2 Hz, 2ArH), 2.69 (2H, m, CH2), 2.57 (4H, m, 2CH2), 2.54 (2H, m, CH2), 1.72 (4H, m, 2CH2), 1.58 (2H, m, CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 171.3 (C=O), 144.9 (ArC), 142.9 (ArC), 125.1 (2ArCH), 118.8 (2ArCH), 54.0 (CH2), 53.5 (2CH2), 32.5 (CH2), 26.3 (2CH2), 24.1 (CH2) ppm. IR (neat) υmax 3455, 3016, 2970, 2946, 1734, 1365, 1216 cm"1. MS m/z calc. Ci4H19N3O3 [M+H]+ 278.1499, found [M+H]+ 278.1509. The synthesis of N-(4-aminophenyl)-3-(piperidin-l-yl)propanamide (96)
Figure imgf000106_0001
N-(4-nitrophenyl)-3-(piperidin-l-yl)propanamide (95) (5.0584 g, 18.240 mmol) was dissolved in MeOH (100 ml) and stirred under N2. 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. The mixture was filtered through celite, the MeOH removed in vacuo and the residue suspended in chloroform (100 ml), washed with 5% NH3 (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4 to yield a pale brown oil (4.2856 g, 17.327 mmol, 95%). Rf 0.32 [MeOH]. 1H NMR (400 MHz, CDCl3) δ 10.96 (IH, s, NH), 7.31 (2H, d, J=8.7 Hz, 2ArH), 6.62 (2H, d, J=8.7 Hz, 2ArH), 3.56 (2H, br s, NH2), 2.62 (2H, t, J=5.9 Hz, CH2), 2.49 (4H, m, 2CH2), 2.45 (2H, t, J=5.9 Hz, CH2), 1.65 (4H, m, 2CH2), 1.51 (2H, m, CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.2 (C=O), 142.5 (ArC), 130.5 (ArC), 120.9 (2ArCH), 115.4 (2ArCH), 54.3 (CH2), 53.5 (2CH2), 32.4 (CH2), 26.1 (2CH2), 24.2 (CH2) ppm. IR (neat) υmax 3215, 3016, 2970, 2935, 1738, 1511, 1365, 1229, 1217, 826 cm"1. MS m/z calc. C14H21N3O [M+H]+ 248.1757, found [M+H]+ 248.1767. The synthesis of N-(4-azidophenyl)-3-(piperidin-l-yl)propanamide (97)
Figure imgf000106_0002
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 0C prior to the addition of cone. HCl (12 M, 3.25 ml, 38.979 mmol, 5.5 equiv.) followed by 1BuONO (2.10 ml, 17.7175 mmol, 2.5 equiv.) and the mixture stirred at 0 0C for 1 hour. After this period NaN3 (1.38 g, 21.2610 mmol, 3 equiv.) was added followed by CAUTIOUS addition of distilled water (10 ml) and the mixture allowed to warm to room temperature and stirred overnight. The reaction was quenched with sat. NaHCθ3 (aq.) (10 ml), the THF removed in vacuo and to the resulting aqueous was added sat. NaHCθ3 (aq.) (50 ml) and the product extracted into EtOAc (3 x 100 ml) prior to drying over MgSO4 and evaporation to yield a brown oil (1.3795 g). Purification was achieved by flash chromatography [5% - 20% MeOH in DCM] to yield a brown oil (500 mg, 1.829 mmol, 26%). Rf 0.32 [MeOH]. 1H NMR (400 MHz, CDCl3) δ 11.40 (IH, s, NH), 7.54 (2H, d, J=8.8 Hz, 2ArH), 6.97 (2H, d, J=8.8 Hz, 2ArH), 2.66 (2H, m CH2), 2.54 (4H, m, 2CH2), 2.50 (2H, m, CH2), 1.69 (4H, m, 2CH2), 1.55 (2H, m, CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.7 (C=O), 136.1 (ArC), 134.8 (ArC), 120.8 (2ArCH), 119.4 (2ArCH), 54.3 (CH2), 53.6(2CH2), 32.4 (CH2), 26.2 (2CH2), 24.2 (CH2) ppm. IR (neat) υmax 3455, 3016, 2970, 2946, 2115, 1718, 1435, 1375, 1226, 1092, 895 cm"1. HRMS m/z calc. Ci4H19N5O [M+H]+ 274.1662, found [M+H]+ 274.1673. The synthesis of 3-morpholino-N-(4-nitrophenyl)propanamide (98)
Figure imgf000107_0001
3-chloro-N-(4-nitrophenyl)propanamide (7) (5.16 g, 22.569 mmol) was dissolved in morpholine (7 ml, 80.672 mmol) prior to the addition of THF (25 ml) and the mixture stirred at 30 0C overnight. The precipitate was filtered from the reaction, and the THF and morpholine removed in vacuo. The resulting solid was suspended in 5% NH3 (aq.) (200 ml) with stirring for 1 hour prior to isolation by filtration, washing with distilled water (10 ml) and oven drying to yield a yellow solid (6.303 g, 22.568 mmol, quant.). Rf 0.53 [5% MeOH in DCM]. Mp. 98 - 100 0C. 1H NMR (400 MHz, CDCl3) δ 11.37 (IH, s, NH), 8.18 (2H, d, J=9.2 Hz, 2ArH), 7.67 (2H, d, J=9.2 Hz, 2ArH), 3.84 (4H, t, J=4.6 Hz, 2CH2), 2.77 (2H, m, CH2), 2.65 (4H, m, 2CH2), 2.58 (2H, m, CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.8 (C=O), 144.4 (ArC), 143.1 (ArC), 125.1 (2ArCH), 118.8 (2ArCH), 67.0 (2CH2), 53.9 (CH2), 52.7 (2CH2), 32.2 (CH2) ppm. IR (neat) υmax 3463, 3016, 2970, 2946, 1734, 1362, 1233, 1107, 1092 cm"1. MS m/z calc. Ci3H17N3O4 [M+H]+ 280.1292, found [M+H]+ 280.1305.
The synthesis of N-(4-aminophenyl)-3-morpholinopropanamide (99)
Figure imgf000108_0001
3-morpholino-N-(4-nitrophenyl)propanamide (98) (5.17 g, 18.511 mmol) was dissolved in MeOH (100 ml) and stirred under N2. To this was added ammonium formate (11.67 g, 185.11 mmol, 10 equiv.) and Pd/C catalyst (500 mg, 0.1 equiv. w/w) and the mixture stirred at room temperature overnight. The mixture was filtered through celite, the MeOH removed in vacuo and the residue suspended in chloroform (100 ml), washed with 5% NH3 (aq.) (2 x 50 ml), brine (50 ml) and dried over MgSO4 to yield an orange solid (4.5812 g, 18.376 mmol, 99%). Rf 0.36 [5% MeOH in DCM]. Mp. 144 - 146 0C. 1H NMR (400 MHz, CDCl3) δ 10.32 (IH, s, NH), 7.29 (2H, d, J=8.7 Ηz, 2ArH), 6.62 (2H, d, 7=8.7 Hz, 2ArH), 3.77 (4H, t, J =4.6 Hz, 2CH2), 3.58 (2H, br s, NH2), 2.70 (2H, t, J=6.0 Hz, CH2), 2.57 (4H, m, 2CH2), 2.49 (2H, t, J=6.0 Hz, CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 169.7 (C=O), 142.8 (ArC), 130.0 (ArC), 121.1 (2ArCH), 115.4 (2ArCH), 67.0 (2CH2), 54.2 (CH2), 52.8 (2CH2), 32.2 (CH2) ppm. IR (neat) υmax 3413, 3333, 3239, 3016, 2970, 1739, 1365, 1354, 1217, 1229, 1107 cm 1. MS m/z calc. C13H19N3O2 [M+H]+ 250.1550, found [M+H]+ 250.1555. The synthesis of N-(4-azidophenyl)-3-morpholinopropanamide (100)
Figure imgf000108_0002
N-(4-aminophenyl)-3-morpholinopropanamide (99) (1.7669 g, 7.087 mmol) was dissolved in THF (100 ml) and cooled to 0 0C prior to the addition of cone. HCl (12 M, 3.25 ml, 38.979 mmol, 5.5 equiv.) followed by 1BuONO (2.10 ml, 17.7175 mmol, 2.5 equiv.) and the mixture stirred at 0 0C for 1 hour. After this period NaN3 (1.38 g, 21.2610 mmol, 3 equiv.) was added followed by CAUTIOUS addition of distilled water (10 ml) and the mixture allowed to warm to room temperature and stirred overnight. The reaction was quenched with sat. NaHCO3 (aq.) (10 ml), the THF removed in vacuo and to the resulting aqueous was added sat. NaHCO3 (aq.) (50 ml) and the product extracted into EtOAc (3 x 100 ml) prior to drying over MgSO4 and evaporation to yield a brown oil (1.6337 g). Purification was achieved by flash chromatography [1% - 5% MeOH in DCM] to yield a brown oil which solidified at room temperature (841.8 mg, 3.058 mmol, 43%). Rf 0.50 [5% MeOH in DCM]. Mp. 67 - 69 0C. 1H NMR (400 MHz, CDCl3) δ 10.76 (IH, s, NH), 7.52 (2H, d, J=8.8 Hz, 2ArH), 6.97 (2H, d, J=8.8 Hz, 2ArH), 3.81 (4H, t, J=4.6 Ηz, 2CH2), 2.73 (2H, m, CH2), 2.61 (4H, m, 2CH2), 2.53 (2H, m, CH2) ppm. 13C NMR (100 MHz, CDCl3) δ 170.1 (C=O), 135.7 (ArC), 135.1 (ArC), 120.8 (2ArCH), 119.5 (2ArCH), 67.0 (2CH2), 54.1 (CH2), 52.8 (2CH2), 32.2 (CH2) ppm. IR (neat) υmax 3455, 3016, 2970, 2952, 2109, 2079, 1734, 1373, 1215, 1112 cm"1. HRMS m/z calc. Ci3H17N5O2 [M+H]+ 276.1455, found [M+H]+ 276.1469.
The synthesis of l,3-bis(3-(4-(3-(dimethylamino)propanamido)phenylcarbamoyl) phenyl)urea (101)
Figure imgf000109_0001
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- (dimethylamino)propanamide (93) (276.1 mg, 1.332 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. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication, and the organic layer decanted. The residue was dissolved in NH3MeOH (1 M; 2 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a white solid (222.8 mg, 0.328 mmol, 99%). A sample (50 mg) was suspended in boiling MeOH (2 ml) and filtered while hot to yield a white solid (37.5 mg). HPLC (method D) 95%, RT 17.28 minutes. Mp. 322 - 324 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.18 (2H, s, 2NH; amide), 10.00 (2H, s, 2NH; amide), 8.94 (2H, s, 2NH; urea), 7.98 (2H, s, 2ArH), 7.70 (6H, m, 6ArH), 7.56 (6H, m, 6ArH), 7.44 (2H, t, 7=7.9 Hz, 2ArH), 2.57 (4H, t, 7=7.6» Hz, 2CH2), 2.44 (4H, t, 7=6.9 Hz, 2CH2), 2.19 (12H, s, 4CH3) ppm. 13C NMR (100 MHz, d6- DMSO) δ 169.9 (2C=O; amide), 165.3 (2C=O; amide), 152.5 (C=O; urea), 139.7 (2ArC), 135.8 (2ArC), 135.1 (2ArC), 134.4 (2ArC), 128.8 (2ArCH), 121.2 (2ArCH), 120.9 (2ArCH), 120.7 (4ArCH), 119.2 (4ArCH), 117.7 (2ArCH), 55.1 (2CH2), 44.9 (4CH3), 34.7 (2CH2) ppm. HRMS m/z calc. C37H42N8O5 [M+H]+ 679.3351, found [M+H]+ 679.3334. Anal. CHN calc. C37H42N8O5 C 65.5%, H 6.2%, N 16.5%, found C 65.6%, H 6.0%, N 16.3%.
The synthesis of l,3-bis(3-(4-(3-(piperidin-l-yl)propanamido)phenylcarbamoyl) phenyl)urea (102)
Figure imgf000110_0001
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.
The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication, and the organic layer decanted. The residue was dissolved in NH3/MeOH (1 M; 2 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a white solid (221.1 mg,
0.291 mmol, 87%). A sample (50 mg) was suspended in boiling MeOH (2 ml) and filtered while hot to yield a white solid (38.4 mg). HPLC (method D) 100%, RT 18.71 minutes.
Mp. 322 - 324 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.18 (2H, s, 2NH; amide), 10.14 (2H, s, 2NH; amide), 8.95 (2H, s, 2NH; urea), 7.97 (2H, s, 2ArH), 7.70 (6H, m, 6ArH),
7.55 (6H, m, 6ArH), 7.44 (2H, t, 7=7.9 Hz, 2ArH), 2.60 (4H, t, 7=7.6» Hz, 2CH2), 2.45
(4H, t, 7=7.6» Hz, 2CH2), 2.39 (8H, m, 4CH2), 1.51 (8H, m, 4CH2), 1.40 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 167.0 (2C=O; amide), 165.3 (2C=O; amide), 152.5 (C=O; urea), 139.7 (2ArC), 135.8 (2ArC), 135.1 (2ArC), 134.4 (2ArC), 128.7 (2ArCH), 121.2 (2ArCH), 120.9 (2ArCH), 120.8 (4ArCH), 119.2 (4ArCH), 117.7 (2ArCH), 54.4 (2CH2), 53.6 (4CH2), 34.0 (2CH2), 25.6 (4CH2), 24.0 (2CH2) ppm. HRMS m/z calc. C43H50N8O5 [M+2H]++ 380.2025, found [M+2H]++ 380.2018. Anal. CHN calc. C43H50N8O5-H2O C 66.5%, H 6.8%, N 14.4%, found C 66.4%, H 6.6%, N 14.4%. The synthesis of l,3-bis(3-(4-(3-(morpholino)propanamido)phenylcarbamoyl) phenyl)urea (103)
Figure imgf000111_0001
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- morpholinopropanamide (99) (332.4 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. The DMF was removed in vacuo and the resulting oil suspended in CHCl3 (20 ml) with sonication, and the organic layer decanted. The residue was dissolved in NH3/MeOH (1 M; 2 ml) with sonication and flooded with 5% NH3 (aq.) (30 ml). The precipitate was filtered, washed with Et2O (2 x 5 ml) and oven dried to yield a pale purple solid (239.2 mg, 0.314 mmol, 94%). A sample (50 mg) was suspended in boiling MeOH (2 ml) and filtered while hot to yield a white solid (38.8 mg). HPLC (method D) 97%, RT 17.20 minutes. Mp. 313 - 315 0C. 1H NMR (400 MHz, dtf-DMSO) δ 10.19 (2H, s, 2NH; amide), 10.00 (2H, s, 2NH; amide), 8.92 (2H, s, 2NH; urea), 7.98 (2H, s, 2ArH), 7.70
(6H, m, 6ArH), 7.56 (6H, m, 6ArH), 7.44 (2H, t, J=7.9 Hz, 2ArH), 3.59 (8H, t, J =4.5 Hz,
4CH2), 2.64 (4H, t, 7=7.6» Hz, 2CH2), 2.48 (4H, t, J=7.1 Hz, 2CH2), 2.42 (8H, m, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 169.8 (2C=O; amide), 165.3 (2C=O; amide), 152.5 (C=O; urea), 139.7 (2ArC), 135.8 (2ArC), 135.1 (2ArC), 134.4 (2ArC), 128.8 (2ArCH), 121.2 (2ArCH), 120.9 (2ArCH), 120.8 (4ArCH), 119.2 (4ArCH), 117.7 (2ArCH), 66.2 (4CH2), 54.2 (2CH2), 53.0 (4CH2), 33.8 (2CH2) ppm. HRMS m/z calc. C4IH46N8O7 [M+H]+ 763.3562, found [M+H]+ 763.3539. Anal. CHN calc. C4IH46N8O7 C 64.6%, H 6.1%, N 14.7%, found C 64.5%, H 6.0%, N 14.6%.
The synthesis of l,3-bis(3-(l-(4-(3-(dimethylamino)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl)urea (104)
Figure imgf000112_0001
N-(4-azidophenyl)-3-(dimethylamino)propanamide (94) (134.6 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (2.5 ml) and dH2O (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 CuSO4.5H2O (4.8 mg, 19.2 μmol, 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. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a pale brown solid (127.1 mg, 0.166 mmol, 87%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield an off white solid (26.33 mg). HPLC (method D) 98%, RT 20.76 minutes. Mp. 265 - 267 0C. 1H NMR (400 MHz, J6-DMSO) δ 10.31 (2H, s, 2NH; amide), 9.19 (2H, s, 2C=CH), 9.00 (2H, s, 2NH; urea), 8.19 (2H, s, 2ArH), 7.92 (4H, d, 7=9.0 Hz, 4ArH), 7.83 (4H, d, 7=9.0 Hz, 4ArH), 7.54 (2H, d, 7=7.4 Hz, 2ArH), 7.47 (2H, d, 7=8.5 Hz, 2ArH), 7.42 (2H, t, 7=7.8 Hz, 2ArH), 2.61 (4H, t, J=6.8 Hz, 2CH2), 2.51 (4H, t, J=6.8 Hz, 2CH2), 2.21 (12H, s, 4CH3) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.5 (2C=O; amide), 152.6 (C=O; urea), 147.2 (2C=CH), 140.3 (2ArC), 139.5 (2ArC), 131.7 (2ArC), 130.9 (2ArC), 129.4 (2ArCH), 120.6 (4ArCH), 119.7 (4ArCH), 119.4 (2C=CH), 119.2 (2ArCH), 118.1 (2ArCH), 115.1 (2ArCH), 54.9 (2CH2), 44.9 (4CH3), 34.7 (2CH2) ppm. HRMS m/z calc. C39H42Ni2O3 [M+H]+ 727.3575, found [M+H]+
727.3610.
The synthesis of l,3-bis(3-(l-(4-(3-(piperidin-l-yl)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (105)
Figure imgf000113_0001
N-(4-azidophenyl)-3-(piperidin-l-yl)propanamide (97) (157.7 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (2.5 ml) and dH2O (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 CuSO4.5H2O (4.8 mg, 19.2 μmol, 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. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a brown solid (147.6 mg, 0.183 mmol, 95%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield an off white solid (20.06 mg) as the 0.12 formate salt. HPLC (method D) 98%, RT 22.51 minutes. Mp. 164 - 166 0C. 1H NMR (400 MHz, dtf-DMSO) δ 10.35 (2H, s, 2NH; amide), 9.11 (2H, s, 2C=CH), 8.93 (2H, s, 2NH; urea), 8.28 (0.12H, s, HCOOH), 8.11 (2H, s, 2ArH), 7.84 (4H, d, J =9.0 Hz, 4ArH), 7.74 (4H, d, 7=9.6» Hz, 4ArH), 7.47 (2H, d, J=7A Hz, 2ArH), 7.39 (2H, d, J=8.4 Hz, 2ArH), 7.37 (2H, t, J=7.8 Hz, 2ArH), 2.57 (4H, t, 7=7.6» Hz, 2CH2), 2.44 (4H, m, 2CH2), 2.35 (8H, m, 4CH2), 1.45 (8H, m, 4CH2), 1.33 (4H, m, 2CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.6 (2C=O; amide), 152.6 (C=O; urea), 147.2 (2C=CH), 140.3 (2ArC), 139.5 (2ArC), 131.7 (2ArC), 130.9 (2ArC), 129.4 (2ArCH), 120.7 (4ArCH), 119.7 (4ArCH), 119.4 (2C=CH), 119.2 (2ArCH), 118.1 (2ArCH), 115.1 (2ArCH), 54.3 (2CH2), 53.6 (4CH2), 34.0 (2CH2), 25.5 (4CH2), 23.9 (2CH2) ppm. HRMS m/z calc. C45H50Ni2O3 [M+H]+ 807.4202, found [M+Hf 807.4229. The synthesis of l,3-bis(3-(l-(4-(3-(morpholino)propanamido)phenyl)-lH-l,2,3- triazol-4-yl)phenyl) urea (106)
Figure imgf000114_0001
N-(4-azidophenyl)-3-morpholinopropanamide (100) (158.9 mg, 0.577 mmol, 3 equiv.) was dissolved in 1BuOH (2.5 ml) and dH2O (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 CuSO4.5H2O (4.8 mg, 19.2 μmol, 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. The crude product was isolated by filtration and washed with ice cold distilled water (2 ml) and Et2O (2 x 2 ml) prior to oven drying as a brown solid (120.3 mg, 0.148 mmol, 77%). Semi-Prep HPLC (method J) was conducted (90 mg) to yield an off white solid (30.32 mg). HPLC (method D) 98%, RT 20.96 minutes. Mp. 230 - 232 0C. 1H NMR (400 MHz, d6-OMSO) δ 10.30 (2H, s, 2NH; amide), 9.19 (2H, s, 2C=CH), 8.89 (2H, s, 2NH; urea), 8.18 (2H, s, 2ArH), 7.92 (4H, d, 7=9.6» Hz, 4ArH), 7.83 (4H, d, 7=9.6» Hz, 4ArH), 7.55 (2H, dt, J=IA, 7.3 Hz, 2ArH), 7.47 (2H, dt, J=1.6, 8.4 Hz, 2ArH), 7.42 (2H, t, 7=7.7 Hz, 2ArH), 3.59 (8H, t, J=4.6 Hz, 4CH2), 2.66 (4H, t, 7=7.6» Hz, 2CH2), 2.54 (4H, t, J =6.9 Hz, 2CH2), 2.43 (8H, t, J=4.4 Hz, 4CH2) ppm. 13C NMR (100 MHz, d6-OMSO) δ 170.4 (2C=O; amide), 152.5 (C=O; urea), 147.2 (2C=CH), 140.3 (2ArC), 139.5 (2ArC), 131.7 (2ArC), 130.9 (2ArC), 129.4 (2ArCH), 120.7 (4ArCH), 119.8 (4ArCH), 119.4 (2C=CH), 119.2 (2ArCH), 118.1 (2ArCH), 115.1 (2ArCH), 66.2 (4CH2), 54.1 (2CH2), 53.0 (4CH2), 34.0 (2CH2) ppm. HRMS m/z calc. C43H46N12O5 [M+H]+ 811.3787, found [M+H]+ 811.3784. Analytical HPLC Purity Analysis
Ligands 101-106 were analysed by HPLC purity analysis. The results are shown in Table 1 below.
Figure imgf000115_0001
Table 1 : Analytical HPLC purities at 254 nm. Method D - 0.1% FA in Acetonitrile and 0.1% aqueous FA, 5% - 50% organic over 28 minutes; Method F - 0.1% FA in MeOH and 0.1% aqueous FA, 25% - 75% organic over 18 minutes. FRET DNA Preparation and Melting Assessment FRET oligonucleotides (Eurogentec Ltd.) have the following sequences:
F21T: 5 FAM-GGG TTA GGG TTA GGG TTA GGG-TAMRA3'; t-loop: 5 FAM-TAT AGC TATA TTT TTT TATA GCT ATA-TAMRA3'; c-kitl: 5 FAM-AGA GGG AGG GCG CTG GGA GGA GGG GCT-
TAMRA3'; c-kit2: FAM-CCC GGG CGG GCG CGA GGG AGG GGA GG-
TAMRA3'.
The required oligonucleotide (stock solution 120 μl, 20 μM) was suspended in FRET buffer (60 rnM KCl, KCacodilate, pH 7.4, 5340 μl, 400 nM DNA) and heated to 85 0C for 10 minuets prior to cooling to room temperature overnight. DNA was distributed (50 μl) 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. Fluorescence values were recorded at 515 - 545 nm at 0.5 0C intervals as the plate was heated from 30 - 100 0C. 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. In the case of the c-kitl oligonucleotide 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 (ΔTm), 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. In the case of a G-quadruplex forming sequence (F21T - telomeric, c-kitl, c-kit2) the compact size of the G-quadruplex structure results in efficient quenching of the donor fluorophore (FAM) by FRET to the acceptor (TAMRA). This quenching is lost upon the unfolding of the DNA by the application of heat. 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 0C) 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 μM in the wells of the RT-PCR plate as required (50 μM). A buffer negative control was also screened. The data was plotted on a logarithmic concentration scale fitted with a dose response curve to allow the concentration of ligand required to elevate the melting temperature by 100C to be determined (Δconci0°c)- The change in melting temperature at a l μM ligand concentration (ΔTmiμM) was calculated by subtraction of the averaged control value from the averaged 1 μM ligand melting temperature ± the maximum s.d. FRET Based Competition Assay
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.
To the F21T oligonucleotide (50 μl) was added CT-DNA (Sigma-Aldrich; 25 μl; 533.3 μM DNA bp stock in 0.5 mM EDTA/30mM Kcacodalate buffer) to afford the CT- DNA bp concentrations of 0.0 (buffer; ΔTmlμM control), 0.6, 6.0, 60.0 and 120.0 μM in the wells of the RT-PCR plate as required. This represents a 1 :0, 1 : 1, 1 : 10, 1 : 100 and 1:200 ratio of G-quartets: CT-DNA bp. The required ligand (20 mM stock solution in d6- DMSO, stored at -20 0C) was diluted to 1 mM in HPLC grade DMSO (Sigma- Aldrich). This stock was used to make three independent dilutions of concentration 1.0 μM in the wells of the RT-PCR plate as required. A buffer negative control was also screened. The data was normalised against the ΔTmiμM control taken as 100% F21T stabilisation, and expressed as the % reduction in F21T ΔTniiμM 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 (KA).
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)2CGCTG3AG2AG3]3' c-myc: 5'biotin - d[(AG3TG4)2A]3'
Duplex: 5'biotin - d[CGAATTCGTCTCCGAATTCG]3' (hairpin)
The experiments were conducted upon a BIAcore 2000 optical biosensor instrument (BIAcore Inc.). Flow cell 1 was left blank as a reference while flow cells 2 - 4 were immobilized with DNA on a streptavidin-derivatized gold chip (SA chip from
BIAcore) by manual injection of DNA stock solutions (flow rate of l μl/min) until the desired value of DNA response was obtained (~ 380 RUs). Typically, a series of different ligand concentrations (InM to lOμM from 20 mM DMSO stock) were injected onto the chip (flow rate of 50μl/min, 5 - 10 min) until a constant steady-state response was obtained followed by a dissociation period (buffer, 10 min). After every cycle, the chip surface was regenerated (20 sec injection of 10 mM glycine solution, pH 2.0) and multiple buffer injections (1 min), followed by running buffer flow. The data was processed as previously described (White et ai, 2007). In short the reference response was subtracted from the response of each DNA containing cell to give a signal response
(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 Cfree, 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:
Figure imgf000118_0001
where K1 and K2 are the macroscopic binding constants for a two-site binding mode. All ligands in this study were found to fit well to a one-site binding model where K2 = 0. Modified Telomerase Repeat Amplification Protocol (TRAP-LIG) Assay The aim of the TRAP-LIG assay is to assess the ability of a ligand to inhibit the processivity of the telomerase enzyme in vitro, either by direct ligand - telomerase interaction or indirectly by G-quadruplex DNA stabilisation of the telomerase substrate DNA.
Telomerase was extracted as total cellular protein into lysis buffer (10 mM Tris- HcI, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 5 mM β- 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 0C) 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 μM, 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 μg; 5'-AATCCGTCGAGCAGAGTT-S '), TRAP buffer (20 mM Tris-HCl, pH 8.3, 68 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 0.05% v/v Tween-20), bovine serium albumin (0.05 μg) dNTPs (125 μM each) and protein extract (500 μg/sample), which was added (40 μl) to the required ligand concentration or control (10 μl) at 4°C. The elongation step was conducted for 30 min at 300C, followed by 5 min at 94°C and final maintenance of the mixture at 200C. The elongation reaction mixture was introduced to a QIA quick nucleotide purification tube (Qiagen) as per the manufacturer's instructions. In short 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. The sample was resuspended in PCR-grade water (40 μl) prior to the addition (10 μl) of the PCR amplification mix containing the ACX reverse primer (1 μM; 5'-GCGCGG(CTTACC)3CTAACC-3'), the TS forward primer (0.1 μg), TRAP buffer (5 μl), BSA (5 μg), 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, 610C for 1 min and 72°C for 1 min. Samples were separated on a 10% PAGE and visualised by staining with SYBR green (Sigma-Aldrich). Gels were quantified using the gene tool software (Sygene, U.K.) followed by negative control subtraction and normalisation of the positive control to 100%. TRAP LIGEC5o (the ligand concentration required for 50% telomerase inhibition) was not observed hence the percentage inhibition observed at 50 μM is reported. General Cell Culture
Cell lines were supplied by ATCC-LGC Promochem and viability maintained in a Heraeus Hera Cell 240 incubator (37 0C, 5% CO2; 75 cm2 plates supplied by TPP). Cells were removed for experimentation as required. Sterile work was conducted in a Heraeus Hera Safe hood. Dulbecco's Modified Eagles Media (DMEM; Invitrogen) supplemented with foetal bovine serium (10% v/v; Invitrogen), hydrocortisone (0.5 μg/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. Sulforhodamine B (SRB) Cytotoxicity Assay The aim of the SRB assay is to assess the cytotoxic effects of ligand exposure on established cell lines in vitro. The cell lines used in this study represent breast (MCF7) and lung (A549) cancers. The ligands were also exposed to a somatic control cell line (WI38) to assess the level of cancer cell selective cytotoxicity.
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 104 cells/ml MCF7 and WI38; 6.25 x 103 cells/ml A549). This was distributed across a 96 well plate as required (160 μl/well; 4000 cells/well MCF7 and WI38; 1000 cells/well A549; Fisher Scientific) and the plate incubated overnight (37 0C, 5% CO2). The required ligand (20 mM stock solution in d6- DMSO, stored at -20 0C) 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 μM or 0.25 - 50 μM as stated, distributed across the wells of a 96 well plate (40 μl) to afford eight repeats of each exposure. Eight positive and negative controls (media, 40 μl) were also screened and the plate incubated for 96 hours (37 0C, 5% CO2). The media was removed and wells incubated with aqueous trichloroacetic acid (TCA; 10% w/v, 200 μl, 30 minuets; Sigma- Aldrich) on ice. The TCA was removed and the plate washed in distilled water (x 5) and oven dried (> 1 hour, 60 0C). SRB solution (0.4% in 1% acetic acid, 80 μl; 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 μl) and oven drying (> 1 hour, 60 0C). Tris-Base (1OmM, 100 μl) 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 (β-Galactosidase Assay) The aim of the β-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.
MCF7 Cells (1 x 105, 10 ml media, ATCC-LGC Promochem) were exposed to two independent sub-cytotoxic concentrations of the required ligand over a 1 week period (37 0C, 5% CO2), with a biweekly treatment. A media negative control was also screened. Cells were counted upon a Neybauer haemocytometer (Assistant, Germany) and the number of cellular population doublings (n) assessed by the equation n = (log Pn - log P0)/log2 where Pn is the number of cells collected and P0 the initial seeding density. Cells were stained for senescence using the β-galacotosidase staining kit (Cell Signalling Technology) according to the manufacturer's instructions. In short, cells were seeded (1 x 105, 2 ml) in a 6 well plate (Fisher- Scientific) with the required ligand concentration and incubated overnight (37 0C, 5% CO2). 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 0C, 5% CO2). Three independent fields of cells were visualised (20Ox magnification) from both repeats, with the mean percentage of blue senescing cells reported ± s.d.
Chromosomal End-End Fusions by Metaphase Spread
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 105, 10 ml media, ATCC-LGC Promochem) were exposed to two independent sub-cytotoxic concentrations of the required ligand over a 1 week period (37 0C, 5% CO2), with a biweekly treatment. A 0.0 μM (media) negative control was also screened. After 1 week exposure, colcemid (lOOμl, GibcoBRL) was added prior to 1 hour incubation (37 0C, 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. Each sample (20 μl) was dropped following vortexing (30 sec) onto a pre-washed superfrost microscope slide (VWR) which was placed on a hotplate (~2 sees, 8O0C), prior to oven drying overnight (6O0C). Slides were dipped in trypsin (50 ml), rinsed in buffer (Gurr pH 6.8, VWR), stained for G-banding in giemsa solution (5%, 3 min, VWR) and washed twice in buffer (Gurr pH 6.8, VWR) prior to over drying overnight (6O0C). Coverslips were mounted using Eukitt mounting medium and slides visualised at 100Ox magnification. Quantification of chromosome fusion frequency was not achieved following assessment of three independent microscope fields within each duplicate, due to the insignificance of their appearance. Results
These are shown in Tables 2a, 2b, 3a, 3b and 4. 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. Conclusions
We have demonstrated that the ligands 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.
Three attributes were demonstrated by the synthesised ligands in the FRET and SPR assays, including; 1) high G-quadruplex DNA stabilising affinity; 2) optimal G-quadruplex stabilising potency coupled to exceptional G-quadruplex selectivity over duplex DNA with a significant correlation observed between FRET and SPR assays; and 3) inter-G-quadruplex selectivity which was demonstrated for some ligands. In addition the TRAP assay was utilised to demonstrate that these ligands do not act as potent telomerase inhibitors in vitro, however some telomerase inhibition is observed. The short term cytotoxic effects of the ligands were assessed by the
Sulforhodamine B (SRB) assay against two cancer cell lines representing common human tumours and a normal somatic cell control. This assay demonstrated that the most potent cytotoxic ligands synthesised in this study had low micromolar cytotoxicity often associated with enhanced cancer cell selective cytotoxic responses. Some of the more potent G-quadruplex stabilising ligands from each series which demonstrated a significant cytotoxic effect against the breast cancer (MCF7) cell line were selected for a telomere uncapping mechanistic study. These 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.
Kt
Figure imgf000124_0001
Table 2a : FRET Results
Figure imgf000125_0001
Table 2b : FRET Results
Kt
Figure imgf000126_0001
Table 3a : SPR, TRAP and SRB Results
Kt
Figure imgf000127_0001
Table 3b : SPR, TRAP and SRB Results
Figure imgf000128_0001
Table 4 : Cytotoxic Effects of Ligands against breast cancer cell line Where:
• n.d. is not determined
• n.r. is no result observed
• #s is sigmoid curve fitting for the c-kitl G-quadruplex in the FRET experiment
• > greater than
• < less than
• Insignificant is no enhancement of telomere end-end fusion observed upon ligand exposure.

Claims

1. A compound of formula (I):
Figure imgf000129_0001
(I) or a salt, solvate or pro-drug thereof; wherein:
R1 and R2 are independently selected from H, NH2, NH(Ci_6 alkyl), N(Ci_6 alkyl)2, NH(C6_2o aryl), NH(C7-2o alkaryl), NH(C7-2o aralkyl), NHC(O)(Ci_6 alkyl), NHC(O)(C6_20 aryl), NHC(O)(C7-20 alkaryl), NHC(O)(C7-20 aralkyl), NHC(0)(Ci_2o heteroaryl), NHC(0)(C2_2o heterocyclyl), NHC(O)(C2_20 heteroaralkyl), NHC(O)(C3-20 heterocyclylalkyl), NHC(O)(C3-20 alkylheterocyclyl), NO2, CN and C1-10 alkyl;
R3, R4, R5 and R6 are independently selected from the group consisting of H, C(O)OH, C(O)O(C1-6 alkyl), C(O)O(C6_20 aryl), C(O)O(C7_20 aralkyl), C(O)O(C7_20 alkaryl), halo, OH, O(Ci_6 alkyl), NH2, NH(Ci_6 alkyl), N(Ci_6 alkyl)2, NH(C6_20 aryl), NH(C7_2o alkaryl), NH(C7_20 aralkyl), NHC(0)(Ci_6 alkyl), NHC(O)(C6_20 aryl), NHC(0)(C7_2o alkaryl), NHC(O)(C7_20 aralkyl), NHC(0)(Ci_2o heteroaryl), NHC(O)(C2_20 heterocyclyl), NHC(O)(C2_20 heteroaralkyl), NHC(O)(C3-20 heterocyclylalkyl), NHC(O)(C3-20 alkylheterocyclyl), NHC(O)(C6_20 arylamino), NHC(O)(C2_20 heteroarylamino), NHC(O)NH(C1-6 alkyl), NHC(O)NH(C6_20 aryl), NHC(O)NH(C7_20 alkaryl), NHC(O)NH(C7_20 aralkyl), NHC(O)NH(C i_20 heteroaryl), NHC(O)NH(C2_20 heterocyclyl), NHC(O)NH(C2_20 heteroaralkyl), NHC(O)NH(C3_20 heterocyclylalkyl), NHC(0)NH(C3_2o alkylheterocyclyl), NHC(O)NH(C6_20 arylamino), NHC(O)NH(C2_20 heteroarylamino), NO2, CN, C(O)H, C(O)(C1-6 alkyl), C(O)(C6_20 aryl), C(O)(C7_20 alkaryl), C(O)(C7_20 aralkyl), C(O)NH(C1-6 alkyl), C(O)NH(C6_20 aryl), C(O)NH(C7_20 alkaryl), C(O)NH(C7_20 aralkyl), C(O)NH(Ci_20 heteroaryl), C(O)NH(C2_20 heterocyclyl), C(0)NH(C2_2o heteroaralkyl), C(O)NH(C3_20 heterocyclylalkyl), C(O)NH(C3_20 alkylheterocyclyl), Ci_20 heteroaryl, C6-2o aryl(Ci_20 heteroaryl) and (Ci_20 heteroaryl)C6-2o aryl, wherein any of the groups alkyl, aryl, arylamino, heteroarylamino, alkaryl, aralkyl, heteroaryl, heterocyclyl, heteroaralkyl, alkylheterocyclyl and heterocyclylalkyl are optionally independently substituted on the backbone with one or more of the groups, preferably 1, 2, 3, 4, 5 or 6 groups, independently selected from C(O)OH, C(0)0(Ci_6 alkyl), C(O)O(C6_20 aryl), C(O)O(C7_20 aralkyl), C(O)O(C7_20 alkaryl), NHC(O)-CH=CH2, -C≡C-H, halo, OH, O(Ci_6 alkyl), O(C6_20 aryl), 0(C7-2o alkaryl), 0(C7-2o aralkyl), =0, NH2, =NH, NH(CL6 alkyl), N(Ci-6 alkyl)2, =N(d_6 alkyl), NH(C6.20 aryl), NH(C7.20 alkaryl), NH(C7-20 aralkyl), NHC(O)(CL6 alkyl), NHC(O)(C6_20 aryl), NHC(O)(C7_20 alkaryl), NHC(O)(C7_20 aralkyl), NHC(O)(d_20 heteroaryl), NHC(O)(C2_20 heterocyclyl), NHC(0)(C2_2o heteroaralkyl), NHC(O)(C3-20 heterocyclylalkyl), NHC(O)(C3-20 alkylheterocyclyl), NO2, CN, C(O)H, C(0)(d_6 alkyl), C(O)(C6_20 aryl), C(O)(C7_20 alkaryl), C(O)(C7_20 aralkyl), Ci_io alkyl, C2_i0 alkoxyalkyl, C7_20 alkoxyaryl, Ci2_20 aryloxyaryl, C7_2o aryloxyalkyl, Ci_io alkoxy, C6_2o aryloxy, C2_io alkenyl, C2_io alkynyl, C3-2o cycloalkyl, C4_2o (cycloalkyl)alkyl, C7_2o aralkyl, C7_2o alkaryl, Ci_2o heteroaryl and C6_20 aryl;
R7 and R8 are independently selected from the group consisting of H and Ci_6 alkyl; and at least one of R3, R4, R5 and R6 is not H.
2. A compound according to claim 1, wherein at least two of R3, R4, R5 and R6 are not H.
3. A compound according to claim 1 or claim 2, wherein R7 and R8 are H.
4. A compound according to claim 1, claim 2 or claim 3, wherein R1 and R2 are independently selected from H and NHC(O)(C3_i2 heterocyclylalkyl).
5. A compound according to any preceding claim, wherein R1 and R2 are independently selected from the group consisting of NHC(O)CL6 alkyl(C3_6 heterocyclyl), the CL6 alkyl is selected from 1 , 1 -methanediyl, 1,2-ethanediyl, 1,3-propanediyl and 1,4- butanediyl.
6. A compound according to claim 5, wherein the heterocyclyl moiety is pyrrolidinyl.
7. A compound according to any preceding claim, wherein R3, R4, R5 and R6 are independently selected from the group consisting of H, C(O)OH, C(0)NH(C6_io aryl), NHC(O)(C6_10 aryl), NHC(O)NH(C6_10 aryl), CLIO heteroaryl, (CLIO heteroaryl)C6_10 aryl and (C6-Io aryl)Ci_io heteroaryl; wherein the groups aryl and heteroaryl are optionally independently substituted on the backbone with one or more of the groups, preferably 1, 2 or 3 groups, independently selected from NHC(O)-CH=CH2, NH2, NHC(O)(CL3 alkyl), NHC(O)(C6_10 aryl), NHC(O)(C7.10 alkaryl), NHC(O)(C7.10 aralkyl), NHC(O)(C3-8 heteroaryl), NHC(O)(C3_8 heterocyclyl), NHC(O)(C3_8 heteroaralkyl), NHC(O)(C3_8 heterocyclylalkyl), NHC(O)(C3_8 alkylheterocyclyl), Ci-4 alkyl, C2_4 alkenyl, C3_i0 cycloalkyl, C4-10 (cycloalkyl)alkyl, C7_io aralkyl, C7_io alkaryl, CLIO heteroaryl and C6_io aryl.
8. A compound according to any preceding claim, wherein when R3 and R4 are not H, R5 and R6 are both H.
9. A compound according to any preceding claim, wherein when R5 and R6 are not H, R3 and R4 are both H.
10. A compound according to any preceding claim, wherein when R1 and R2 are not H, R3 and R4 are both H.
11. A compound according to any preceding claim, wherein when R1 and R2 are not H, R5 and R6 are not H.
12. A compound according to any preceding claim, wherein when R3, R4, R5 and R6 are not H, they are are independently selected from the group consisting of (a), (b), (c), (d), (e) and (f):
Figure imgf000131_0001
wherein, R9 is selected from the group consisting of-(CH2)n-X, and n is 0, 1, 2, 3, 4 or 5; X is selected from NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2,
Figure imgf000131_0002
R10 and R11 are independently selected from the group consisting of H, NHC(O)-(CH2)n- pyrrolidinyl and NHC(O)-(CH2)n-CH=CH2, and n is 0, 1, 2, 3, 4 or 5; and wherein the wavy line indicates the point of attachment of the group to the rest of the molecule.
13. A compound according to any preceding claim, wherein when R1, R2, R3, R4, R5 and R6 are not H, one or more of these groups may comprise at least one terminal group having the structure (III):
Figure imgf000132_0001
wherein, n is 0, 1, 2, 3, 4 or 5, and X is selected from NH2, NH(Ci-C6 alkyl), N(Ci-C6 alkyl)2,
Figure imgf000132_0002
14. A compound according to claim 13, wherein the compound (I) contains at least one terminal group having the structure (III).
15. A pharmaceutical composition comprising a compound, salt, solvate or pro-drug according to any preceding claim and a pharmaceutically acceptable diluent or carrier.
16. A method of making a pharmaceutical composition according to claim 15, comprising mixing said compound, salt, solvate or pro-drug with a pharmaceutically acceptable diluent or carrier.
17. A compound, salt, solvate or pro-drug according to any of claims 1 to 14, for use in therapy.
18. A method for the treatment of a disease, selected from the group consisting of cardiovascular diseases, disorders of the peripheral and central nervous system, inflammation, urological diseases, developmental disorders, cancer, metabolic diseases, viral, bacterial and endocrinological diseases and disorders of the gastroenterology system in a mammal, comprising administering to a patient in need thereof a therapeutically effective amount of a compound, salt, solvate or pro-drug according to any of claims 1 to 14 or a pharmaceutical composition according to claim 26.
19. A method according to claim 18, wherein the disease is cancer.
20. A method according claim 18, wherein the disease is metastases following a primary tumour.
21. A method according to any of claims 19, wherein the disease is disease selected from the group consisting of 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.
22. Use of a compound, salt, solvate or pro-drug according to any of claims 1 to 14, or a pharmaceutical composition according to claim 15, in the manufacture of a medicament for the prophylaxis or treatment of a disease as defined in any of claims 18 to 21.
23. A compound, salt, solvate or pro-drug according to any of claim 1 to 14, for use in the prophylaxis or treatment of a disease as defined in any of claims 18 to 21.
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