WO2004080481A1 - Novel nph insulin preparations - Google Patents

Novel nph insulin preparations Download PDF

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Publication number
WO2004080481A1
WO2004080481A1 PCT/DK2004/000160 DK2004000160W WO2004080481A1 WO 2004080481 A1 WO2004080481 A1 WO 2004080481A1 DK 2004000160 W DK2004000160 W DK 2004000160W WO 2004080481 A1 WO2004080481 A1 WO 2004080481A1
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Prior art keywords
alkyl
independently selected
aryl
pharmaceutical preparation
preparation according
Prior art date
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PCT/DK2004/000160
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French (fr)
Inventor
Per Balschmidt
Helle Birk Olsen
Niels C. Kaarsholm
Peter Madsen
Palle Jakobsen
Svend Ludvigsen
Gerd Schluckebier
Dorte Bjerre Steensgaard
Anders Klarskov Petersen
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Novo Nordisk A/S
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Priority to JP2006504321A priority Critical patent/JP2006519791A/en
Priority to EP04719932A priority patent/EP1605967A1/en
Publication of WO2004080481A1 publication Critical patent/WO2004080481A1/en
Priority to US11/226,870 priority patent/US20060258561A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids

Definitions

  • This invention relates to novel NPH insulin crystalline preparations comprising high-affinity ligands for the HisBlO Zn 2 ⁇ -sites of the R-state insulin hexamer.
  • Diabetes mellitus is a common disorder of glucose metabolism.
  • the disease is characterized by hyperglycemia and may be classified as type 1 diabetes, sometimes termed insulin- dependent diabetes mellitus, or type 2 diabetes, which is sometimes termed non-insulin- dependent.
  • Insulin dependent diabetes mellitus is characterized by severely diminished or absent production of endogenous insulin. This chronic condition must be treated with daily subcutaneous injections of insulin to maintain a reasonably normal blood glucose level. Similar injections are also common in later stage type 2 diabetes.
  • the use of insulin as a therapeutic agent for this treatment is usually considered one of the outstanding successes of modern medicine.
  • the therapy has its associated problems mainly because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood.
  • the kinetics of absorption from the subcutaneous tissue of fast acting human insulin is too slow and lasts too long to precisely mimic the peak of insulin which is normally secreted within minutes in response to carbohydrate ingestion during a meal. More importantly, the action profile of the most commonly used crystalline long-acting basal insulin show a spike, i.e. a high concentration of relatively short duration of insulin in the blood, within a few hours after injection. Also, the total duration of action is somewhat too short for once daily injection, and the absorption times show some fluctuation from day to day leading to poor reproducibil- ity of the basal insulin level.
  • the most widely used long acting insulin is a neutral crystalline suspension, i.e. NPH insulin, comprising a crystalline complex of human insulin (or an analogue thereof), zinc ion and protamine sulphate together with a suitable preservative such as phenol, m-cresol, or mixtures thereof.
  • NPH insulin neutral crystalline suspension
  • the preparations usually contain a buffering substance such as phosphate and an isotonicity agent such as glycerol, mannitol or sodium chloride.
  • the delayed action is believed to originate from the rate-limiting dissolution of the NPH-insulin crystals in the subcutaneous tissue fluids.
  • the main determinant for the spike in the action profile as well as the total length of duration of action is thought to be the inherent solubility of the NPH- insulin crystal in the subcutis.
  • the poorly reproducible absorption times often encountered with NPH insulin are thought to originate from difficulties in resuspending the vial before injection which may lead to variations in the dose actually delivered from one injection to another.
  • the rate of dissolution at the site of injection depends to some extent on the local blood flow which is influenced by e.g. exercise and temperature adding further elements to the poorly reproducible absorption times. Taken together, these factors are considered to limit the inherent quality of the action profile obtained from NPH-insulin.
  • NPH-insulin (crystalline preparations) may be prepared in the presence of certain high-affinity ligands for the HisB10 Zn 2+ -sites of the R-state insulin hexamer.
  • Preparation of NPH-insulin in the presence of high-affinity ligand results in crystalline NPH-insulin suspensions that are absorbed more slowly from subcutis than regular NPH-insulin.
  • the resulting action profile is longer and the spike is less pronounced than observed with regular NPH-insulin.
  • the novel NPH-insulin also shows better physical and chemical stability than regular NPH-insulin.
  • Figure 1 is a graphic representation of glucose utilization after subcutaneous injection of a NPH preparation showing the effects of stoichiometric and excess concentration of 4-[3-(1 H-Tetrazol-5-yl)-carbazol-9-ylmethyl]-benzoic acid compared to Zn 2+ .
  • Halogen designates an atom selected from the group consisting of F, Cl, Br and I.
  • C ⁇ -C 6 -alkyl represents a saturated, branched or straight hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, feft-butyl, n-pentyl, isopentyl, neopentyl, fert-pentyl, n-hexyl, isohexyl and the like.
  • CrC 6 -alkylene represents a saturated, branched or straight bivalent hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methylene, 1 ,2-ethylene, 1 ,3-propylene, 1 ,2-propylene, 1 ,4-butylene, 1 ,5- pentylene, 1 ,6-hexylene, and the like.
  • C 2 -C 6 -alkenyl represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one double bond.
  • groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, iso-propenyl, 1,3-buta- dienyi, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 1-pentenyl, 2-pentenyl, 3- pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 2,4-hexadienyl, 5- hexenyl and the like.
  • C 2 -C 6 -alkyny represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one triple bond.
  • groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4- hexynyl, 5-hexynyl, 2,4-hexadiynyl and the like.
  • CrC 6 -alkoxy refers to the radical -O-CrCe-alkyl, wherein C ⁇ -C 6 -alkyl is as defined above. Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tetf-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.
  • C 3 -C 8 -cycloalkyl represents a saturated, carbocyclic group having from 3 to 8 carbon atoms. Representative examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
  • C 4 - 8 -cycloalkenyl represents a non-aromatic, carbocyclic group having from 4 to 8 carbon atoms containing one or two double bonds.
  • Representative examples are 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl, 3- cyclohexenyl, 2-cycloheptenyl, 3-cycloheptenyl, 2-cyclooct ⁇ nyl, 1 ,4-cyclooctadienyl and the like.
  • heterocyclyl represents a non-aromatic 3 to 10 membered ring containing one or more heteroatoms selected from nitrogen, oxygen and sulphur and optionally containing one or two double bonds. Representative examples are pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, aziridinyl, tetrahydrofuranyl and the like.
  • aryl as used herein is intended to include carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9 to 14 membered bi- and tricyclic, carbocyclic, aromatic ring systems.
  • Aryl is also intended to include the partially hydrogenaled derivatives of the ring systems enumerated above.
  • Non-limiting examples of such partially hydrogenaled derivatives are 1,2,3,4-tetrahydronaphthyl, 1,4- dihydronaphthyl and the like.
  • arylene as used herein is intended to include divalent, carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9 to 14 membered bi- and tricyclic, divalent, carbocyclic, aromatic ring systems. Representative examples are phenylene, biphenylylene, naphthylene, anthracenylene, phenanthrenylene, fluorenylene, indenylene, azulenylene and the like. Arylene is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above.
  • Non-limiting examples of such partially hydrogenated derivatives are 1 ,2,3,4-tetrahydronaphthylene, 1 ,4-dihydronaphthylene and the like.
  • aryloxy denotes a group -O-aryl, wherein aryl is as defined above.
  • aroyl as used herein denotes a group -C(O)-aryl, wherein aryl is as defined above.
  • heteroaryl as used herein is intended to include aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as 5 to 7 membered monocyclic and 8 to 14 membered bi- and tricyclic aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur.
  • Representative examples are furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1 ,2,3-triazolyl, 1 ,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, 1 ,3,5- triazinyl, 1,2,3- oxadiazolyl, 1,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazolyl, 1,2,4- thiadiazolyl, 1 ,2,5-thiadiazolyl, 1 ,3,4-thiadiazolyl
  • Heteroaryl is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above.
  • Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.
  • heteroarylene as used herein is intended to include divalent, aromatic, heterocyc- lic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as 5 to 7 membered monocyclic and 8 to 14 membered bi- and tricyclic aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur.
  • Heteroaryl is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above.
  • Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydro- benzofuranylene, pyrrolinylene, pyrazolinylene, indolinylene, oxazolidinylene, oxazolinylene, oxazepinylene and the like.
  • ArG1 as used herein is intended to include an aryl or arylene radical as applicable, where aryl or arylene are as defined above but limited to phenyl, biphenylyl, naphthyl, anthra- cenyl, phenanthrenyl, fluorenyl, indenyl, and azulenyl as well as the corrresponding divalent radicals.
  • ArG2 as used herein is intended to include an aryl or arylene radical as applicable, where aryl or arylene are as defined above but limited to phenyl, biphenylyl, naphthyl, fluorenyl, and indenyl, as well as the corrresponding divalent radicals.
  • Het1 is intended to include a heteroaryl or heteroarylene radical as applicable, where heteroaryl or heteroarylene are as defined above but limited to furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3- triazinyl, 1 ,2,4-triazinyl, 1,3,5- triazinyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazoly
  • Het2 as used herein is intended to include a heteroaryl or heteroarylene radical as applicable, where heteroaryl or heteroarylene are as defined above but limited to furyl, thienyl pyrrolyl, pyrazolyl, 3-oxopyra ⁇ olyl, oxazolyl, thia ⁇ olyl, imida ⁇ olyl, isoxa ⁇ olyl, isothia ⁇ olyl 1,2,3-tria ⁇ olyl, 1,2,4-tria ⁇ olyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyra ⁇ inyl, 1,2,3 triazinyl, 1 ,2,4-triazinyl, 1,3,5- triazinyl, 1 ,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl 1 ,3,4-oxadiazolyl, 1 ,
  • Het3 is intended to include a heteroaryl or heteroarylene radical as applicable, where heteroaryl or heteroarylene are as defined above but limited to furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyridyl, tetrazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, quinolyl, isoqui- nolyl, quinoxalinyl, carbazolyl, thiazolidinyl, 2-thiooxothiazolidinyl, as well as
  • Aryl-CrCe-alkyl is intended to mean C C 6 - alkyl or C 2 -C 6 -alkenyl as defined above, substituted by an aryl or heteroaryl as defined above, for example:
  • protamine refers to a mixture of strongly basic proteins usually obtained from fish sperm
  • protamine can refer to a relatively salt-free preparation of the proteins, sometimes termed protamine base.
  • protamine also refers to preparations comprising salts of the proteins. Even though concentrations are commonly given as concentration of protamine sulphate in this application, the person skilled in the art will readily be able to convert this to other protamine preparations.
  • treatment means the management and care of a patient for the purpose of combating a disease, disorder or condition.
  • the term is intended to include the delaying of the progression of the disease, disorder or condition, the alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition.
  • the patient to be treated is preferably a mammal, in particular a human being.
  • fragment as used herein is intended to mean a bivalent chemical group
  • Neutral amino acid as used herein is intended to mean any natural (codable) and non-natural amino acid, including ⁇ - or ⁇ -aminocarboxylic acids, including D-isomers of these (when applicable) without charges at physiologically relevant pH in the side chain, such as glycine, alanine, ⁇ -alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, aspargine, glutamine, cysteine, methionine, 3-aminobenzoic acid, 4-aminobenzoic acid or the like.
  • positively charged group as used herein is intended to mean any pharmaceutically acceptable group that contains a positive charge at physiologically relevant pH, such as amino (primary, secondary and tertiary), ammonium and guanidino groups.
  • amino acid as used herein is intended to mean mean any natural (codable) and non-natural ⁇ -aminocarboxylic acid, including D-isomers of these.
  • ⁇ amino acid as used herein is intended to mean any ⁇ -aminocarboxylic acid, such as ⁇ -alanine, isoserine or the like.
  • groups of compounds such as car- boxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imi- dazoles, triazoles, 4-cyano-1 ,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thia- zolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, naphthoic acids and salicylic acids, these groups of compounds are intended to include also derivatives of the compounds from which the groups take their name.
  • insulin refers to naturally produced insulin or recombinantly produced insulin.
  • Recombinant insulin may be produced in any suitable host cell, for example the host cells may be bacterial, fungal (including yeast), insect, animal or plant cells.
  • analogue of human insulin as used herein (and related expressions) is meant human insulin in which one or more amino acids have been deleted and/or replaced by other amino acids, including non-codeable amino acids, or human insulin comprising additional amino acids, i.e. more than 51 amino acids, such that the resulting analogue possesses insulin activity
  • insulin derivative refers to human insulin or an analogue thereof in which at least one organic substituent is bound to one or more of the amino acids.
  • desBSO and the like as used herein is intended to mean meant a natural insulin B chain or an analogue thereof lacking the B30 amino acid residue.
  • amino acid residues are indicated in the three letter amino acid code or the one letter amino code.
  • B1 is intended to mean the amino acid residue in position 1 in the B chain of insulin or analogue thereof (counted from the N-terminal end) and the amino acid residue in position 1 in the A chain of insulin or analogue thereof (counted from the N-terminal end), respectively.
  • phenolic compound or similar expressions as used herein refers to a chemical compound in which a hydroxyl group is bound directly to a benzene or substituted benzene ring. Examples of such compounds include, but are not limited to, phenol, o-cresol, m-cresol and p-cresol.
  • physiologically relevant pH as used herein is intended to mean a pH of about 7.1 to 7.9.
  • the term "putative insulin hexamer" or similar expressions as used herein is refers to six insulin molecules which may combine to form an insulin hexamer.
  • the chemical environment the insulin is in may determine that the insulin is not always in hexamer form.
  • a ratio of e.g. 2 moles of Zinc ions per mole putative insulin hexamer corresponds to a ratio of 1 mole per 3 moles insulin monomer regardless of the state of the insulin.
  • Regular NPH-insulin is a crystalline complex between the R-state insulin hexamer and protamine (usually originating from salmon or herring).
  • the hexamer component of the complex normally has additional small molecules bound to the known binding sites of the R6 insulin, i.e., preservative molecules such as phenol or m-cresol bind to six hydrophobic pockets formed in the dimer-dimer interfaces and anions from added buffers and salts (e.g. chloride) may bind to the two His 610 Zn 2+ sites residing on the 3-fold symmetry axis of the hexamer.
  • preservative molecules such as phenol or m-cresol
  • anions such as chloride bind to the R-state His Zn .2+ -site with modest affinity hence providing I little stabilization of the hexamer.
  • ligands with substantially higher affinity for the H ,BIO 2n 2+ -site may be found and characterized by using a fluorescence based competit ; ⁇ on assay which is based on the displacement of 5-(4- dimethylaminobenzylidene)-thiazolidine-2,4-dione from the R-state His 810 Zn 2+ -site by the incoming ligand in question.
  • the present invention is based on the discovery that NPH-insulin crystals may be formed in the presence of certain high-affinity ligands for the His 810 Zn 2+ sites of the R-state hexamer.
  • the ligands When the ligands are present along with insulin, Zn 2+ , and optionally phenolic preservative, buffers and isotonicity agents, the NPH-insulin crystals still form upon combination with protamine.
  • regular NPH-insulin crystals without presence of high-affinity ligands for the His 810 Zn 24 sites of the R-stafe hexamer may be formed initially and the ligand may then be incorporated by subsequent addition of the ligand to the crystalline suspension.
  • the novel NPH-insulin complex has several advantages over regular NPH-insulin: When the crystalline suspension is injected subcutaneously into pigs, the absorption half-life is significantly increased compared to regular NPH-insulin (see example 1011). Moreover, the action profile of the novel NPH-preparation is longer and smoother than that obtained with regular NPH- insulin. Finally, the physical and chemical stability is significantly enhanced over the reference preparation.
  • Suitable ligands according to this invention are characterized by a) having high affinity to His B1 ° Zn + site of the R-state hexamer (e.g. K ⁇ 10 ⁇ M) as measured in the TZD-assay for quantitation of ligands binding to the R-state His 610 Zn 2+ or the 4H3N-assay and b) being capable of forming NPH crystals when included along with the zinc-insulin in the preparation, i.e. the presence of the bound ligand does not impede normal complex formation with protamine (co-crystallization mode).
  • the R-state hexamer e.g. K ⁇ 10 ⁇ M
  • the regular insulin-protamine crystalline complex without presence of high-affinity ligands for the His 810 Zn + sites of the R-state hexamer may be formed initially and the ligand incorporated subsequently by addition of the ligand to the crystalline suspension (soaking mode)
  • the present invention thus provides in embodiment 1 a pharmaceutical preparation comprising
  • ® ligand that binds reversibly to a His 810 Zn 2+ site of an R-state insulin hexamer, wherein said ligand is selected from the group consisting of carboxylates, dithiocar- boxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imida ⁇ oles, tria- zoles, 4-cyano-1 ,2,3-triazoIes, benzimidazoles, benzotriazoles, purines, thymines, thiazolidinediones, fetrazoles, 5-mercaptotetra ⁇ oles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, naphthoic acids and salicylic acids, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
  • Embodiment 2 A pharmaceutical preparation according to embodiment 1 wherein the insulin preparation comprises 60 to 3000 nmol/ml of insulin.
  • Embodiment 3 A pharmaceutical preparation according to embodiment 2 wherein the insulin preparation comprises 240 to 1200 nmol/ml of insulin.
  • Embodiment 4 A pharmaceutical preparation according to embodiment 3 wherein the insulin preparation comprises about 600 nmol/ml of insulin.
  • Embodiment 5 A pharmaceutical preparation according to any one of the embodiments 1 to 4.
  • the insulin is selected from the group consisting of human insulin, an analogue of human insulin, a derivative of human insulin, and combinations of any of these.
  • Embodiment 6 A pharmaceutical preparation according to embodiment 5 wherein the insulin is an analogue of human insulin selected from the group consisting of i.An analogue wherein position B28 is Asp, Glu, Lys, Leu, Val, or Ala and position B29 is Lys or Pro; ii.An analogue wherein position B3 is Lys and position B29 is Glu; and iii.des(B28-B30), des(B27) or des(B30) human insulin.
  • Embodiment 7 A pharmaceutical preparation according to embodiment 6, wherein the insulin is an analogue of human insulin wherein position B28 is Asp or Lys, and position B29 is Lys or Pro.
  • Embodiment 8 A pharmaceutical preparation according to embodiment 6 wherein the insulin is des(B30) human insulin.
  • Embodiment 9 A pharmaceutical preparation according to embodiment 5 wherein the insulin is a derivative of human insulin having one or more lipophilic substituents.
  • Embodiment 10. A pharmaceutical preparation according to embodiment 9 wherein the insulin derivative is selected from the group consisting of B29-N ⁇ -myristoyl-des(B30) human insulin, B29-N ⁇ -palmitoyl-des(B30) human insulin, B29-N ⁇ -myristoyl human insulin, B29-N ⁇ - palmitoyl human insulin, B28-N ⁇ -myristoyl Lys 628 Pro 629 human insulin, B28-N ⁇ -palmitoyl Lys 828 Pro 829 human insulin, B30-N ⁇ -myristoyl-Thr 829 Lys 830 human insulin, B30-N ⁇ -palmitoyl- Thr ⁇ Lys 830 human insulin, B29-N ⁇ -(N-palmitoyl- ⁇ -glutamyl)-des
  • Embodiment 12 A pharmaceutical preparation according to any one of the embodiments 1 to 11 wherein the protamine is protamine sulphate.
  • Embodiment 13 A pharmaceutical preparation according to embodiment 13 wherein the concentration of protamine sulphate is from 0.05-3 mg/mL.
  • Embodiment 14 A pharmaceutical preparation according to embodiment 14 wherein the concentration of protamine sulphate is from 0.1-0.6 mg/mL.
  • Embodiment 15 A pharmaceutical preparation according to any one of the embodiments 1 to 15 wherein the amount of zinc ions is 2-6 moles per mole putative insulin hexamer.
  • Embodiment 16 A pharmaceutical preparation according to embodiment 16 wherein the amount of zinc ions is 2 to 3 moles per mole putative insulin hexamer.
  • Embodiment 17 A pharmaceutical preparation according to any one of the embodiments 1 to 17 wherein the ratio of ligand that binds reversibly to a His 810 Zn 2+ site of an R-state insulin hexamer to zinc ions is 1:3 to 3:1.
  • Embodiment 18 A pharmaceutical preparation according to embodiment 18 wherein the ratio of ligand that binds reversibly to a His 810 Zn 2+ site of an R-state insulin hexamer to zinc ions is 1:2 to 2:1.
  • Embodiment 19 A pharmaceutical preparation according to embodiment 19 wherein the ratio of ligand that binds reversibly to a His 810 Zn 2+ site of an R-state insulin hexamer to zinc ions is 1 :.2 to 1.2:1.
  • Embodiment 20 A pharmaceutical preparation according to any one of the embodiments 1 to 20 wherein the ligand that binds reversibly to a His 810 Zn 2+ site of an R-state insulin hexamer is a chemical structure selected from the group consisting of carboxylates, dithiocarboxy- lates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles, 4- cyano-1 ,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thymines, thiazolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines, N-hydroxyazoles, hydantoines, thiohydan- toines, naphthoic acids and salicylic acids.
  • Embodiment 21 A pharmaceutical preparation according to embodiment 21 wherein the ligand that binds reversibly to a His 810 Zn 2+ site of an R-state insulin hexamer is a chemical structure selected from the group consisting of benzotria ⁇ oles, 3-hydroxy 2-napthoic acids, salicylic acids, tetrazoles or thiazolidinediones.
  • Embodiment 22 A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn 2+ site of an R-state insulin hexamer is wherein
  • Y is -S-, -O- or -NH-
  • R 1 and R 4 are independently selected from hydrogen or GrC 6 -alkyl
  • R 2 is hydrogen or C ⁇ -C 6 -alkyl or aryl, R 1 and R 2 may optionally be combined to form a double bond,
  • R 3 and R 5 are independently selected from hydrogen, halogen, aryl, CrC 6 -alkyl, or
  • a and B are independently selected from CrCe-alkyl, aryl, aryl-C ⁇ -C 6 -alkyl, aryl-C 2 -C 6 -alkenyl or heteroaryl, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R 6 and the aryl or heteroaryl is optionally substituted with up to four substituents R 7 , R 8 , R 9 , and R 10 ,
  • a and R 3 may be connected through one or two valence bonds
  • B and R 5 may be connected through one or two valence bonds
  • R 6 is independently selected from halogen, -CN, -CF 3 , -OCF 3) aryl, -COOH and -NH 2
  • R 7 , R 8 , R 9 and R 10 are independently selected from
  • oC C 6 -alkyl C 2 -C 6 -alkenyl or C 2 -C 6 -alkynyl, each of which may optionally be substituted with one or more substituents independently selected from R 13 , • aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C ⁇ -C 6 -alkoxy, aryl-C-i-C ⁇ -alkyl, aryl-C 2 -C 6 -alkenyl, aroyl-Ca-Ce-alkenyl, aryI-C 2 -C 6 -alkynyl, heteroaryl, heteroaryl-Cr Ce-alkyl, heteroaryl-C 2 -C 6 -alkenyl, heteroaryl-C 2 -C 6 -alkynyl, or C 3 -C 6 cycloalkyl,
  • each cyclic moiety may optionally be substituted with one or more substituents independently selected from R 14 ,
  • R 11 and R 12 are independently selected from hydrogen, OH, C ⁇ -C 2 o-alkyl, aryl-d-Ce-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R 15 , and the aryl groups may optionally be substituted one or more substituents independently selected from R 16 ; R 11 and R 12 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
  • R 13 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OR 11 , -C(O)OR 11 , -NR 11 R 12 , and -C(O)NR 11 R 12 ,
  • R 14 is independently selected from halogen, -C(O)OR 1 ⁇ -CH 2 C(O)OR 11 , -CH 2 OR 11 , -CN, - CF 3 , -OCF 3 , -NO 2 , -OR 11 , -NR 11 R 12 , S(O) 2 R 1 ⁇ aryl and C C 6 -alkyl,
  • R 15 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OC r C 6 -alkyl, -C(O)OC C 6 - alkyl, -COOH and -NH 2)
  • Embodiment 26 A pharmaceutical composition according to any one of the embodiments 23 to 26 wherein Y is -O- or -S-.
  • Embodiment 27 A pharmaceutical composition according to embodiment 27 wherein Y is
  • Embodiment 28 A pharmaceutical composition according to embodiment 27 wherein Y is
  • Embodiment 29 A pharmaceutical composition according to any one of the embodiments 23 to 30 wherein A is aryl optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 30 A pharmaceutical composition according to embodiment 31 wherein A is selected from ArG1 optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 31 A pharmaceutical composition according to embodiment 32 wherein A is phenyl or naphtyl optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 32 A pharmaceutical composition according to embodiment 33 wherein A is
  • Embodiment 34 A pharmaceutical composition according to any one of the embodiments 23 to 30 wherein A is heteroaryl optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 35 A pharmaceutical composition according to embodiment 36 wherein A is selected from Het1 optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 36 A pharmaceutical composition according to embodiment 37 wherein A is selected from Het2 optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 37 A pharmaceutical composition according to embodiment 38 wherein A is selected from Het3 optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 38 A pharmaceutical composition according to embodiment 39 wherein A is selected from the group consisting of indolyl, benzofuranyl, quinolyl, furyl, thienyl, or pyrrolyl, wherein each heteroaryl may optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 39 A pharmaceutical composition according to embodiment 39 wherein A is benzofuranyl optionally substituted with up to four substituents R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 40 A pharmaceutical composition according to embodiment 41 wherein A is
  • Embodiment 41 A pharmaceutical composition according to embodiment 39 wherein A is carbazolyl optionally substituted with up to four substituents R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 42 A pharmaceutical composition according to embodiment 43 wherein A is
  • Embodiment 43 A pharmaceutical composition according to embodiment 39 wherein A is quinolyl optionally substituted with up to four substituents R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 44 A pharmaceutical composition according to embodiment 45 wherein A is
  • Embodiment 45 A pharmaceutical composition according to embodiment 39 wherein A is indolyl optionally substituted with up to four substituents R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 46 A pharmaceutical composition according to embodiment 47 wherein A is
  • Embodiment 47 A pharmaceutical composition according to any one of the embodiments 23 to 48 wherein R 1 is hydrogen.
  • Embodiment 48 A pharmaceutical composition according to any one of the embodiments 23 to 49 wherein R 2 is hydrogen.
  • Embodiment 49 A pharmaceutical composition according to any one of the embodiments 23 to 48 wherein R 1 and R 2 are combined to form a double bond.
  • Embodiment 50 A pharmaceutical composition according to any one of the embodiments 23 to 51 wherein R 3 is d-C ⁇ -alkyl, halogen, or C(O)NR 16 R 17 .
  • Embodiment 51 A pharmaceutical composition according to embodiment 52 wherein R 3 is d-Ce-alkyl or C(O)NR 16 R 17 .
  • Embodiment 52 A pharmaceutical composition according to embodiment 53 wherein R 3 is methyl.
  • Embodiment 53 A pharmaceutical composition according to any one of the embodiments 23 to 30 wherein B is phenyl optionally substituted with up to four substituents, R 7 , R 8 , R 9 , and R 10 which may be the same or different.
  • Embodiment 54 A pharmaceutical composition according to any one of the embodiments 23 to 30 or 55 wherein R 4 is hydrogen.
  • Embodiment 55 A pharmaceutical composition according to any one of the embodiments 23 to 30 or 55 to 56 wherein R 5 is hydrogen.
  • Embodiment 56 A pharmaceutical composition according to any one of the embodiments 23 to 57 wherein R 6 is aryl.
  • Embodiment 57 A pharmaceutical composition according to embodiment 58 wherein R 6 is phenyl.
  • Embodiment 58 A pharmaceutical composition according to any one of the embodiments 23 to 59 wherein R 7 , R 8 , R 9 and R 0 are independently selected from
  • Embodiment 59 A pharmaceutical composition according to embodiment 60 wherein R 7 , R 8 , R 9 and R 10 are independently selected from
  • each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R 14
  • Embodiment 60 A pharmaceutical composition according to embodiment 61 wherein R 7 , R 8 , R 9 and R 10 are independently selected from
  • oC ⁇ -C 6 -alkyl or d-C 6 - which may each optionally be substituted with one or more substituents independently selected from R 13 • aryl, aryloxy, aroyl, aryl-C C 6 -alkoxy, aryl-d-Ce-alkyl, heteroaryl,
  • each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R 14 .
  • Embodiment 61 A pharmaceutical composition according to embodiment 62 wherein R 7 , R 8 , R 9 and R 10 are independently selected from ohydrogen, halogen, -OR 11 , -OC r C 6 -alkyl-C(0)OR 11 , or -C(O)OR 11 ,
  • Embodiment 62 A pharmaceutical composition according to embodiment 63 wherein R 7 , R 8 , R 9 and R 10 are independently selected from
  • Embodiment 63 A pharmaceutical composition according to any one of the embodiments 23 to 65 wherein R 11 and R 12 are independently selected from hydrogen, C ⁇ -C 20 -alkyl, aryl or aryl-C ⁇ -C 6 -alkyl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R 15 , and the aryl groups may optionally be substituted one or more substituents independently selected from R 16 ; R 11 and R 12 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds.
  • R 11 and R 12 are independently selected from hydrogen, C ⁇ -C 20 -alkyl, aryl or aryl-C ⁇ -C 6 -alkyl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected
  • Embodiment 64 A pharmaceutical composition according to embodiment 66 wherein R 11 and R 12 are independently selected from hydrogen, d-do-alkyl, aryl or aryl-d-Ce-alkyl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R 15 , and the aryl groups may optionally be substituted one or more substituents independently selected from R 16 .
  • Embodiment 65 A pharmaceutical composition according to embodiment 67 wherein R 11 and R 12 are independently selected from phenyl or phenyl-d-C 6 -alkyl.
  • Embodiment 66 A pharmaceutical composition according to embodiment 67 wherein one or both of R 11 and R 12 are methyl.
  • Embodiment 67 A pharmaceutical composition according to any one of the embodiments 23 to 69 wherein R 13 is independently selected from halogen, CF 3 , OR 11 or NR 11 R 12 .
  • Embodiment 68 A pharmaceutical composition according to embodiment 70 wherein R 13 is independently selected from halogen or OR 11 .
  • Embodiment 69 A pharmaceutical composition according to embodiment 71 wherein R 3 is OR 11 .
  • Embodiment 70 A pharmaceutical composition according to any one of the embodiments 23 to 72 wherein R 14 is independently selected from halogen, -C(O)OR 11 , -CN, -CF 3 , -OR 11 , S(O) 2 R 11 , and C C 6 -alkyl.
  • Embodiment 71 A pharmaceutical composition according to embodiment 73 wherein R 14 is independently selected from halogen, -C(O)OR 11 , or -OR 11 .
  • Embodiment 72 A pharmaceutical composition according to any one of the embodiments 23 to 74 wherein R 15 is independently selected from halogen, -CN, -CF 3 , -C(O)OC C 6 -alkyl,and -COOH.
  • Embodiment 73 A pharmaceutical composition according to embodiment 75 wherein R 15 is independently selected from halogen or -C(O)Od-C 6 -alkyl.
  • Embodiment 75 A pharmaceutical composition according to embodiment 77 wherein R 16 is independently selected from halogen, -C(O)Od-G6-alkyl, -COOH, -NO 2 , or d-G 6 -alkyl.
  • Embodiment 76 A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn 2+ site of an R-state insulin hexamer is
  • R 19 is hydrogen or C ⁇ -C 6 -aIkyl
  • R 20 is hydrogen or d-C ⁇ -alkyl
  • D and F are a valence bond or C C 6 -alkylene optionally substituted with one or more substituents independently selected from R 72 ,
  • R 72 is independently selected from hydroxy, d-C 6 -alkyl, or aryl,
  • E is C-i-C ⁇ -alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents R 21 , R 22 and R 23 ,
  • G is d-C ⁇ -alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents R 24 , R 25 and R 26 ,
  • R 17 , R 18 , R 21 , R 22 , R 23 , R 24 , R 25 and R 26 are independently selected from
  • R 29 which may optionally be substituted with one or more substituents independently selected from R 29 , • aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C ⁇ -C 6 -alkoxy, aryl-d-C 6 -alkyl, aryl-C 2 - C 6 -alkenyl, aryl-C 2 -C 6 -aIkynyl, heteroaryl, heteroaryl-C ⁇ -C 6 -alkyl, heteroaryl-C 2 -C 6 - alkenyl or heteroaryl-C 2 -C 6 -alkynyl,
  • R 27 and R 28 are independently selected from hydrogen, d-G 6 -alkyl, aryl-d-C ⁇ -alkyl or aryl, or R 27 and R 28 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
  • R 29 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OR 27 , and -NR 7 R 28 ,
  • R 30 is independently selected from halogen, -C(O)OR 27 , -CN, -CF 3 , -OCF 3 , -NO 2 , -OR 27 ,
  • Embodiment 77 A pharmaceutical composition according to embodiment 79 wherein D is a valence bond.
  • Embodiment 78 A pharmaceutical composition according to embodiment 79 wherein D is d-C 6 -alkylene optionally substituted with one or more hydroxy, d-C ⁇ -alkyl, or aryl.
  • Embodiment 79 A pharmaceutical composition according to any one of the embodiments 79 to 81 wherein E is aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents independently selected from R 21 , R 22 and R 23 .
  • Embodiment 80 A pharmaceutical composition according to embodiment 82 wherein E is aryl optionally substituted with up to three substituents independently selected from R 21 , R 22 and R 23 .
  • Embodiment 81 A pharmaceutical composition according to embodiment 83 wherein E is selected from ArG1 and optionally substituted with up to three substituents independently selected from R 21 , R 22 and R 23 .
  • Embodiment 82 A pharmaceutical composition according to embodiment 84 wherein E is phenyl optionally substituted with up to three substituents independently selected from R 21 ,
  • Embodiment 83 A pharmaceutical composition according to embodiment 85 wherein the ligand that binds reversibly to a HisBlO Zn 2+ site of an R-state insulin hexamer is
  • Embodiment 84 A pharmaceutical composition according to any one of the embodiments 79 to 86 wherein R 2 ⁇ R 22 and R 23 are independently selected from
  • the cyclic moieties optionally may be substituted with one or more substituents selected from R 30 .
  • Embodiment 85 A pharmaceutical composition according to embodiment 87 wherein R 21 ,
  • R 22 and R 23 are independently selected from
  • the cyclic moieties optionally may be substituted with one or more substituents selected from R 30 .
  • Embodiment 86 A pharmaceutical composition according to embodiment 88 wherein R 21 ,
  • R 22 and R 23 are independently selected from
  • Embodiment 87 A pharmaceutical composition according to embodiment 89 wherein R 21 ,
  • R 22 and R 23 are independently selected from
  • Embodiment 88 A pharmaceutical composition according to embodiment 90 wherein R 21 , R 22 and R 23 are independently selected from
  • Embodiment 89 A pharmaceutical composition according to any one of the embodiments 79 to 91 wherein R 19 is hydrogen or methyl.
  • Embodiment 90 A pharmaceutical composition according to embodiment 92 wherein R 19 is hydrogen.
  • Embodiment 91 A pharmaceutical composition according to any one of the embodiments 79 to 93 wherein R 27 is Hydrogen, d-C ⁇ -alkyl or aryl.
  • Embodiment 92 A pharmaceutical composition according to embodiment 94 wherein R 27 is hydrogen or d-C 6 -alkyl.
  • Embodiment 93 A pharmaceutical composition according to any one of the embodiments 79 to 95 wherein R 28 is hydrogen or G C 6 -alkyl.
  • Embodiment 94 A pharmaceutical composition according to embodiment 79 wherein F is a valence bond.
  • Embodiment 95 A pharmaceutical composition according to embodiment 79 wherein F is d- Ce-alkylene optionally substituted with one or more hydroxy, CrC 6 -alkyI f or aryl.
  • Embodiment 96 A pharmaceutical composition according to any one of the embodiments 79 or 97 to 98 wherein G is C C 6 -aIkyl or aryl, wherein the aryl is optionally substituted with up to three substituents R 24 , R 25 and R 26 .
  • Embodiment 97 A pharmaceutical composition according to any one of the embodiments 79 or 97 to 98 wherein G is d-G 6 -alkyl or ArG1, wherein the aryl is optionally substituted with up to three substituents R 24 , R 25 and R 26 .
  • Embodiment 98 A pharmaceutical composition according to embodiment 99 wherein G is d-C ⁇ -alkyl.
  • Embodiment 99 A pharmaceutical composition according to embodiment 101 wherein G is phenyl optionally substituted with up to three substituents R 24 , R 25 and R 26 .
  • Embodiment 100 A pharmaceutical composition according to any one of the embodiments.
  • R 24 , R 25 and R 26 are independently selected from
  • Embodiment 101 A pharmaceutical composition according to embodiment 103 wherein R 24 ,
  • the cyclic moieties optionally may be substituted with one or more substituents selected from R 30 .
  • Embodiment 102 A pharmaceutical composition according to embodiment 104 wherein R 24 ,
  • R 25 and R 26 are independently selected from
  • the cyclic moieties optionally may be substituted with one or more substituents selected from R 30 .
  • Embodiment 103 A pharmaceutical composition according to embodiment 105 wherein R 21 ,
  • Embodiment 104 A pharmaceutical composition according to embodiment 106 wherein R 21 , R 22 and R 23 are independently selected from
  • Embodiment 105 A pharmaceutical composition according to embodiment 107 wherein R 21 ,
  • R 22 and R 23 are independently selected from
  • cyclic moieties optionally may be substituted with one or more substituents selected from R 30 .
  • Embodiment 106 A pharmaceutical composition according to any one of the embodiments 79 or 97 to 108 wherein R 20 is hydrogen or methyl.
  • Embodiment 107 A pharmaceutical composition according to embodiment 109 wherein R 20 is hydrogen.
  • Embodiment 108 A pharmaceutical composition according to any one of the embodiments 79 or 97 to 110 wherein R 27 is hydrogen, d-C 6 -alkyl or aryl.
  • Embodiment 109 A pharmaceutical composition according to embodiment 111 wherein R 27 is hydrogen or d-C 6 -alkyl or ArG
  • Embodiment 110 A pharmaceutical composition according to embodiment 112 wherein R 27 is hydrogen or d-C ⁇ -alkyl.
  • Embodiment 111 A pharmaceutical composition according to any one of the embodiments 79 or 97 to 112 wherein R 28 is hydrogen or C r C 6 -alkyl.
  • Embodiment 112. A pharmaceutical composition according to embodiment 79 wherein R 17 and R 18 are independently selected from
  • the cyclic moieties optionally may be substituted with one or more substituents selected from R 30 .
  • Embodiment 113 A pharmaceutical composition according to embodiment 115 wherein R 17 and R 18 are independently selected from • hydrogen, halogen, -CN, -CF 3 , -NO 2) -OR 27 , -NR 27 R 28 , or -C(O)OR 27 ,
  • the cyclic moieties optionally may be substituted with one or more substituents selected from R 30 .
  • Embodiment 114 A pharmaceutical composition according to embodiment 116 wherein R 17 and R 18 are independently selected from
  • Embodiment 115 A pharmaceutical composition according to embodiment 117 wherein R 17 and R 18 are independently selected from
  • Embodiment 116 A pharmaceutical composition according to embodiment 118 wherein R 17 and R 18 are independently selected from o hydrogen, halogen, -CH, -CF 3 , -NO 2 , -OR 27 , -NR 27 R 28 , or-C(O)OR 27 o d-Ce-alkyl optionally substituted with one or more substituents independently selected from R 29
  • Embodiment 117 A pharmaceutical composition according to any one of the embodiments 79 to 119 wherein R 27 is hydrogen or CrC ⁇ -alkyl.
  • Embodiment 118 A pharmaceutical composition according to embodiment 120 wherein R 27 is hydrogen, methyl or ethyl.
  • Embodiment 119 A pharmaceutical composition according to any one of the embodiments 79 to 121 wherein R 28 is hydrogen or C G 6 -aIkyl.
  • Embodiment 120 A pharmaceutical composition according to embodiment 122 wherein R 28 is hydrogen, methyl or ethyl.
  • Embodiment 121 A pharmaceutical composition according to any one of the embodiments 79 to 123 wherein R 72 is -OH or phenyl.
  • Embodiment 122 A pharmaceutical composition according to embodiment 79 wherein the ligand that binds reversibly to a HisBlO Zn 2+ site of an R-state insulin hexamer is
  • Embodiment 123 A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn 2+ site of an R-state insulin hexamer is of the form H-l-J
  • J is o d-Ce-alkyl, C 2 -C 6 -alkenyl or C 2 -C 6 -alkynyl, which may each optionally be substituted with one or more substituents selected from R 34 , o ryl, aryloxy, aryl-oxycarbonyl-, aroyl, aryl-d-Ce-alkoxy-, aryl-C 2 - C 6 -alkenyl-, aryl-C 2 -Ge-aIkynyl-, heteroaryl, heteroaryl-CrGe-alkyl-, heteroaryl-C 2 -C 6 - alkenyl- or heteroaryl-C 2 -C 6 -alkynyl-, wherein the cyclic moieties are optionally substituted with one or more substituents selected from R 37 , • Hydrogen,
  • R 31 is independently selected from hydrogen, halogen, -CN, -CH 2 CN, -CHF 2 , -CF 3 , -OCF 3 , -OCHF 2 , -OCH 2 CF 3 , -OCF 2 CHF 2 , -S(O) 2 CF 3 , -SCF 3 , -NO 2 , -OR 35 , -C(O)R 35 , -NR 35 R 36 , -SR 35 , -NR 35 S(O) 2 R 36 , -S(O) 2 NR 35 R 36 , -S(O)NR 35 R 36 , -S(O)NR 35 R 36 , -S(O)R 35 , -S(O) 2 R 35 , -C(O)NR 35 R 36 , -OC(O)NR 35 R 36 , -NR 35 C(O)R 36 , -CH 2 C(O)NR 35 R 36 , -OCH 2 C(
  • R 32 and R 33 are independently selected from hydrogen, C C 6 -alkyl or d-C 6 -alkanoyl,
  • R 34 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OR 35 , and -NR 35 R 36 ,
  • R 35 and R 36 are independently selected from hydrogen, d-C 6 -alkyl, aryl-d-C 6 -alkyl or aryl, or R 35 and R 36 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
  • R 37 is independently selected from halogen, -G(O)OR 35 , -C(O)H, -CN, -CF 3 , -OCF 3 , -NO 2 , - OR 35 , -NR 35 R 36 , d-Ce-alkyl or C C 6 -aIkanoyl, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
  • Embodiment 124 A pharmaceutical composition according to embodiment 126 wherein the ligand that binds reversibly to a HisBlO Zn 2+ site of an R-state insulin hexamer is of the form H-l-J, wherein H is
  • phenyl, naphthalene or benzocarbazole rings are optionally substituted with one or more substituents independently selected from R 31 ,
  • n 1 or 2
  • R 31 is independently selected from hydrogen, halogen, -CN, -CH 2 CN, -CHF 2 , -CF 3 , -OCF 3 ,
  • R 32 and R 33 are independently selected from hydrogen, CrC ⁇ -alkyl or G C 6 -alkanoyl,
  • R 34 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OR 35 , and -NR 35 R 36 ,
  • R 35 and R 36 are independently selected from hydrogen, Ci-Ge-alkyl, aryl-d-Ce-alkyl or aryl, or R 35 and R 36 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
  • R 37 is independently selected from halogen, -C(O)OR 35 , -C(O)H, -CN, -CF 3 , -OCF 3 , -NO 2 , - OR 35 , -NR 35 R 38 , d-Ce-alkyl or C r C 6 -alkanoyl,
  • Embodiment 125 A pharmaceutical composition according to any one of the embodiments
  • Embodiment 126 A pharmaceutical composition according to embodiment 128 wherein H is
  • Embodiment 127 A pharmaceutical composition according to embodiment 128 wherein H is
  • Embodiment 128 A pharmaceutical composition according to any one of the embodiments 126 to 130wherein I is a valence bond, -CH 2 N(R 32 )-, or -S0 2 N(R 33 )-.
  • Embodiment 129 A pharmaceutical composition according to embodiment 131 wherein I is a valence bond.
  • Embodiment 130 A pharmaceutical composition according to any one of the embodiments 126 to 132 wherein J is o hydrogen,
  • Embodiment 131 A pharmaceutical composition according to embodiment 133 wherein J is
  • Embodiment 132 A pharmaceutical composition according to embodiment 133 wherein J is
  • Embodiment 133 A pharmaceutical composition according to embodiment 135 wherein J is o hydrogen, o phenyl or naphthyl optionally substituted with one or more substituents independently selected from R 37 .
  • Embodiment 134 A pharmaceutical composition according to embodiment 136 wherein J is hydrogen.
  • Embodiment 135. A pharmaceutical composition according to any one of the embodiments 126 to 137 wherein R 32 and R 33 are independently selected from hydrogen or C ⁇ -C 6 -alkyl.
  • Embodiment 136. A pharmaceutical composition according to any one of the embodiments 126 to 138 wherein R 34 is hydrogen, halogen, -CN, -CF 3 , -OCF 3 , -SCF 3 , -NO 2 , -OR 35 , -C(O)R 35 , -NR 35 R 36 , -SR 35 , -C(O)NR 35 R 36 , -OC(O)NR 35 R 36 , -NR 35 C(O)R 36 , -OC(O)R 35 , -OC
  • Embodiment 137 A pharmaceutical composition according to embodiment 139 wherein R 34 is hydrogen, halogen, -CF 3 , -N0 2 , -OR 35 , -NR 35 R 36 , -SR 35 , -NR 35 C(O)R 36 , or -C(O)OR 35 .
  • Embodiment 138 A pharmaceutical composition according to embodiment 140 wherein R 34 is hydrogen, halogen, -CF 3 , -N0 2 , -OR 35 , -NR 35 R 36 , or -NR 35 C(0)R 36 .
  • Embodiment 139 A pharmaceutical composition according to embodiment 141 wherein R 34 is hydrogen, halogen, or -OR 35 .
  • Embodiment 140 A pharmaceutical composition according to any one of the embodiments.
  • R 35 and R 36 are independently selected from hydrogen, C C 6 -alkyl, or aryl.
  • Embodiment 141 A pharmaceutical composition according to embodiment 143 wherein R 35 and R 36 are independently selected from hydrogen or d-C 6 -alkyl.
  • Embodiment 142 A pharmaceutical composition according to any one of the embodiments
  • R 37 is halogen, -C(O)OR 35 , -CN, -CF 3 , -OR 35 , -NR 35 R 36 , C ⁇ -C 6 -alkyl or C
  • Embodiment 143 A pharmaceutical composition according to embodiment 145 wherein R 37 is halogen, -C(O)OR 35 , -OR 35 , -NR 35 R 36 , d-C 6 -alkyl or d-C 6 -alkanoyl.
  • Embodiment 144 A pharmaceutical composition according to embodiment 146 wherein R 37 is halogen, -C(O)OR 35 or -OR 35 .
  • Embodiment 145 A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisB10 Zn 2+ site of an R-state insulin hexamer is
  • U is a valence bond, d-C 6 -alkenylene, -C C 6 -alkyl-O- or C C 6 -alkylene wherein any C C 6 -alkyl moiety is optionally substituted with d-C 6 -alkyl,
  • R 38 is GrG ⁇ -alkyl, aryl, wherein the alkyl or aryl moieties are optionally substituted with one or more substituents independently selected from R 39 , R 39 is independently selected from halogen, cyano, nitro, amino,
  • M is a valence bond, arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted with one or more substituents independently selected from R 40 ,
  • R 40 is selected from o hydrogen, halogen, -CN, -CH 2 CN, -CHF 2 , -CF 3 , -OCF 3 , -OCHF 2 , -OCH 2 CF 3 , -OCF 2 CHF 2 , -S(O) 2 CF 3 , -OS(0) 2 CF 3 , -SCF 3 , -N0 2 , -OR 41 , -NR 41 R 42 , -SR 41 , -NR 41 S(0) 2 R 42 , -S(0) 2 NR 41 R 42 , -S(O)NR 41 R 42 , -S(0)R 41 , -S(0) 2 R 41 , -OS(O) 2 R 41 , -C(O)NR 41 R 42 , -OC(0)NR 41 R 42 , -NR 41 C(O)R 42 , -CH 2 C(O)NR 41 R 42 , -OC C 6 - alkyl-C(O)NR
  • R 41 and R 42 are independently selected from hydrogen, -OH, d-Ce-alkyl, d-Ce-alkenyl, aryl- d-Ce-alkyl or aryl, wherein the alkyl moieties may optionally be substituted with one or more substituents independently selected from R 45 , and the aryl moieties may optionally be substituted with one or more substituents independently selected from R 46 ; R 41 and R 42 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
  • R 43 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OR 41 , and -NR 41 R 42 R 44 is independently selected from halogen, -C(O)OR 41 , -CH 2 C(O)OR 41 , -CH 2 OR 41 , -CN, -
  • R 45 is independently selected from halogen, -CN, -CF 3 , -OGF 3 , -O-C Ce-alkyl, -C(0)-O-C
  • R 46 is independently selected from halogen, -G(0)Od-G 6 -alkyl, -COOH, -CN, -CF 3 , -OCF 3 , -
  • R 47 and R 48 are independently selected from hydrogen, d-C 6 -alkyl, aryl optionally substituted with one or more R 49 ,
  • R 49 is independently selected from halogen and -COOH
  • any alkyl, alkenyl , alkynyl, aryl and heteroaryl moiety is optionally substituted with one or more substituents independently selected from R so ,
  • R 53 is independently selected from d-Ce-alkyl, d-C 6 -alkoxy, -d-Ce-alkyl-COOH, -C 2 -
  • Embodiment 149 A pharmaceutical composition according to embodiment 151 wherein K is a valence bond or C C 6 -alkylene, wherein any d-C 6 -alkyl moiety is optionally substituted with R 38 .
  • Embodiment 151 A pharmaceutical composition according to embodiment 152 wherein K is a valence bond.
  • Embodiment 152 A pharmaceutical composition according to any one of the embodiments
  • U is a valence bond or -C C 6 -alkyl-O-.
  • Embodiment 153 A pharmaceutical composition according to embodiment 155 wherein U is a valence bond.
  • Embodiment 154 A pharmaceutical composition according to any one of the embodiments
  • M is arylene or heteroarylene, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from
  • Embodiment 155 A pharmaceutical composition according to embodiment 157 wherein is
  • Embodiment 156 A pharmaceutical composition according to embodiment 158 wherein M is
  • Embodiment 157 A pharmaceutical composition according to embodiment 159 wherein M is
  • Embodiment 158 A pharmaceutical composition according to embodiment 160 wherein M is phenylene optionally substituted with one or more substituents independently selected from
  • Embodiment 159 A pharmaceutical composition according to embodiment 160 wherein M is indolylene optionally substituted with one or more substituents independently selected from
  • Embodiment 160 A pharmaceutical composition according to embodiment 162 wherein M is
  • Embodiment 161 A pharmaceutical composition according to embodiment 160 wherein M is carbazolylene optionally substituted with one or more substituents independently selected from R 40 .
  • Embodiment 162. A pharmaceutical composition according to embodiment 164 wherein M is
  • Embodiment 163 A pharmaceutical composition according to any one of the embodiments.
  • R 40 is selected from
  • a pharmaceutical composition according to embodiment 166 wherein R 40 is selected from o hydrogen, halogen, -CN, -CF 3 , -OCF 3 , -N0 2 , -OR 41 , -NR 41 R 42 , -SR 41 , -S(0) 2 R 41 , -NR 41 C(0)R 42 , -Od-C 6 -alkyl-C(0)NR 41 R 42 , -C 2 -C 6 -alkenyl-C( O)OR 41 !
  • Ci-C ⁇ -alkyl or C 2 -C 6 - alkenyl which may each optionally be substituted with one or more substituents independently selected from R 43 ,
  • Embodiment 165 A pharmaceutical composition according to embodiment 167 wherein R 40 is selected from
  • Embodiment 166 A pharmaceutical composition according to embodiment 168 wherein R 40 is selected from
  • Embodiment 167 A pharmaceutical composition according to any one of the embodiments 148 to 170 wherein R 41 and R 42 are independently selected from hydrogen, d-Ce-alkyl, or aryl, wherein the aryl moieties may optionally be substituted with halogen or -COOH.
  • Embodiment 168. A pharmaceutical composition according to embodiment 171 wherein R 41 and R 42 are independently selected from hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties may optionally be substituted with halogen or -COOH.
  • Embodiment 169 A pharmaceutical composition according to any one of the embodiments 148 to 170 wherein R 41 and R 42 are independently selected from hydrogen, d-Ce-alkyl, or aryl, wherein the aryl moieties may optionally be substituted with halogen or -COOH.
  • Embodiment 171. A pharmaceutical composition according to any one of the embodiments 148 to 174 wherein R 4 ' and R 48 are independently selected from hydrogen, methyl and phenyl.
  • Embodiment 172 A pharmaceutical composition according to any one of the embodiments 148 to 178 wherein T is o Hydrogen,
  • Embodiment 173 A pharmaceutical composition according to embodiment 179 wherein T is
  • Embodiment 174 A pharmaceutical composition according to embodiment 180 wherein T is
  • Embodiment 176 A pharmaceutical composition according to embodiment 183 wherein R 50 is CrCe-alkyl, GrC 6 -alkoxy, aryl, aryloxy, aryl-d-Ce-alkoxy , -OR 51 , -NO 2 , halogen, -COOH, -CF 3 , wherein any aryl moiety is optionally substituted with one or more R 53 .
  • R 50 is CrCe-alkyl, GrC 6 -alkoxy, aryl, aryloxy, aryl-d-Ce-alkoxy , -OR 51 , -NO 2 , halogen, -COOH, -CF 3 , wherein any aryl moiety is optionally substituted with one or more R 53 .
  • Embodiment 177 Embodiment 177.
  • Embodiment 178 A pharmaceutical composition according to embodiment 185 wherein R 50 is d-C ⁇ -alkyl, ArG1-0-, ArG1-C C ⁇ -alkoxy , -OR 51 , halogen, -COOH, -CF 3 , wherein any aryl moiety is optionally substituted with one or more R 53 .
  • Embodiment 179 A pharmaceutical composition according to embodiment 186 wherein R 50 is phenyl, methyl or ethyl.
  • Embodiment 180 A pharmaceutical composition according to embodiment 188 wherein R 50 is methyl or ethyl.
  • Embodiment 18 A pharmaceutical composition according to any one of the embodiments.
  • Embodiment 182 A pharmaceutical composition according to any one of the embodiments.
  • R 53 is CrC 6 -alkyl, CrC 6 -alkoxy, -OR 51 , halogen.or -CF 3 .
  • Embodiment 183 A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn 2+ site of an R-state insulin hexamer is
  • V is CrCe-alkyl, aryl, heteroaryl, aryl-C - 6 -alkyl- or aryl-C 2 . 6 -alkenyl-, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R 54 , and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R 55 ,
  • R 54 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , aryl, -COOH and -NH 2)
  • R 55 is independently selected from
  • R 58 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OR 56 , and -NR 56 R 57 ,
  • R 59 is independently selected from halogen, -C(O)OR 56 , -CH 2 C(O)OR 56 , -CH 2 OR 56 , -CN, - CF 3 , -OCF 3 , -NO 2 , -OR 56 , -NR 56 R 57 and d-C 6 -alkyl,
  • R 62 is d-Ce-alkyl, aryl optionally substituted with one or more substituents independently selected from halogen, or heteroaryl optionally substituted with one or more d-C 6 -alkyl independently, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
  • Embodiment 184 A pharmaceutical composition according to embodiment 196 wherein V is aryl, heteroaryl, or aryl-d- 6 -alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected R 54 , and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R 55 .
  • Embodiment 185 A pharmaceutical composition according to embodiment 197 wherein V is aryl, Het1 , or aryl-C ⁇ . 6 -alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from R 54 , and the aryl or heteroaryl moiety is optionally substituted with one or more substituents independently selected from R 55 .
  • Embodiment 186 A pharmaceutical composition according to embodiment 198 wherein V is aryl, Het2, or aryl-d.
  • Embodiment 187 A pharmaceutical composition according to embodiment 199 wherein V is aryl, Het3, or aryl-C ⁇ . 6 -alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from R 54 , and the aryl or heteroaryl moiety is optionally substituted with one or more substituents independently selected from R 55 .
  • Embodiment 191 A pharmaceutical composition according to embodiment 203 wherein V is phenyl optionally substituted with one or more substituents independently selected from R 55 .
  • Embodiment 192. A pharmaceutical composition according to any one of the embodiments 196 to 204 wherein R 55 is independently selected from o halogen, d-Ce-alkyl, -CN, -OCF 3 ,-CF 3 , -NO 2 , -OR 56 , -NR 56 R 57 , -NR 56 G(O)R 57
  • Embodiment 195 A pharmaceutical composition according to embodiment 207 wherein R 55 is independently selected from halogen, -OR 56 , -NR 56 R 57 , -C(O)OR 56 , -OC C 8 - alkyl-C(O)OR 56 , -NR 56 C(O)R 57 , methyl or ethyl.
  • Embodiment 197 A pharmaceutical composition according to embodiment 209 wherein R 56 and R 57 are independently selected from hydrogen or d-C 2 -alkyl, R 56 and R 57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom.
  • Embodiment 198 A pharmaceutical composition according to embodiment 210 wherein R 56 and R 57 are independently selected from hydrogen or methyl, ethyl, propyl butyl, R 56 and R 57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom.
  • Embodiment 199 A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn 2+ site of an R-state insulin hexamer is
  • AA is d-Ce-alkyl, aryl, heteroaryl, aryl-G - 6 -alkyl- or aryl-G 2 - 6 -alkenyl-, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R 63 , and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R 64 ,
  • R 63 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , aryl, -COOH and -NH 2 ,
  • R 64 is independently selected from
  • R 67 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OR 65 , and -NR 65 R 66 ,
  • R 68 is independently selected from halogen, -G(O)OR 65 , -CH 2 C(O)OR 65 , -CH 2 OR 65 , -CN, - CF 3 , -OCF 3 , -N0 2 , -OR 65 , -NR 65 R 66 and d-C ⁇ - ⁇
  • R 69 is independently selected from d-C ⁇ -alkyl, aryl optionally substituted with one or more halogen, or heteroaryl optionally substituted with one or more d-C ⁇ -alkyl,
  • R 70 is independently selected from halogen, -CN, -CF 3 , -OCF 3 , -OCrC 6 -alkyl, -C(O)Od-C 6 - alkyl, -COOH and -NH 2 ,
  • Embodiment 200 A pharmaceutical composition according to embodiment 212 wherein AA is aryl, heteroaryl or aryl-Ci- ⁇ -alkyl-, wherein the alkyl is optionally substituted with one or more R 63 , and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R 64 .
  • Embodiment 201 A pharmaceutical composition according to embodiment 213 wherein AA is aryl or heteroaryl optionally substituted with one or more substituents independently selected from R 64 .
  • Embodiment 202 A pharmaceutical composition according to embodiment 214 wherein AA is ArG1 or Het1 optionally substituted with one or more substituents independently selected from R 64 .
  • Embodiment 203 A pharmaceutical composition according to embodiment 215 wherein AA is ArG1 or Het2 optionally substituted with one or more substituents independently selected from R 64 .
  • Embodiment 204 A pharmaceutical composition according to embodiment 216 wherein AA is ArG1 or Het3 optionally substituted with one or more substituents independently selected from R 64 .
  • Embodiment 205 A pharmaceutical composition according to embodiment 217 wherein AA is phenyl, naphtyl, anthryl, carbazolyl, thienyl, pyridyl, or benzodioxyl optionally substituted with one or more substituents independently selected from R 64 .
  • Embodiment 206 A pharmaceutical composition according to embodiment 218 wherein AA is phenyl or naphtyl optionally substituted with one or more substituents independently selected from R 64 .
  • Embodiment 207 A pharmaceutical composition according to any one of the embodiments 212 to 219 wherein R 64 is independently selected from hydrogen, halogen, -CF 3 , -OCF 3 , -OR 65 , -NR 65 R 66 , d-Ce-alkyl , -OC(O)R 65 , -OCrC 6 -alkyl-C(O)OR 65 , aryl-C 2 -C 6 -alkenyl, aryloxy or aryl, wherein C C 6 -alkyl is optionally substituted with one or more substituents independently selected from R 67 , and the cyclic moieties optionally are substituted with one or more substituents independently selected from R 68 .
  • R 64 is independently selected from hydrogen, halogen, -CF 3 , -OCF 3 , -OR 65 , -NR 65 R 66 , d-Ce-alkyl , -OC(O)R 65 , -
  • Embodiment 208 A pharmaceutical composition according to embodiment 220 wherein R 64 is independently selected from halogen, -CF 3 , -OCF 3 , -OR 65 , -NR 65 R 66 , methyl, ethyl, propyl, -OC(O)R 65 , -OCH 2 -C(O)OR 65 , -OCH 2 -CH 2 -C(O)OR 65 , phenoxy optionally substituted with one or more substituents independently selected from R 68 .
  • Embodiment 209 A pharmaceutical composition according to any one of the embodiments 212 to 221 wherein R 65 and R 66 are independently selected from hydrogen, CF 3 , C C ⁇ 2 -alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R 71 .
  • Embodiment 210 A pharmaceutical composition according to embodiment 222 wherein R 65 and R 66 are independently hydrogen, d-C ⁇ 2 -alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R 71 .
  • Embodiment 211 A pharmaceutical composition according to embodiment 223 wherein R 65 and R 66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, ArG1 or Het1 optionally substituted with one or more substituents independently selected from R 71 .
  • Embodiment 212. A pharmaceutical composition according to embodiment 224 wherein R 65 and R 66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, ArG1 or Het2 optionally substituted with one or more substituents independently selected from R 71 .
  • Embodiment 215. A pharmaceutical composition according to any one of the embodiments 212 to 227 wherein R 71 is halogen or d-Ce-alkyl.
  • Embodiment 216 A pharmaceutical composition according to embodiment 228 wherein R 71 is halogen or methyl.
  • Embodiment 217 Method of prolonging the action of an insulin preparation comprising insulin, protamine and zinc ions wherein said method comprises adding a zinc-binding ligand according to any of embodiments 21 to 216 to the insulin preparation.
  • Embodiment 218 A method of treating type 1 or type 2 diabetes comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical preparation according to any one of the embodiments 1 to216.
  • Embodiment 219. Use of a preparation according to any one of the embodiments 1 to 216for the preparation of a medicament for treatment of type 1 or type 2 diabetes.
  • Embodiment 220. A method of preparing a pharmaceutical preparation comprising the steps of mixing
  • Embodiment 22 A method according to embodiment 233 wherein the ligand for the His 810
  • Zn 2+ site is added to the mixture before crystal growth.
  • Embodiment 222 A method according to embodiment 233 wherein the ligand for the His 810
  • novel NPH-insulin preparations disclosed here can be used for parenteral or pulmonal administration.
  • NPH preparations of the present invention are used in connection with pen-like injection devices, which may be prefilled and disposable, or the insulin preparations may be supplied from a reservoir which is removable.
  • pen-like injection devices are FlexPen ® , InnoLet ® , InDuoTM, Innovo ® .
  • NPH preparations of the present invention may be used in connection with devices for pulmonary administration of aqueous insulin preparations.
  • the NPH preparation of the invention is dried to form a powder.
  • suitable devices used in pulmonary administration of a NPH preparation according to the present invention may be the dry powder formulation and delivery devices being developed by Inhale Therapeutic Systems, Inc., and the Spiros® dry powder inhaler system being developed by Dura Pharmaceuticals, Inc.
  • the zinc-binding ligand for the His 810 Zn 2+ site is present in the preparation in a smaller concentration than that of Zn + -
  • not all of the insulin hexamers will have zinc-binding ligand for the His 810 Zn 2+ site present, and thus insulin from these hexamers will be released rapidly.
  • Such a preparation will therefore have a dual-release profile after administration, i.e. the administration will result in a both a rapid release of insulin and a protracted release.
  • Insulin formulations of the invention are usually administered from multi-dose containers where a preservative effect is desired. Since phenolic preservatives also stabilize the R-state hexamer the formulations may contain up to 50 mM of phenolic molecules.
  • the phenolic molecules in the insulin formulation may be selected from the group consisting of phenol, m- cresol, chloro-cresol, thymol, m-chlor-phenol, resorcinole, 7-hydroxyindole or any mixture thereof.
  • 0.5 to 5.0 mg/ml of phenolic compound may be employed.
  • 0.6 to 5.0 mg/ml of m-cresol may be employed.
  • 0.5 to 5.0 mg/ml of phenol may be employed.
  • 1.4 to 5.0 mg/ml of phenol may be employed.
  • 0.5 to 5.0 mg/ml of a mixture of m-cresol or phenol may be employed.
  • 1.4 to 5.0 mg/ml of a mixture of m-cresol or phenol may be employed.
  • the pharmaceutical preparation may further comprises a buffer substance, such as a TRIS, phosphate, glycine or glycylglycine (or another ⁇ witterionic substance) buffer, an isotonicity agent, such as NaCl, glycerol, mannitol and/or lactose.
  • a buffer substance such as a TRIS, phosphate, glycine or glycylglycine (or another ⁇ witterionic substance) buffer
  • an isotonicity agent such as NaCl, glycerol, mannitol and/or lactose.
  • Chloride would be used at moderate concentrations (e.g. up to 50 mM) to avoid competition with the zinc-site ligands of the present invention.
  • the action of insulin may further be slowed down in vivo by the addition of physiologically acceptable agents that increase the viscosity of the pharmaceutical preparation.
  • the pharmaceutical preparation according to the invention may furthermore comprise an agent which increases the viscosity, such as polyethylene glycol, polypropylene glycol, copolymers thereof, dextrans and/or polylactides.
  • the insulin preparation of the invention comprises between 0.001 % by weight and 1 % by weight of a non-ionic surfactant, for example tween 20 or Poloxamer 188.
  • a non-ionic surfactant for example tween 20 or Poloxamer 188.
  • the insulin preparation of the present invention may have a pH value in the range of 3.5 to 8.5, more preferably 7.1 to 7.9.
  • the invention furthermore relates to treatment of a patient in which the pharmaceutical preparation of the invention, i.e. comprising zinc ions, acid-stabilised insulin analogue and a ligand for the R-state His 810 Zn 2+ site, is combined with another form of treatment.
  • the pharmaceutical preparation of the invention i.e. comprising zinc ions, acid-stabilised insulin analogue and a ligand for the R-state His 810 Zn 2+ site
  • treatment of a patient with the pharmaceutical preparation of the invention is combined with diet and/or exercise.
  • the pharmaceutical preparation of the invention is administered in combination with one or more further active substances in any suitable ratios.
  • Such further active substances may e.g. be selected from antiobesity agents, antidiabetics, antihypertensive agents, agents for the treatment of complications resulting from or associated with diabetes and agents for the treatment of complications and disorders resulting from or associated with obesity.
  • the pharmaceutical preparation of the invention may be administered in combination with one or more antiobesity agents or appetite regulating agents.
  • Such agents may be selected from the group consisting of CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, ⁇ V1C4 (rnelano- cortin 4) agonists, MC3 (melanocortin 3) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corlicotropin releasing factor binding protein) antagonists, urocortin agonists, ⁇ 3 adrenergic agonists such as CL-316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140, MSH (melanoeyte- stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CGK (cholecystokinin) agonists, serotonin re-uptake inhibitors such
  • the antiobesity agent is leptin.
  • the antiobesity agent is dexamphetamine or amphetamine.
  • the antiobesity agent is fenfluramine or dexfenfluramine.
  • the antiobesity agent is sibutramine.
  • the antiobesity agent is orlistat.
  • the antiobesity agent is mazindol or phentermine.
  • the antiobesity agent is phendimetrazine, diethylpropion, fluoxetine, bupropion, topiramate or ecopipam.
  • the orally active hypoglycemic agents comprise imidazolines, sulphonylureas, biguanides, meglitinides, oxadiazolidinediones, thiazolidinediones, insulin sensitizers, insulin secretagogues such as glimepride, ⁇ -glucosidase inhibitors, agents acting on the ATP- dependent potassium channel of the ⁇ -cells eg potassium channel openers such as those disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (Novo Nordisk A/S) which are incorporated herein by reference, or mitiglinide, or a potassium channel blocker, such as BTS-67582, nateglinide, glucagon antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference, GLP-1 agonists such as those disclosed in WO
  • the pharmaceutical preparation of the invention is administered in combination with a sulphonylurea e.g. tolbutamide, chlorpropa- mide, tolazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide.
  • a sulphonylurea e.g. tolbutamide, chlorpropa- mide, tolazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide.
  • the pharmaceutical preparation of the invention is administered in combination with a biguanide, e.g. metformin.
  • a biguanide e.g. metformin.
  • the pharmaceutical preparation of the invention is administered in combination with a meglitinide eg repaglinide or nateglinide.
  • the pharmaceutical preparation of the invention is administered in combination with a thiazolidinedione insulin sensitizer, e.g. trogli- tazone, ciglitazone, pioglitazone, rosiglitazone, isaglitazone, darglitazone, englitazone, CS- 011/CI-1037 or T 174 or the compounds disclosed in WO 97/41097, WO 97/41119, WO 97/41120, WO 00/41121 and WO 98/45292 (Dr. Reddy's Research Foundation), which are incorporated herein by reference.
  • a thiazolidinedione insulin sensitizer e.g. trogli- tazone, ciglitazone, pioglitazone, rosiglitazone
  • the pharmaceutical preparation of the invention may be administered in combination with an insulin sensitizer, e.g. such as GI 262570, YM-440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW-409544, CRE-16336, AR-H049020, LY510929, MBX- 02, CLX-0940, GW-501516 or the compounds disclosed in WO 99/19313, WO 00/50414, WO 00/63191, WO 00/63192, WO 00/63193 (Dr.
  • an insulin sensitizer e.g. such as GI 262570, YM-440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW-409544, CRE-16336, AR-H049020, LY510929, MBX- 02, CLX-0940, GW-501516 or the compounds disclosed in WO 99/19313, WO 00/50414
  • the pharmaceutical preparation of the invention is administered in combination with an ⁇ -glucosidase inhibitor, e.g. voglibose, emigli- tate, miglitol or acarbose.
  • an agent acting on the ATP-dependent potassium channel of the ⁇ -cells e.g. tolbutamide, glibenclamide, glipizide, glicazide, BTS-67582 or re- paglinide.
  • the pharmaceutical preparation of the invention may be administered in combination with nateglinide.
  • the pharmaceutical preparation of the invention is administered in combination with an antilipidemic agent, e.g. cholestyramine, coleslipol, clofibrate, gemfibro ⁇ il, lovastatin, pravaslatin, simvastatin, probucol or dextrothy- roxine.
  • an antilipidemic agent e.g. cholestyramine, coleslipol, clofibrate, gemfibro ⁇ il, lovastatin, pravaslatin, simvastatin, probucol or dextrothy- roxine.
  • the pharmaceutical preparation of the invention is administered in combination with more than one of the above-mentioned compounds, e.g. in combination with metformin and a sulphonylurea such as glyburide; a sulphonylurea and acarbose; nateglinide and metformin; acarbose and metformin; a sulphonylurea, metformin and troglitazone; metformin and a sulphonylurea; etc.
  • metformin and a sulphonylurea such as glyburide
  • a sulphonylurea and acarbose such as glyburide
  • a sulphonylurea and acarbose such as glyburide
  • a sulphonylurea and acarbose such as glyburide
  • the pharmaceutical preparation of the invention may be administered in combination with one or more antihypertensive agents.
  • antihypertensive agents are ⁇ -blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and ⁇ - blockers such as doxazosin, urapidil, prazosin and terazosin.
  • the pharmaceutical preparation of the invention may also be combined with NEP inhibitors such as candoxatril.
  • the HPLC pump was connected to two eluent reservoirs containing:
  • the analysis was performed at 40 °C by injecting an appropriate volume of the sample (preferably 1 ⁇ L) onto the column, which was eluted with a gradient of acetonitrile.
  • an appropriate volume of the sample preferably 1 ⁇ L
  • the HPLC conditions, detector settings and mass spectrometer settings used are given in the following table.
  • Sciex AP1 100 Single quadropole mass spectrometer
  • a Valco column switch with a Valco actuator controlled by timed events from the pump is a Valco column switch with a Valco actuator controlled by timed events from the pump.
  • the Sciex Sample control software running on a Macintosh PowerPC 7200 computer was used for the instrument control and data acquisition.
  • the HPLC pump was connected to four eluent reservoirs containing:
  • samples contain approximately 500 ⁇ g/mL of the compound to be analysed in an acceptable solvent such as methanol, ethanol, acetonitrile, THF, water and mixtures thereof. (High concentrations of strongly eluting solvents will interfere with the chromatography at low acetonitrile concentrations.)
  • an acceptable solvent such as methanol, ethanol, acetonitrile, THF, water and mixtures thereof.
  • the analysis was performed at room temperature by injecting 20 ⁇ L of the sample solution on the column, which was eluted with a gradient of acetonitrile in either 0.05% TFA or 0.002 M ammonium acetate. Depending on the analysis method varying elution conditions were used.
  • the eluate from the column was passed through a flow splitting T-connector, which passed approximately 20 ⁇ L/min through approx. 1 m. 75 ⁇ fused silica capillary to the API interface of AP1 100 spectrometer.
  • the remaining 1.48 mL/min was passed through the UV detector and to the ELS detector.
  • the detection data were acquired concurrently from the mass spectrometer, the UV detector and the ELS detector.
  • the HPLC pump is connected to two eluent reservoirs containing:
  • ELS analogue output from ELS
  • a Valco column switch with a Valco actuator controlled by timed events from the pump is a Valco column switch with a Valco actuator controlled by timed events from the pump.
  • the Sciex Sample control software running on a Macintosh Power G3 computer was used for the instrument control and data acquisition.
  • the HPLC pump was connected to two eluent reservoirs containing:
  • the analysis was performed at room temperature by injecting 20 ⁇ l of the sample solution on the column, which was eluted with a gradient of acetonitrile in 0.05% TFA
  • the eluate from the column was passed through a flow splitting T-connector, which passed approximately 20 ⁇ l/min through approx. 1 m 75 ⁇ fused silica capillary to the API interface of AP1 150 spectrometer.
  • the remaining 1.48 ml/min was passed through the UV detector and to the ELS detector.
  • the detection data were acquired concurrently from the mass spectrometer, the UV detector and the ELS detector.
  • LG conditions, detector settings and mass spectrometer settings used for the different methods are given in the following table.
  • D, E and R 19 are as defined above, and E is optionally substituted with up to three substituents R 21 , R 22 and R 23 independently as defined above.
  • the carboxylic acid of 1 H-ben ⁇ otriazoIe-5-carboxylic acid is activated, ie the OH functionality is converted into a leaving group L (selected from eg fluorine, chlorine, bromine, iodine, 1- imida ⁇ olyl, 1,2,4-tria ⁇ oIyl, 1-ben ⁇ otriazolyloxy, 1-(4-aza benzotriazolyl)oxy, pentafluoro- phenoxy, N-succinyloxy 3,4-dihydro-4-oxo-3-(1,2,3-benzotriazinyl)oxy, benzotriazole 5-COO, or any other leaving group known to act as a leaving group in acylation reactions.
  • L selected from eg fluorine, chlorine, bromine, iodine, 1- imida ⁇ olyl, 1,2,4-tria ⁇ oIyl, 1-ben ⁇ otriazolyloxy, 1-(4-aza benzotriazolyl)
  • the activated benzotriazole-5-carboxylic acid is then reacted with R 2 -(CH 2 ) n -B' in the presence of a base.
  • the base can be either absent (i.e. R 2 -(CH 2 ) n -B' acts as a base) or triethylamine, N- ethyl-N,N.-diisopropylamine, N-methylmorpholine, 2,6-lutidine, 2,2,6,6-tetramethylpiperidine, potassium carbonate, sodium carbonate, caesium carbonate or any other base known to be useful in acylation reactions.
  • the reaction is performed in a solvent solvent such as THF, di- oxane, toluene, dichloromethane, DMF, NMP or a mixture of two or more of these.
  • a solvent solvent such as THF, di- oxane, toluene, dichloromethane, DMF, NMP or a mixture of two or more of these.
  • the reaction is performed between 0 °C and 80 °C, preferably between 20 °C and 40 °C.
  • the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art.
  • Benzotria ⁇ ole-5-carboxylic acid (856 mg), HOAt (715 mg) and EDAC (1.00 g) were dissolved in DMF (17.5 mL) and the mixture was stirred at room temperature 1 hour. A 0.5 mL aliqot of this mixture was added to aniline (13.7 L, 0.15 mmol) and the resulting mixture was vigorously shaken at room temperature for 16 hours. 1N hydrochloric acid (2 mL) and ethyl ace- tate (1 mL) were added and the mixture was vigorously shaken at room temperature for 2 hours. The organic phase was isolated and concentrated in vacuo to afford the title compound.
  • the compounds in the following examples were similarly made.
  • the compounds may be isolated by filtration or by chromatography.
  • a base such as sodium acetate, potassium acetate, ammonium a
  • the compounds in the following examples were similarly prepared.
  • the compounds can be further purified by filtration and washing with water, ethanol and / or heptane instead of concentration in vacuo.
  • the compounds can be purified by washing with ethanol, water and/or heptane, or by chromatography, such as preparative HPLC.
  • Example 67 General procedure (B)) 5-(4-Dimethylaminobenzylidene)thiazolidine-2,4-dione

Abstract

This invention relates to NPH-insulin ( crystalline preparations) that are prepared in the presence of certain high-affinity ligands for the HisB10 Zn2+-, sites of the R-state insulin hexamer. Preparation of NPH-insulin in the presence of high-affinity ligand results in crystal-line NPH-insulin suspensions that are absorbed more slowly from subcutis than regular NPH-insulin. Hence the resulting action profile is longer and the spike is less pronounced than observed with regular NPH-insulin.

Description

NOVEL NPH INSULIN PREPARATIONS
FIELD OF THE INVENTION
This invention relates to novel NPH insulin crystalline preparations comprising high-affinity ligands for the HisBlO Zn2÷-sites of the R-state insulin hexamer.
BACKGROUND OF THE INVENTION
Diabetes mellitus is a common disorder of glucose metabolism. The disease is characterized by hyperglycemia and may be classified as type 1 diabetes, sometimes termed insulin- dependent diabetes mellitus, or type 2 diabetes, which is sometimes termed non-insulin- dependent. Insulin dependent diabetes mellitus is characterized by severely diminished or absent production of endogenous insulin. This chronic condition must be treated with daily subcutaneous injections of insulin to maintain a reasonably normal blood glucose level. Similar injections are also common in later stage type 2 diabetes. The use of insulin as a therapeutic agent for this treatment is usually considered one of the outstanding successes of modern medicine. However, the therapy has its associated problems mainly because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood.
The kinetics of absorption from the subcutaneous tissue of fast acting human insulin is too slow and lasts too long to precisely mimic the peak of insulin which is normally secreted within minutes in response to carbohydrate ingestion during a meal. More importantly, the action profile of the most commonly used crystalline long-acting basal insulin show a spike, i.e. a high concentration of relatively short duration of insulin in the blood, within a few hours after injection. Also, the total duration of action is somewhat too short for once daily injection, and the absorption times show some fluctuation from day to day leading to poor reproducibil- ity of the basal insulin level.
Long-term studies have shown that the complications of diabetes such as retinopathy and nephropathy can only be prevented or delayed by an intensive treatment regimen aiming at normalization of blood glucose. Consequently, the major challenge of the insulin-replacement therapy consists in reproducing the complex pattern of insulin secretion dynamics in healthy individuals, to achieve constant blood glucose in both basal and meal-related situations. The most widely used long acting insulin is a neutral crystalline suspension, i.e. NPH insulin, comprising a crystalline complex of human insulin (or an analogue thereof), zinc ion and protamine sulphate together with a suitable preservative such as phenol, m-cresol, or mixtures thereof. In addition, the preparations usually contain a buffering substance such as phosphate and an isotonicity agent such as glycerol, mannitol or sodium chloride.
When the suspension is injected into the subcutaneous tissue, the delayed action is believed to originate from the rate-limiting dissolution of the NPH-insulin crystals in the subcutaneous tissue fluids. Thus the main determinant for the spike in the action profile as well as the total length of duration of action is thought to be the inherent solubility of the NPH- insulin crystal in the subcutis. On the other hand, the poorly reproducible absorption times often encountered with NPH insulin are thought to originate from difficulties in resuspending the vial before injection which may lead to variations in the dose actually delivered from one injection to another. Moreover, the rate of dissolution at the site of injection depends to some extent on the local blood flow which is influenced by e.g. exercise and temperature adding further elements to the poorly reproducible absorption times. Taken together, these factors are considered to limit the inherent quality of the action profile obtained from NPH-insulin.
SUMMARY OF THE INVENTION
It has now surprisingly been found that NPH-insulin (crystalline preparations) may be prepared in the presence of certain high-affinity ligands for the HisB10 Zn2+-sites of the R-state insulin hexamer. Preparation of NPH-insulin in the presence of high-affinity ligand results in crystalline NPH-insulin suspensions that are absorbed more slowly from subcutis than regular NPH-insulin. Hence the resulting action profile is longer and the spike is less pronounced than observed with regular NPH-insulin. The novel NPH-insulin also shows better physical and chemical stability than regular NPH-insulin.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 (example 1011) is a graphic representation of glucose utilization after subcutaneous injection of a NPH preparation showing the effects of stoichiometric and excess concentration of 4-[3-(1 H-Tetrazol-5-yl)-carbazol-9-ylmethyl]-benzoic acid compared to Zn2+ . DEFINITIONS
The following is a detailed definition of the terms used to describe the invention: "Halogen" designates an atom selected from the group consisting of F, Cl, Br and I. The term "Cι-C6-alkyl" as used herein represents a saturated, branched or straight hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, feft-butyl, n-pentyl, isopentyl, neopentyl, fert-pentyl, n-hexyl, isohexyl and the like.
The term "CrC6-alkylene" as used herein represents a saturated, branched or straight bivalent hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methylene, 1 ,2-ethylene, 1 ,3-propylene, 1 ,2-propylene, 1 ,4-butylene, 1 ,5- pentylene, 1 ,6-hexylene, and the like.
The term "C2-C6-alkenyl" as used herein represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, iso-propenyl, 1,3-buta- dienyi, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 1-pentenyl, 2-pentenyl, 3- pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 2,4-hexadienyl, 5- hexenyl and the like.
The term "C2-C6-alkyny as used herein represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one triple bond. Examples of such groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4- hexynyl, 5-hexynyl, 2,4-hexadiynyl and the like.
The term "CrC6-alkoxy" as used herein refers to the radical -O-CrCe-alkyl, wherein Cι-C6-alkyl is as defined above. Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tetf-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like. The term "C3-C8-cycloalkyl" as used herein represents a saturated, carbocyclic group having from 3 to 8 carbon atoms. Representative examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
The term "C4-8-cycloalkenyl" as used herein represents a non-aromatic, carbocyclic group having from 4 to 8 carbon atoms containing one or two double bonds. Representative examples are 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl, 3- cyclohexenyl, 2-cycloheptenyl, 3-cycloheptenyl, 2-cyclooctβnyl, 1 ,4-cyclooctadienyl and the like.
The term "heterocyclyl" as used herein represents a non-aromatic 3 to 10 membered ring containing one or more heteroatoms selected from nitrogen, oxygen and sulphur and optionally containing one or two double bonds. Representative examples are pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, aziridinyl, tetrahydrofuranyl and the like. The term "aryl" as used herein is intended to include carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9 to 14 membered bi- and tricyclic, carbocyclic, aromatic ring systems. Representative examples are phenyl, biphenylyl, naphthyl, anthracenyl, phe- nanthrenyl, fluorenyl, indenyl, a∑ulenyl and the like. Aryl is also intended to include the partially hydrogenaled derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenaled derivatives are 1,2,3,4-tetrahydronaphthyl, 1,4- dihydronaphthyl and the like.
The term "arylene" as used herein is intended to include divalent, carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9 to 14 membered bi- and tricyclic, divalent, carbocyclic, aromatic ring systems. Representative examples are phenylene, biphenylylene, naphthylene, anthracenylene, phenanthrenylene, fluorenylene, indenylene, azulenylene and the like. Arylene is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 1 ,2,3,4-tetrahydronaphthylene, 1 ,4-dihydronaphthylene and the like. The term "aryloxy" as used herein denotes a group -O-aryl, wherein aryl is as defined above. The term "aroyl" as used herein denotes a group -C(O)-aryl, wherein aryl is as defined above. The term "heteroaryl" as used herein is intended to include aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as 5 to 7 membered monocyclic and 8 to 14 membered bi- and tricyclic aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur. Representative examples are furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1 ,2,3-triazolyl, 1 ,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, 1 ,3,5- triazinyl, 1,2,3- oxadiazolyl, 1,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazolyl, 1,2,4- thiadiazolyl, 1 ,2,5-thiadiazolyl, 1 ,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, ben- zofuryl, benzothienyl, indazolyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl, thiazolidinyl, 2- thiooxothiazolidinyl and the like. Heteroaryl is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like. The term "heteroarylene" as used herein is intended to include divalent, aromatic, heterocyc- lic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as 5 to 7 membered monocyclic and 8 to 14 membered bi- and tricyclic aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur. Representative examples are furylene, thienylene, pyrrolylene, oxa- zolylene, thiazolylene, imidazolylene, isoxazolylene, isothia∑olylene, 1 ,2,3-tria∑olylene, 1 ,2,4- triazolylene, pyranylene, pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene, 1 ,2,3- triazinylene, 1 ,2,4-triazinylene, 1 ,3,5- tria∑inylene, 1 ,2,3-oxadia∑olyIene, 1 ,2,4-oxadia∑olylene, 1 ,2,5-oxadia∑olylene, 1 ,3,4-oxadiazolylene, 1,2,3-thiadia∑olylene, 1 ,2,4-thiadia∑olylene, 1,2,5- thiadiazolylene, 1,3,4-thiadiazolylene, tetrazolylene, thiadiazinylene, indolylene, isoindolylene, benzofurylene, ben∑othienylene, indazolylene, benzimidazolylene, benzthiazolylene, ben- zisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene, quinazolinylene, quinoliz- inylene, quinolinylene, isoquinolinylene, quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene, azepinylene, diazepinylene, acridinylene and the like. Heteroaryl is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydro- benzofuranylene, pyrrolinylene, pyrazolinylene, indolinylene, oxazolidinylene, oxazolinylene, oxazepinylene and the like.
The term "ArG1" as used herein is intended to include an aryl or arylene radical as applicable, where aryl or arylene are as defined above but limited to phenyl, biphenylyl, naphthyl, anthra- cenyl, phenanthrenyl, fluorenyl, indenyl, and azulenyl as well as the corrresponding divalent radicals.
The term "ArG2" as used herein is intended to include an aryl or arylene radical as applicable, where aryl or arylene are as defined above but limited to phenyl, biphenylyl, naphthyl, fluorenyl, and indenyl, as well as the corrresponding divalent radicals.
The term "Het1" as used herein is intended to include a heteroaryl or heteroarylene radical as applicable, where heteroaryl or heteroarylene are as defined above but limited to furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3- triazinyl, 1 ,2,4-triazinyl, 1,3,5- triazinyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadia∑olyl, 1 ,2,5-thiadiazolyl, 1 ,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, ben∑othienyl, indazolyl, benzimidazolyl, benzthiazolyl, benzisothia∑olyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carba∑olyl, a∑epinyl, di- azepinyl, acridinyl, thiazolidinyl, 2-thiooxothiazolidinyl, as well as the corrresponding divalent radicals.
The term "Het2" as used herein is intended to include a heteroaryl or heteroarylene radical as applicable, where heteroaryl or heteroarylene are as defined above but limited to furyl, thienyl pyrrolyl, pyrazolyl, 3-oxopyra∑olyl, oxazolyl, thia∑olyl, imida∑olyl, isoxa∑olyl, isothia∑olyl 1,2,3-tria∑olyl, 1,2,4-tria∑olyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyra∑inyl, 1,2,3 triazinyl, 1 ,2,4-triazinyl, 1,3,5- triazinyl, 1 ,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadia∑olyl, 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadia∑olyl, 1 ,3,4-thiadiazolyl tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, ben∑othienyl, ben∑imida∑olyl, ben∑thia zolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, quinolinyl, isoquinolinyl, quinoxaliny carbazolyl, thiazolidinyl, 2-thiooxothiazolidinyl, as well as the corrresponding divalent rad cals.
The term "Het3" as used herein is intended to include a heteroaryl or heteroarylene radical as applicable, where heteroaryl or heteroarylene are as defined above but limited to furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyridyl, tetrazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, quinolyl, isoqui- nolyl, quinoxalinyl, carbazolyl, thiazolidinyl, 2-thiooxothiazolidinyl, as well as the corrrespond' ing divalent radicals.
"Aryl-CrCe-alkyl", "heteroaryl-C-rCe-alkyl", "aryl-C2-C-6-alkenyr' etc. is intended to mean C C6- alkyl or C2-C6-alkenyl as defined above, substituted by an aryl or heteroaryl as defined above, for example:
Figure imgf000007_0001
The term "optionally substituted" as used herein means that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent the substituents may be the same or different.
Certain of the above defined terms may occur more than once in the structural formulae, and upon such occurrence each term shall be defined independently of the other. Furthermore, when using the terms "independently are" and "independently selected from" it should be understood that the groups in question may be the same or different. The term "protamine" as used herein refers to a mixture of strongly basic proteins usually obtained from fish sperm, "protamine" can refer to a relatively salt-free preparation of the proteins, sometimes termed protamine base. "Protamine" also refers to preparations comprising salts of the proteins. Even though concentrations are commonly given as concentration of protamine sulphate in this application, the person skilled in the art will readily be able to convert this to other protamine preparations.
The terms "treatment" and "treating" as used herein means the management and care of a patient for the purpose of combating a disease, disorder or condition. The term is intended to include the delaying of the progression of the disease, disorder or condition, the alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition. The patient to be treated is preferably a mammal, in particular a human being. The term "fragment" as used herein is intended to mean a bivalent chemical group The term "Neutral amino acid" as used herein is intended to mean any natural (codable) and non-natural amino acid, including α- or β-aminocarboxylic acids, including D-isomers of these (when applicable) without charges at physiologically relevant pH in the side chain, such as glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, aspargine, glutamine, cysteine, methionine, 3-aminobenzoic acid, 4-aminobenzoic acid or the like. The term "positively charged group" as used herein is intended to mean any pharmaceutically acceptable group that contains a positive charge at physiologically relevant pH, such as amino (primary, secondary and tertiary), ammonium and guanidino groups. The term " amino acid" as used herein is intended to mean mean any natural (codable) and non-natural α-aminocarboxylic acid, including D-isomers of these. The term "β amino acid" as used herein is intended to mean any β-aminocarboxylic acid, such as β-alanine, isoserine or the like.
When in the specification or claims mention is made of groups of compounds such as car- boxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imi- dazoles, triazoles, 4-cyano-1 ,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thia- zolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, naphthoic acids and salicylic acids, these groups of compounds are intended to include also derivatives of the compounds from which the groups take their name.
The term insulin as used herein refers to naturally produced insulin or recombinantly produced insulin. Recombinant insulin may be produced in any suitable host cell, for example the host cells may be bacterial, fungal (including yeast), insect, animal or plant cells. By "analogue of human insulin" as used herein (and related expressions) is meant human insulin in which one or more amino acids have been deleted and/or replaced by other amino acids, including non-codeable amino acids, or human insulin comprising additional amino acids, i.e. more than 51 amino acids, such that the resulting analogue possesses insulin activity
The expression "insulin derivative" as used herein (and related expressions) refers to human insulin or an analogue thereof in which at least one organic substituent is bound to one or more of the amino acids.
The term "desBSO" and the like as used herein is intended to mean meant a natural insulin B chain or an analogue thereof lacking the B30 amino acid residue.
The amino acid residues are indicated in the three letter amino acid code or the one letter amino code.
The terms "B1", "A1" and the like as used herein is intended to mean the amino acid residue in position 1 in the B chain of insulin or analogue thereof (counted from the N-terminal end) and the amino acid residue in position 1 in the A chain of insulin or analogue thereof (counted from the N-terminal end), respectively..
The term "phenolic compound" or similar expressions as used herein refers to a chemical compound in which a hydroxyl group is bound directly to a benzene or substituted benzene ring. Examples of such compounds include, but are not limited to, phenol, o-cresol, m-cresol and p-cresol.
The term "physiologically relevant pH" as used herein is intended to mean a pH of about 7.1 to 7.9.
The term "putative insulin hexamer" or similar expressions as used herein is refers to six insulin molecules which may combine to form an insulin hexamer. The chemical environment the insulin is in may determine that the insulin is not always in hexamer form. Thus, a ratio of e.g. 2 moles of Zinc ions per mole putative insulin hexamer corresponds to a ratio of 1 mole per 3 moles insulin monomer regardless of the state of the insulin.
Abbreviations:
4H3N 4-hydroxy-3-nitrobenzoic acid
AcOH acetic acid BT Benzotriazol-5-oyl
DMF N,N-Dimethylformamide
DMSO Dimethylsulfoxide
DIC Diisopropylcarbodiimide
EDAC 1-efhyl-3-(3'-dimethylarnino-propyI)carbodiimide,
Fmoc 9H-Fluorene-9-ylmethoxycarbonyl
HOAt 1 -hydroxy-7-azabenzotriazole
HOBT 1 -Hydroxyben∑otriazole
NMP N-methyl-2-pyrrolidone
TFA Trifluoroacetic acid
Abbreviations for non-natural amino acid residues:
Figure imgf000010_0001
DESCRIPTION OF THE INVENTION
Regular NPH-insulin is a crystalline complex between the R-state insulin hexamer and protamine (usually originating from salmon or herring). The hexamer component of the complex normally has additional small molecules bound to the known binding sites of the R6 insulin, i.e., preservative molecules such as phenol or m-cresol bind to six hydrophobic pockets formed in the dimer-dimer interfaces and anions from added buffers and salts (e.g. chloride) may bind to the two His610 Zn2+ sites residing on the 3-fold symmetry axis of the hexamer.
In solution, anions such as chloride bind to the R-state His Zn .2+ -site with modest affinity hence providing I little stabilization of the hexamer. However, ligands with substantially higher affinity for the H ,BIO 2n2+-site may be found and characterized by using a fluorescence based competit ;ιon assay which is based on the displacement of 5-(4- dimethylaminobenzylidene)-thiazolidine-2,4-dione from the R-state His810 Zn2+-site by the incoming ligand in question. The present invention is based on the discovery that NPH-insulin crystals may be formed in the presence of certain high-affinity ligands for the His810 Zn2+ sites of the R-state hexamer. When the ligands are present along with insulin, Zn2+, and optionally phenolic preservative, buffers and isotonicity agents, the NPH-insulin crystals still form upon combination with protamine. Alternatively, regular NPH-insulin crystals without presence of high-affinity ligands for the His810 Zn24 sites of the R-stafe hexamer may be formed initially and the ligand may then be incorporated by subsequent addition of the ligand to the crystalline suspension. The novel NPH-insulin complex has several advantages over regular NPH-insulin: When the crystalline suspension is injected subcutaneously into pigs, the absorption half-life is significantly increased compared to regular NPH-insulin (see example 1011). Moreover, the action profile of the novel NPH-preparation is longer and smoother than that obtained with regular NPH- insulin. Finally, the physical and chemical stability is significantly enhanced over the reference preparation.
Suitable ligands according to this invention are characterized by a) having high affinity to HisB1° Zn + site of the R-state hexamer (e.g. K < 10 μM) as measured in the TZD-assay for quantitation of ligands binding to the R-state His610 Zn2+ or the 4H3N-assay and b) being capable of forming NPH crystals when included along with the zinc-insulin in the preparation, i.e. the presence of the bound ligand does not impede normal complex formation with protamine (co-crystallization mode). Alternatively, the regular insulin-protamine crystalline complex without presence of high-affinity ligands for the His810 Zn + sites of the R-state hexamer may be formed initially and the ligand incorporated subsequently by addition of the ligand to the crystalline suspension (soaking mode)
The present invention thus provides in embodiment 1 a pharmaceutical preparation comprising
• Insulin
• Protamine
• Zinc ions
® ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer, wherein said ligand is selected from the group consisting of carboxylates, dithiocar- boxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imida∑oles, tria- zoles, 4-cyano-1 ,2,3-triazoIes, benzimidazoles, benzotriazoles, purines, thymines, thiazolidinediones, fetrazoles, 5-mercaptotetra∑oles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, naphthoic acids and salicylic acids, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
Embodiment 2. A pharmaceutical preparation according to embodiment 1 wherein the insulin preparation comprises 60 to 3000 nmol/ml of insulin.
Embodiment 3. A pharmaceutical preparation according to embodiment 2 wherein the insulin preparation comprises 240 to 1200 nmol/ml of insulin.
Embodiment 4. A pharmaceutical preparation according to embodiment 3 wherein the insulin preparation comprises about 600 nmol/ml of insulin.
Embodiment 5. A pharmaceutical preparation according to any one of the embodiments 1 to
4 wherein the insulin is selected from the group consisting of human insulin, an analogue of human insulin, a derivative of human insulin, and combinations of any of these.
Embodiment 6. A pharmaceutical preparation according to embodiment 5 wherein the insulin is an analogue of human insulin selected from the group consisting of i.An analogue wherein position B28 is Asp, Glu, Lys, Leu, Val, or Ala and position B29 is Lys or Pro; ii.An analogue wherein position B3 is Lys and position B29 is Glu; and iii.des(B28-B30), des(B27) or des(B30) human insulin. Embodiment 7. A pharmaceutical preparation according to embodiment 6, wherein the insulin is an analogue of human insulin wherein position B28 is Asp or Lys, and position B29 is Lys or Pro.
Embodiment 8. A pharmaceutical preparation according to embodiment 6 wherein the insulin is des(B30) human insulin.
Embodiment 9. A pharmaceutical preparation according to embodiment 5 wherein the insulin is a derivative of human insulin having one or more lipophilic substituents. Embodiment 10. A pharmaceutical preparation according to embodiment 9 wherein the insulin derivative is selected from the group consisting of B29-Nε-myristoyl-des(B30) human insulin, B29-Nε-palmitoyl-des(B30) human insulin, B29-Nε-myristoyl human insulin, B29-Nε- palmitoyl human insulin, B28-Nε-myristoyl Lys628 Pro629 human insulin, B28-Nε-palmitoyl Lys828 Pro829 human insulin, B30-Nε-myristoyl-Thr829Lys830 human insulin, B30-Nε-palmitoyl- Thr^Lys830 human insulin, B29-Nε-(N-palmitoyl-γ-glutamyl)-des(B30) human insulin, B29-Nε- (N-lithocholyI-γ-gluiamyI)-des(B30) human insulin, B29-Nε-(ω-carboxyheptadecanoyl)- des(B30) human insulin and B29-Nε-(ω-carboxyheptadecanoyl) human insulin. Embodiment 11. A pharmaceutical preparation according to embodiment 10 wherein the insulin derivative is B29-Nε-myristoyl-des(B30) human insulin.
Embodiment 12. A pharmaceutical preparation according to any one of the embodiments 1 to 11 wherein the protamine is protamine sulphate.
Embodiment 13. A pharmaceutical preparation according to embodiment 13 wherein the concentration of protamine sulphate is from 0.05-3 mg/mL.
Embodiment 14. A pharmaceutical preparation according to embodiment 14 wherein the concentration of protamine sulphate is from 0.1-0.6 mg/mL.
Embodiment 15. A pharmaceutical preparation according to any one of the embodiments 1 to 15 wherein the amount of zinc ions is 2-6 moles per mole putative insulin hexamer. Embodiment 16. A pharmaceutical preparation according to embodiment 16 wherein the amount of zinc ions is 2 to 3 moles per mole putative insulin hexamer. Embodiment 17. A pharmaceutical preparation according to any one of the embodiments 1 to 17 wherein the ratio of ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer to zinc ions is 1:3 to 3:1.
Embodiment 18. A pharmaceutical preparation according to embodiment 18 wherein the ratio of ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer to zinc ions is 1:2 to 2:1.
Embodiment 19. A pharmaceutical preparation according to embodiment 19 wherein the ratio of ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer to zinc ions is 1 :.2 to 1.2:1.
Embodiment 20. A pharmaceutical preparation according to any one of the embodiments 1 to 20 wherein the ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer is a chemical structure selected from the group consisting of carboxylates, dithiocarboxy- lates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles, 4- cyano-1 ,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thymines, thiazolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines, N-hydroxyazoles, hydantoines, thiohydan- toines, naphthoic acids and salicylic acids.
Embodiment 21. A pharmaceutical preparation according to embodiment 21 wherein the ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer is a chemical structure selected from the group consisting of benzotria∑oles, 3-hydroxy 2-napthoic acids, salicylic acids, tetrazoles or thiazolidinediones.
Embodiment 22. A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn2+ site of an R-state insulin hexamer is
Figure imgf000014_0001
wherein
X is =O, =S or =NH
Y is -S-, -O- or -NH-
R1 and R4 are independently selected from hydrogen or GrC6-alkyl,
R2 is hydrogen or Cι-C6-alkyl or aryl, R1 and R2 may optionally be combined to form a double bond,
R3 and R5 are independently selected from hydrogen, halogen, aryl, CrC6-alkyl, or
-C(0)NR11R12,
A and B are independently selected from CrCe-alkyl, aryl, aryl-Cι-C6-alkyl, aryl-C2-C6-alkenyl or heteroaryl, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R6 and the aryl or heteroaryl is optionally substituted with up to four substituents R7, R8, R9, and R10,
A and R3 may be connected through one or two valence bonds, B and R5 may be connected through one or two valence bonds,
R6 is independently selected from halogen, -CN, -CF3, -OCF3) aryl, -COOH and -NH2, R7, R8, R9 and R10 are independently selected from
• hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3) -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(O)2CF3, -OS(0)2CF3, -SCF3, -NO2, -OR11, -NR11R12, -SR11, -NR11S(O)2R12, -S(O)2NR11R12, -S(O)NR11R12, -S(O)R11, -S(O)2R11, -OS(O)2 R11, -C(O)NR11R12, -OC(O)NR 1R12, -NR11C(O)R12, -CH2C(O)NR11R12,
-OC C6-alkyl-C(O)NR11R12, -CH2OR11, -CH2OC(O)R11, -CH2NR11R12, -OC(O)R11, -OC C15-alkyl-C(O)OR11, -OC C6-aIkyl-OR11, -SC C6-alkyl-C(O)OR11 -C2-C6-aIkenyl-C(=O)OR11 , -NR11-C(=O)-CrC6-alkyl-C(=O)OR11 ,
-NR11-C(=O)-C C6-aIkenyl-C(=O)OR11 , -C(O)OR11, C(O)R11, or -C2-C6-alkenyl- C(=O)R11, =0, or -C2-C6-alkenyl-C(=O)-NR11R12,
oC C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, each of which may optionally be substituted with one or more substituents independently selected from R13, • aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-Cι-C6-alkoxy, aryl-C-i-Cβ-alkyl, aryl-C2-C6-alkenyl, aroyl-Ca-Ce-alkenyl, aryI-C2-C6-alkynyl, heteroaryl, heteroaryl-Cr Ce-alkyl, heteroaryl-C2-C6-alkenyl, heteroaryl-C2-C6-alkynyl, or C3-C6 cycloalkyl,
of which each cyclic moiety may optionally be substituted with one or more substituents independently selected from R14,
R11 and R12 are independently selected from hydrogen, OH, Cι-C2o-alkyl, aryl-d-Ce-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R15, and the aryl groups may optionally be substituted one or more substituents independently selected from R16; R11 and R12 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R13 is independently selected from halogen, -CN, -CF3, -OCF3, -OR11, -C(O)OR11 , -NR11R12, and -C(O)NR11R12,
R14 is independently selected from halogen, -C(O)OR1\ -CH2C(O)OR11, -CH2OR11, -CN, - CF3, -OCF3, -NO2, -OR11, -NR11R12, S(O)2R1\ aryl and C C6-alkyl,
R15 is independently selected from halogen, -CN, -CF3, -OCF3, -OCrC6-alkyl, -C(O)OC C6- alkyl, -COOH and -NH2)
R16 is independently selected from halogen, -0(0)0^ -C6-alkyl, -COOH, -CN, -CF3, -OCF3, - NO2, -OH, -OCrC6-alkyl, -NH2, C(=O) or C C6-alkyl, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
Embodiment 23. A pharmaceutical composition according to embodiment 23 wherein X is =O or =S.
Embodiment 24. A pharmaceutical composition according to embodiment 24 wherein X is =O.
Embodiment 25. A pharmaceutical composition according to embodiment 24 wherein X is =S. Embodiment 26. A pharmaceutical composition according to any one of the embodiments 23 to 26 wherein Y is -O- or -S-.
Embodiment 27. A pharmaceutical composition according to embodiment 27 wherein Y is
-O-.
Embodiment 28. A pharmaceutical composition according to embodiment 27 wherein Y is
-S-.
Embodiment 29. A pharmaceutical composition according to any one of the embodiments 23 to 30 wherein A is aryl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
Embodiment 30. A pharmaceutical composition according to embodiment 31 wherein A is selected from ArG1 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
Embodiment 31. A pharmaceutical composition according to embodiment 32 wherein A is phenyl or naphtyl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
Embodiment 32. A pharmaceutical composition according to embodiment 33 wherein A is
Figure imgf000016_0001
33. A pharmaceutical composition according to embodiment 33 wherein A is phenyl.
Embodiment 34. A pharmaceutical composition according to any one of the embodiments 23 to 30 wherein A is heteroaryl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
Embodiment 35. A pharmaceutical composition according to embodiment 36 wherein A is selected from Het1 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
Embodiment 36. A pharmaceutical composition according to embodiment 37 wherein A is selected from Het2 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
Embodiment 37. A pharmaceutical composition according to embodiment 38 wherein A is selected from Het3 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different. Embodiment 38. A pharmaceutical composition according to embodiment 39 wherein A is selected from the group consisting of indolyl, benzofuranyl, quinolyl, furyl, thienyl, or pyrrolyl, wherein each heteroaryl may optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
Embodiment 39. A pharmaceutical composition according to embodiment 39 wherein A is benzofuranyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
Embodiment 40. A pharmaceutical composition according to embodiment 41 wherein A is
Figure imgf000017_0001
Embodiment 41. A pharmaceutical composition according to embodiment 39 wherein A is carbazolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different. Embodiment 42. A pharmaceutical composition according to embodiment 43 wherein A is
Figure imgf000017_0002
Embodiment 43. A pharmaceutical composition according to embodiment 39 wherein A is quinolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different. Embodiment 44. A pharmaceutical composition according to embodiment 45 wherein A is
Figure imgf000017_0003
Embodiment 45. A pharmaceutical composition according to embodiment 39 wherein A is indolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different. Embodiment 46. A pharmaceutical composition according to embodiment 47 wherein A is
Figure imgf000018_0001
Embodiment 47. A pharmaceutical composition according to any one of the embodiments 23 to 48 wherein R1 is hydrogen.
Embodiment 48. A pharmaceutical composition according to any one of the embodiments 23 to 49 wherein R2 is hydrogen.
Embodiment 49. A pharmaceutical composition according to any one of the embodiments 23 to 48 wherein R1 and R2 are combined to form a double bond.
Embodiment 50. A pharmaceutical composition according to any one of the embodiments 23 to 51 wherein R3 is d-Cβ-alkyl, halogen, or C(O)NR16R17.
Embodiment 51. A pharmaceutical composition according to embodiment 52 wherein R3 is d-Ce-alkyl or C(O)NR16R17.
Embodiment 52. A pharmaceutical composition according to embodiment 53 wherein R3 is methyl.
Embodiment 53. A pharmaceutical composition according to any one of the embodiments 23 to 30 wherein B is phenyl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
Embodiment 54. A pharmaceutical composition according to any one of the embodiments 23 to 30 or 55 wherein R4 is hydrogen.
Embodiment 55. A pharmaceutical composition according to any one of the embodiments 23 to 30 or 55 to 56 wherein R5 is hydrogen.
Embodiment 56. A pharmaceutical composition according to any one of the embodiments 23 to 57 wherein R6 is aryl.
Embodiment 57. A pharmaceutical composition according to embodiment 58 wherein R6 is phenyl.
Embodiment 58. A pharmaceutical composition according to any one of the embodiments 23 to 59 wherein R7, R8, R9 and R 0 are independently selected from
o hydrogen, halogen, -N02l -OR11, -NR11R12, -SR11, -NR11S(O)2R12, -S(0)2NR11R12, -S(O)NR11R12, -S(O)R1\ -S(O)2R11, -OS(O)2 R11, -NR11C(O)R12, -CH2OR11, - CH2OC(O)R1\ -CH2NR11R12, -OC(O)R11, -OC C6-alkyl-C(O)OR11, -OC C6- alkyl-C(0)NR11R12, -OC C6-alkyI-OR11, -SC C6-alkyl-C(O)OR11, -C2-C6-alkenyl- C(=O)OR11, -C(O)OR11, or-C2-C6-alkenyl-C(=O)R11,
oC C6-alkyl, C2-C6-aIkenyl or C2-C6-alkynyl, which may each optionally be substituted with one or more substituents independently selected from R13
oaryl, aryloxy, aroyl, arylsulfanyl, aryl-d-Gβ-alkoxy, aryl-Cι-Cβ-alkyl, aryl-C2- Cβ-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-d-Ge-alkyl, wherein each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14
Embodiment 59. A pharmaceutical composition according to embodiment 60 wherein R7, R8, R9 and R10 are independently selected from
• hydrogen, halogen, -NO2, -OR11, -NR11R12, -SR11, -S(O)2R11, -OS(O)2 R , - CH2OC(O)R11, -OC(O)R11, -OCrC6-alkyl-C(O)OR11, -OCrC6-alkyl-OR11, -SCrC6- alkyl-C(O)OR11, -C(O)OR11, or-C2-C6-alkenyl-C(=O)R11,
• d-Ce-alkyl or d-C6-alkenyl which may each optionally be substituted with one or more substituents independently selected from R13
• aryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-d-Ce-alkyl, heteroaryl,
of which each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14
Embodiment 60. A pharmaceutical composition according to embodiment 61 wherein R7, R8, R9 and R10 are independently selected from
• hydrogen, halogen, -NO2> -OR11, -NR11R12, -SR11, -S(O)2R11, -OS(0)2 R11, - CH2OC(O)R11, -OC(O)R11, -Od-C6-alkyl-C(O)OR11, -OC C6-alkyl-OR11, -SCrC6- alkyl-C(0)OR11, -C(0)OR11, or-C2-C6-alkenyl-C(=O)R11,
oCι-C6-alkyl or d-C6- which may each optionally be substituted with one or more substituents independently selected from R13 • aryl, aryloxy, aroyl, aryl-C C6-alkoxy, aryl-d-Ce-alkyl, heteroaryl,
of which each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14.
Embodiment 61. A pharmaceutical composition according to embodiment 62 wherein R7, R8, R9 and R10 are independently selected from ohydrogen, halogen, -OR11, -OCrC6-alkyl-C(0)OR11, or -C(O)OR11,
o C G6-alkyl which may each optionally be substituted with one or more substituents independently selected from R13
• aryl, aryloxy, aryI-d-C6-aIkoxy,
of which each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14. Embodiment 62. A pharmaceutical composition according to embodiment 63 wherein R7, R8, R9 and R10 are independently selected from
• hydrogen, halogen, -OR11, -OC C6-alkyl-C(O)OR11, or -C(O)OR11,
• d-C6-alkyl which may optionally be substituted with one or more substituents independently selected from R13
• phenyl, phenyloxy, phenyl-Cι-C6-alkoxy, wherein each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14.
Embodiment 63. A pharmaceutical composition according to any one of the embodiments 23 to 65 wherein R11 and R12 are independently selected from hydrogen, Cι-C20-alkyl, aryl or aryl-Cι-C6-alkyl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R15, and the aryl groups may optionally be substituted one or more substituents independently selected from R16; R11 and R12 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds. Embodiment 64. A pharmaceutical composition according to embodiment 66 wherein R11 and R12 are independently selected from hydrogen, d-do-alkyl, aryl or aryl-d-Ce-alkyl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R15, and the aryl groups may optionally be substituted one or more substituents independently selected from R16.
Embodiment 65. A pharmaceutical composition according to embodiment 67 wherein R11 and R12 are independently selected from phenyl or phenyl-d-C6-alkyl.
Embodiment 66. A pharmaceutical composition according to embodiment 67 wherein one or both of R11 and R12 are methyl.
Embodiment 67. A pharmaceutical composition according to any one of the embodiments 23 to 69 wherein R13 is independently selected from halogen, CF3, OR11 or NR11R12. Embodiment 68. A pharmaceutical composition according to embodiment 70 wherein R13 is independently selected from halogen or OR11.
Embodiment 69. A pharmaceutical composition according to embodiment 71 wherein R 3 is OR11.
Embodiment 70. A pharmaceutical composition according to any one of the embodiments 23 to 72 wherein R14 is independently selected from halogen, -C(O)OR11, -CN, -CF3, -OR11, S(O)2R11, and C C6-alkyl.
Embodiment 71. A pharmaceutical composition according to embodiment 73 wherein R14 is independently selected from halogen, -C(O)OR11, or -OR11.
Embodiment 72. A pharmaceutical composition according to any one of the embodiments 23 to 74 wherein R15 is independently selected from halogen, -CN, -CF3, -C(O)OC C6-alkyl,and -COOH.
Embodiment 73. A pharmaceutical composition according to embodiment 75 wherein R15 is independently selected from halogen or -C(O)Od-C6-alkyl.
Embodiment 74. A pharmaceutical composition according to any one of the embodiments 23 to 76 wherein R16 is independently selected from halogen, -C(O)Od-C6-alkyl, -COOH, -NO2, -pC C6-alkyl, -NH2, C(=O) or C C6-alkyl.
Embodiment 75. A pharmaceutical composition according to embodiment 77 wherein R16 is independently selected from halogen, -C(O)Od-G6-alkyl, -COOH, -NO2, or d-G6-alkyl. Embodiment 76. A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn2+ site of an R-state insulin hexamer is
Figure imgf000022_0001
wherein
R19 is hydrogen or Cι-C6-aIkyl,
R20 is hydrogen or d-Cβ-alkyl,
D and F are a valence bond or C C6-alkylene optionally substituted with one or more substituents independently selected from R72,
R72 is independently selected from hydroxy, d-C6-alkyl, or aryl,
E is C-i-Cβ-alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents R21, R22 and R23,
G is d-Cβ-alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents R24, R25 and R26,
R17, R18, R21, R22, R23, R24, R25 and R26 are independently selected from
• hydrogen, halogen, -CN, -CH2CN, -CHF2> -CF3, -OCF3, -OCHF2> -OCH2CF3, -OCF2CHF2) -S(O)2CF3, -SCF3, -NO2, -OR27, -NR27R28, -SR27, -NR27S(O)2R28, -S(O)2NR27R28, -S(O)NR27R28, -S(O)R27, -S(O)2R27, -C(O)NR27R28, -OC(O)NR27R28, -NR27C(O)R28, -NR27C(O)OR28, -CH2C(O)NR27R28, -OCH2C(O)NR27R28, -CH2OR27, -CH2NR27R28, -OC(O)R27, -OC C6-alkyl-C(O)OR27, -Sd-C6-alkyl-C(O)OR27, -C2-C6- alkenyl-C(=O)OR27, -NR27-C(=O)-C C6-alkyl-C(=O)OR27, -NR27-C(=O)-C C6- alkenyl-C(=O)OR27, -C(=O)NR27-C1-C6-alkyl-C(=O)OR27, -d-C6-alkyl-C(=O)OR27,or -C(O)OR27,
• d-Ce-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
which may optionally be substituted with one or more substituents independently selected from R29, • aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-Cι-C6-alkoxy, aryl-d-C6-alkyl, aryl-C2- C6-alkenyl, aryl-C2-C6-aIkynyl, heteroaryl, heteroaryl-Cι-C6-alkyl, heteroaryl-C2-C6- alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30,
R27 and R28 are independently selected from hydrogen, d-G6-alkyl, aryl-d-Cθ-alkyl or aryl, or R27 and R28 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R29is independently selected from halogen, -CN, -CF3, -OCF3, -OR27, and -NR 7R28,
R30 is independently selected from halogen, -C(O)OR27, -CN, -CF3, -OCF3, -NO2, -OR27,
-NR27R28 and d-Cβ-aIkyl, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
Embodiment 77. A pharmaceutical composition according to embodiment 79 wherein D is a valence bond.
Embodiment 78. A pharmaceutical composition according to embodiment 79 wherein D is d-C6-alkylene optionally substituted with one or more hydroxy, d-Cβ-alkyl, or aryl.
Embodiment 79. A pharmaceutical composition according to any one of the embodiments 79 to 81 wherein E is aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents independently selected from R21, R22 and R23.
Embodiment 80. A pharmaceutical composition according to embodiment 82 wherein E is aryl optionally substituted with up to three substituents independently selected from R21, R22 and R23.
Embodiment 81. A pharmaceutical composition according to embodiment 83 wherein E is selected from ArG1 and optionally substituted with up to three substituents independently selected from R21, R22 and R23.
Embodiment 82. A pharmaceutical composition according to embodiment 84 wherein E is phenyl optionally substituted with up to three substituents independently selected from R21,
R22 and R23. Embodiment 83. A pharmaceutical composition according to embodiment 85 wherein the ligand that binds reversibly to a HisBlO Zn2+ site of an R-state insulin hexamer is
Figure imgf000024_0001
Embodiment 84. A pharmaceutical composition according to any one of the embodiments 79 to 86 wherein R2\ R22 and R23 are independently selected from
• hydrogen, halogen, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -SCF3, - NO2, -OR27, -NR27R28, -SR27, -C(O)NR27R28, -OC(O)NR27R28, -NR27C(O)R28, -NR27C(O)OR28, -CH2C(O)NR27R28, -OCH2C(O)NR27R28, -CH2OR27, -CH2NR27R28, -OC(O)R27, -Od-C6-alkyl-C(O)OR27, -SC C6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=O)OR27, -NR27-C(=O)-C C6-alkyl-C(=O)OR27, -NR2 -C(=O)-d-C6- alkenyl-C(=O)OR27-, -C(=O)NR27-d-C6-alkyl-C(=O)OR27, -Cι-C6-aIkyl-C(=O)OR27, or -C(O)OR27,
• d-d-alky!, C2-C6-alkenyl or C2-C6-alkynyl,
which may optionally be substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-Ci-Ce-alkoxy, ary|-d-C6-alkyl, aryl-C2- Ce-alkenyl, aryl-C2-C6-aIkynyl, heteroaryl, heteroaryl-Cι-C6-alkyl, heteroaryl-C2-C6- alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 85. A pharmaceutical composition according to embodiment 87 wherein R21,
R22 and R23 are independently selected from
o hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(O)R28, -NR27C(O)OR28, -OC(O)R27, -OC C6-alkyl-C(O)OR27, -SC C6-alkyl-C(O)OR27, -C2-C6-aIkenyl- C(=O)OR27, -C(=O)NR27-C C6-alkyl-C(=O)OR27, -C C6-alkyl-C(=O)OR27, or -C(O)OR27,
o d-Cβ-alkyl optionally substituted with one or more substituents independently selected from R29
©aryl, aryloxy, aroyl, aryl-d-G6-alkoxy, aryl-d-C6-alkyl, heteroaryl, heteroaryl-GrCβ-
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 86. A pharmaceutical composition according to embodiment 88 wherein R21,
R22 and R23 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(O)R28, -NR27C(O)OR28, -OC(O)R27, -OC1-C6-alkyl-C(O)OR27, -SCrC6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=O)OR27, -C(=O)NR27-d-C6-alkyl-C(=O)OR27, -CrC6-alkyl-C(=O)OR27, or -C(O)OR27,
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aroyl, aryl-CrC6-alkoxy, aryl-Crd-alkyl, heteroaryl, heteroaryl-CrC6- alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 87. A pharmaceutical composition according to embodiment 89 wherein R21,
R22 and R23 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(O)R28, -NR27C(O)OR28, -OC(O)R27, -OC C6-alkyl-C(O)OR27, -SCrC6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=O)OR27, -C(=O)NR27-CrC6-alkyl-C(=O)OR27, -d-C6-alkyI-C(=O)OR27, or -C(O)OR27, • methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
oArG1, ArG1-O-, ArG1-C(O)-, ArG1-C C6-alko) y, ArG1-d-Ce-alkyi, Het3, Het3-d-
C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 88. A pharmaceutical composition according to embodiment 90 wherein R21, R22 and R23 are independently selected from
o hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27G(0)R28, -NR27C(0)OR28, -OC(O)R27, -Od-C6-alkyl-C(O)OR27, -Sd-C6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=O)OR27, -C(=O)NR27-Cι-C6-alkyl-C(=O)OR27, -d-C6-alkyl-C(=O)OR27, or -C(O)OR27,
• d-C6-alkyl optionally substituted with one or more substituents independently selected from R29
• phenyl, phenyloxy, phenyl-d-C6-alkoxy, phenyl-CrCβ-alkyl, of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 89. A pharmaceutical composition according to any one of the embodiments 79 to 91 wherein R19 is hydrogen or methyl.
Embodiment 90. A pharmaceutical composition according to embodiment 92 wherein R19 is hydrogen.
Embodiment 91. A pharmaceutical composition according to any one of the embodiments 79 to 93 wherein R27 is Hydrogen, d-Cβ-alkyl or aryl.
Embodiment 92. A pharmaceutical composition according to embodiment 94 wherein R27 is hydrogen or d-C6-alkyl.
Embodiment 93. A pharmaceutical composition according to any one of the embodiments 79 to 95 wherein R28 is hydrogen or G C6-alkyl.
Embodiment 94. A pharmaceutical composition according to embodiment 79 wherein F is a valence bond.
Embodiment 95. A pharmaceutical composition according to embodiment 79 wherein F is d- Ce-alkylene optionally substituted with one or more hydroxy, CrC6-alkyIf or aryl. Embodiment 96. A pharmaceutical composition according to any one of the embodiments 79 or 97 to 98 wherein G is C C6-aIkyl or aryl, wherein the aryl is optionally substituted with up to three substituents R24, R25 and R26.
Embodiment 97. A pharmaceutical composition according to any one of the embodiments 79 or 97 to 98 wherein G is d-G6-alkyl or ArG1, wherein the aryl is optionally substituted with up to three substituents R24, R25 and R26.
Embodiment 98. A pharmaceutical composition according to embodiment 99 wherein G is d-Cβ-alkyl.
Embodiment 99. A pharmaceutical composition according to embodiment 101 wherein G is phenyl optionally substituted with up to three substituents R24, R25 and R26.
Embodiment 100. A pharmaceutical composition according to any one of the embodiments
79 to 102 wherein R24, R25 and R26 are independently selected from
• hydrogen, halogen, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -SCF3, - NO2, -OR27, -NR27R28, -SR27, -C(O)NR27R28, -OC(O)NR27R28, -NR27C(O)R28, -NR27C(O)OR28, -CH2C(O)NR27R28, -OCH2C(O)NR27R28, -CH2OR27, -CH2NR27R28, -OC(O)R27, -OC1-C6-alkyl-C(O)OR27, -SCrC6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=O)OR27, -NR27-C(=O)-d-C6-alkyl-C(=O)OR27, -NR27-C(=O)-C C6- alkenyl-C(=O)OR27-, -C(=O)NR27-C1-C6-alkyl-C(=O)OR27, -d-C6-alkyl-C(=O)OR27, or -C(O)OR27,
• C C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
which may optionally be substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-Cι-C6-alkoxy, aryl-C Ce-alkyl, aryl-C2- C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-CrCe-alkyl, heteroaryl-C2-C6- alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30. Embodiment 101. A pharmaceutical composition according to embodiment 103 wherein R24,
R25 and R26 are independently selected from • hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(O)R28, -NR27C(O)OR28, -OC(O)R27, -Od-C6-alkyl-C(O)OR27, -Sd-C6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=0)OR27, -C(=O)NR27-d-C6-alkyl-C(=O)OR27, -C1-C6-aIkyl-C(=O)OR27, or -C(0)OR27,
oC Cβ-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
which may optionally be substituted with one or more substituents independently selected from R29
oaryl, aryloxy, aryloxycarbonyl, aroyl, aryl-Cι-C6-alkoxy, aryl-GrGβ-alkyl, aryl-C2- C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-Cι-C6-alkyl, heteroaryl-C2-C6- alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 102. A pharmaceutical composition according to embodiment 104 wherein R24,
R25 and R26 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(O)R28, -NR27C(O)OR28, -OC(O)R27, -OC C6-alkyl-C(O)OR27, -Sd-Ce-alkyl-CfO R27, -C2-C6-alkenyl- C(=O)OR27, -C(=O)NR27-Cι-C6-alkyl-C(=O)OR27, -d-C6-alkyl-C(=O)OR27, or -C(O)OR27,
• CrC6-alkyl optionally substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aroyl, aryl-CrCe-alkoxy, aryl-d-Ce-alkyl, heteroaryl, heteroaryl-C C6- alkyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 103. A pharmaceutical composition according to embodiment 105 wherein R21,
R22 and R23 are independently selected from • hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(O)R28, -NR27C(O)OR28, -OC(O)R27, -OC1-C6-alkyl-C(O)OR27, -SC1-C6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=O)OR27, -C(=O)NR27-C1-C6-alkyl-C(=O)OR27, -d-C6-alkyl-C(=O)OR27, or -C(0)OR27,
o methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
°ArG1, ArG1-0-, ArG1-G(0)-, ArGI-d-Ce-alkoxy, ArG1-d-Cβ-al yl, Het3, Hβt3-d-
C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 104. A pharmaceutical composition according to embodiment 106 wherein R21, R22 and R23 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(O)R28, -NR27C(O)OR28, -OC(O)R27, -OC C6-alkyl-C(O)OR27, -Sd-C6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=O)OR27, -C(=O)NR27-d-C6-alkyl-C(=O)OR27, -d-C6-alkyl-C(=O)OR27, or -C(O)OR27,
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• ArG1 , ArG1-O-, ArG1-C(O)-, ArG1-d-C6-alkoxy, ArG1-C C6-alkyl, Het3, Het3-C C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 105. A pharmaceutical composition according to embodiment 107 wherein R21,
R22 and R23 are independently selected from
o hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(O)R28, -NR27C(O)OR28, -OC(O)R27. -OC1-C6-aIkyl-C(O)OR27 I -SC C6-alkyl-C(O)OR27, -C2-C6-alkenyl- C(=O)OR27, -C(=O)NR27-C1-C6-alkyI-C(=O)OR27, -CrC6-alkyl-C(=O)OR27, or -C(O)OR27, • methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
oArG1, ArG1-0-, ArG1-C C6-alkoxy, ArG1-d-C6-alkyl, of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 106. A pharmaceutical composition according to any one of the embodiments 79 or 97 to 108 wherein R20 is hydrogen or methyl.
Embodiment 107. A pharmaceutical composition according to embodiment 109 wherein R20 is hydrogen.
Embodiment 108. A pharmaceutical composition according to any one of the embodiments 79 or 97 to 110 wherein R27 is hydrogen, d-C6-alkyl or aryl.
Embodiment 109. A pharmaceutical composition according to embodiment 111 wherein R27 is hydrogen or d-C6-alkyl or ArG
Embodiment 110. A pharmaceutical composition according to embodiment 112 wherein R27 is hydrogen or d-Cβ-alkyl.
Embodiment 111. A pharmaceutical composition according to any one of the embodiments 79 or 97 to 112 wherein R28 is hydrogen or CrC6-alkyl.
Embodiment 112. A pharmaceutical composition according to embodiment 79 wherein R17 and R18 are independently selected from
• hydrogen, halogen, -CN, -CF3, -OCF3, -NO2, -OR27, -NR27R28, -SR27, -S(O)R27, -S(O)2R27, -C(O)NR27R28, -CH2OR27, -OC(O)R27, -OCrC6-alkyl-C(O)OR27, -Sd-C6- alkyl-C(O)OR27, or -C(O)OR27,
• d-Ce-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, optionally substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-d-Ce-alkyl, heteroaryl, heteroaryl-C C6- alkyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 113. A pharmaceutical composition according to embodiment 115 wherein R17 and R18are independently selected from • hydrogen, halogen, -CN, -CF3, -NO2) -OR27, -NR27R28, or -C(O)OR27,
od-C6-alkyl optionally substituted with one or more substituents independently selected from R29
oaryl, aryloxy, aroyl, aryl-d-G6-alkoxy, aryl-CrCβ-alkyl, heteroaryl, heteroaryl-d-G6- alkyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 114. A pharmaceutical composition according to embodiment 116 wherein R17 and R18 are independently selected from
• hydrogen, halogen, -CN, -CF3, -NO2, -OR27, -NR27R28, or -C(O)OR27
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aroyl, aryl-d-C6-alkoxy, aryl-CrC6-alkyl, heteroaryl, heteroaryl-CrCe- alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 115. A pharmaceutical composition according to embodiment 117 wherein R17 and R18 are independently selected from
• hydrogen, halogen, -CN, -CF3, -NO2, -OR27, -NR27R28, or -C(O)OR27
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• ArG1, ArG1-O-, ArG1-C(O)-, ArG1-C C6-alkoxy, ArG1-d-C6-alkyl, Het3, Het3-C C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 116. A pharmaceutical composition according to embodiment 118 wherein R17 and R18 are independently selected from o hydrogen, halogen, -CH, -CF3, -NO2, -OR27, -NR27R28, or-C(O)OR27 o d-Ce-alkyl optionally substituted with one or more substituents independently selected from R29
• phenyl, phenyloxy, phenyl-d-C6-alkoxy, phenyl-C C6-alkyl, of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
Embodiment 117. A pharmaceutical composition according to any one of the embodiments 79 to 119 wherein R27 is hydrogen or CrCβ-alkyl.
Embodiment 118. A pharmaceutical composition according to embodiment 120 wherein R27 is hydrogen, methyl or ethyl.
Embodiment 119. A pharmaceutical composition according to any one of the embodiments 79 to 121 wherein R28 is hydrogen or C G6-aIkyl.
Embodiment 120. A pharmaceutical composition according to embodiment 122 wherein R28 is hydrogen, methyl or ethyl.
Embodiment 121. A pharmaceutical composition according to any one of the embodiments 79 to 123 wherein R72 is -OH or phenyl.
Embodiment 122. A pharmaceutical composition according to embodiment 79 wherein the ligand that binds reversibly to a HisBlO Zn2+ site of an R-state insulin hexamer is
Figure imgf000032_0001
Embodiment 123. A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn2+ site of an R-state insulin hexamer is of the form H-l-J
wherein H is
Figure imgf000032_0002
wherein the phenyl, naphthalene or benzocarbazole rings are optionally substituted with one or more substituents independently selected from R31
I is selected from
° a valence bond,
• -CH2N(R32)- or -SO2N(R33)-, o
Figure imgf000033_0001
Z1 is S(0)2 or GH2, Z2 is -NH-, -O-or -S-, and n is 1 or 2,
J is o d-Ce-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may each optionally be substituted with one or more substituents selected from R34, o ryl, aryloxy, aryl-oxycarbonyl-, aroyl, aryl-d-Ce-alkoxy-,
Figure imgf000033_0002
aryl-C2- C6-alkenyl-, aryl-C2-Ge-aIkynyl-, heteroaryl, heteroaryl-CrGe-alkyl-, heteroaryl-C2-C6- alkenyl- or heteroaryl-C2-C6-alkynyl-, wherein the cyclic moieties are optionally substituted with one or more substituents selected from R37, • Hydrogen,
R31 is independently selected from hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(O)2CF3, -SCF3, -NO2, -OR35, -C(O)R35, -NR35R36, -SR35, -NR35S(O)2R36, -S(O)2NR35R36, -S(O)NR35R36, -S(O)R35, -S(O)2R35, -C(O)NR35R36, -OC(O)NR35R36, -NR35C(O)R36, -CH2C(O)NR35R36, -OCH2C(O)NR35R36, -CH2OR35, -CH2NR35R36, -OC(O)R35, -Od-C6-alkyl-C(O)OR35, -SC1-C6-alkyl-C(O)OR35 -C2-C6-alkenyl- C(=O)OR35, -NR35-C(=O)-d-C6-alkyl-C(=O)OR35, -NR35-C(=O)-d-C6-alkenyl-C(=O)OR35-, d-Ce-alkyl, d-C6-alkanoyl or -C(0)OR35,
R32 and R33 are independently selected from hydrogen, C C6-alkyl or d-C6-alkanoyl,
R34 is independently selected from halogen, -CN, -CF3, -OCF3, -OR35, and -NR35R36,
R35 and R36 are independently selected from hydrogen, d-C6-alkyl, aryl-d-C6-alkyl or aryl, or R35 and R36 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R37 is independently selected from halogen, -G(O)OR35, -C(O)H, -CN, -CF3, -OCF3, -NO2, - OR35, -NR35R36, d-Ce-alkyl or C C6-aIkanoyl, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
Embodiment 124. A pharmaceutical composition according to embodiment 126 wherein the ligand that binds reversibly to a HisBlO Zn2+ site of an R-state insulin hexamer is of the form H-l-J, wherein H is
Figure imgf000034_0001
wherein the phenyl, naphthalene or benzocarbazole rings are optionally substituted with one or more substituents independently selected from R31,
I is selected from
• a valence bond,
• -CH2N(R32)- or -SO2N(R33)-,
Figure imgf000034_0002
is S(O)2 or CH2, Z2 is N,-O-or -S-, and n is 1 or 2,
J is
• d-d-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may each optionally be substituted with one or more substituents selected from R34,
• Aryl, aryloxy, aryl-oxycarbonyl-, aroyl, aryl-d-C6-alkoxy-, aryl-d-Ce-alkyl-, aryl-C2- C6-alkenyl-, aryl-d-d-alkynyh heteroaryl, heteroaryl-d-Ce-alkyl-, heteroaryl-C2-C6- alkenyl- or heteroaryl-C2-C6-alkynyl-, wherein the cyclic moieties are optionally substituted with one or more substituents selected from R37,
• hydrogen,
R31 is independently selected from hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3,
) -OCHF2, -OCH2CF3, -OCF2CHF2, -S(0)2CF3, -SCF3, -N02, -OR35, -C(0)R35, -NR35R36, -SR35,
-NR35S(0)2R36, -S(0)2NR35R36, -S(0)NR35R36, -S(0)R35, -S(0)2R35, -C(0)NR35R36,
-OC(O)NR35R36, -NR35C(O)R36, -CH2C(0)NR35R36, -OCH2C(0)NR35R36, -CH2OR35, -CH2NR35R36, -OC(O)R35, -OCrC6-alkyl-C(O)OR35, -SC1-C6-alkyl-C(O)OR35 -C2-C6-alkenyl- C(=O)OR35, -NR35-C(=O)-d-Ce-alkyl-C(=O)OR35, -NR35-C(=O)-C C6-alkenyl-C(=O)OR35-, d-Ce-alkyl, CrC6-alkanoyl or -C(O)OR35,
R32 and R33 are independently selected from hydrogen, CrCβ-alkyl or G C6-alkanoyl,
R34 is independently selected from halogen, -CN, -CF3, -OCF3, -OR35, and -NR35R36,
R35 and R36 are independently selected from hydrogen, Ci-Ge-alkyl, aryl-d-Ce-alkyl or aryl, or R35 and R36 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R37 is independently selected from halogen, -C(O)OR35, -C(O)H, -CN, -CF3, -OCF3, -NO2, - OR35, -NR35R38, d-Ce-alkyl or CrC6-alkanoyl,
or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base,
With the proviso that R31 and J cannot both be hydrogen.
Embodiment 125. A pharmaceutical composition according to any one of the embodiments
126 or 127 wherein H is
Figure imgf000035_0001
Embodiment 126. A pharmaceutical composition according to embodiment 128 wherein H is
Figure imgf000035_0002
Embodiment 127. A pharmaceutical composition according to embodiment 128 wherein H is
Figure imgf000036_0001
Embodiment 128. A pharmaceutical composition according to any one of the embodiments 126 to 130wherein I is a valence bond, -CH2N(R32)-, or -S02N(R33)-. Embodiment 129. A pharmaceutical composition according to embodiment 131 wherein I is a valence bond.
Embodiment 130. A pharmaceutical composition according to any one of the embodiments 126 to 132 wherein J is o hydrogen,
• d-Cβ-alkyl. C2-C6-alkenyl or C2-C6-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR35, and -NR35R36,
• aryl, or heteroaryl, wherein the cyclic moieties are optionally substituted with one or more substituents independently selected from R37.
Embodiment 131. A pharmaceutical composition according to embodiment 133 wherein J is
• hydrogen,
• aryl or heteroaryl, wherein the cyclic moieties are optionally substituted with one or more substituents independently selected from R37.
Embodiment 132. A pharmaceutical composition according to embodiment 133 wherein J is
• hydrogen,
• ArG1 or Het3, wherein the cyclic moieties are optionally substituted with one or more substituents independently selected from R37.
Embodiment 133. A pharmaceutical composition according to embodiment 135 wherein J is o hydrogen, o phenyl or naphthyl optionally substituted with one or more substituents independently selected from R37. Embodiment 134. A pharmaceutical composition according to embodiment 136 wherein J is hydrogen.
Embodiment 135. A pharmaceutical composition according to any one of the embodiments 126 to 137 wherein R32 and R33 are independently selected from hydrogen or Cι-C6-alkyl. Embodiment 136. A pharmaceutical composition according to any one of the embodiments 126 to 138 wherein R34 is hydrogen, halogen, -CN, -CF3, -OCF3, -SCF3, -NO2, -OR35, -C(O)R35, -NR35R36, -SR35, -C(O)NR35R36, -OC(O)NR35R36, -NR35C(O)R36, -OC(O)R35, -OC
C6-alkyl-C(O)OR35, -SCrC6-alkyI-C(O)OR35 or -C(O)OR35.
Embodiment 137. A pharmaceutical composition according to embodiment 139 wherein R34 is hydrogen, halogen, -CF3, -N02, -OR35, -NR35R36, -SR35, -NR35C(O)R36, or -C(O)OR35.
Embodiment 138. A pharmaceutical composition according to embodiment 140 wherein R34 is hydrogen, halogen, -CF3, -N02, -OR35, -NR35R36, or -NR35C(0)R36.
Embodiment 139. A pharmaceutical composition according to embodiment 141 wherein R34 is hydrogen, halogen, or -OR35.
Embodiment 140. A pharmaceutical composition according to any one of the embodiments
126 to 142 wherein R35 and R36 are independently selected from hydrogen, C C6-alkyl, or aryl.
Embodiment 141. A pharmaceutical composition according to embodiment 143 wherein R35 and R36 are independently selected from hydrogen or d-C6-alkyl.
Embodiment 142. A pharmaceutical composition according to any one of the embodiments
126 to 144 wherein R37 is halogen, -C(O)OR35, -CN, -CF3, -OR35, -NR35R36, Cι-C6-alkyl or C
Ce-alkanoyl.
Embodiment 143. A pharmaceutical composition according to embodiment 145 wherein R37 is halogen, -C(O)OR35, -OR35, -NR35R36, d-C6-alkyl or d-C6-alkanoyl.
Embodiment 144. A pharmaceutical composition according to embodiment 146 wherein R37 is halogen, -C(O)OR35 or -OR35.
Embodiment 145. A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisB10 Zn2+ site of an R-state insulin hexamer is
Figure imgf000037_0001
wherein K is a valence bond, C C6-alkylene, ~NH-C(=O)-U-, -Cι-C6-alkyl-S-, -C C6-alkyl-O-, -C(=O)-, or -C(=O)-NH-, wherein any CrC6-alkyl moiety is optionally substituted with R38,
U is a valence bond, d-C6-alkenylene, -C C6-alkyl-O- or C C6-alkylene wherein any C C6-alkyl moiety is optionally substituted with d-C6-alkyl,
R38 is GrGβ-alkyl, aryl, wherein the alkyl or aryl moieties are optionally substituted with one or more substituents independently selected from R39, R39 is independently selected from halogen, cyano, nitro, amino,
M is a valence bond, arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted with one or more substituents independently selected from R40,
R40 is selected from o hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(O)2CF3, -OS(0)2CF3, -SCF3, -N02, -OR41, -NR41R42, -SR41, -NR41S(0)2R42, -S(0)2NR41R42, -S(O)NR41R42, -S(0)R41, -S(0)2R41, -OS(O)2 R41, -C(O)NR41R42, -OC(0)NR41R42, -NR41C(O)R42, -CH2C(O)NR41R42, -OC C6- alkyl-C(O)NR41R42, -CH2OR41, -CH2OC(O)R41, -CH2NR41R42, -OC(0)R41, -OC Cβ- alkyl-C(O)OR41, -Od-C6-alkyl-OR41, -S-C C6-alkyl-C(O)OR41, -C2-C6-alkenyl- C(=O)OR41, -NR41-C(=O)-d-C6-alkyl-C(=O)OR41, -NR41-C(=O)-d-C6- alkenyl-C(=O)OR41 , -C(O)OR41, -C2-C6-alkenyl-C(=O)R41, =O, -NH-C(=O)-O-d- C6-alkyl, or -NH-C(=O)-C(=O)-O-d-C6-alkyl,
• C C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may each optionally be substituted with one or more substituents selected from R43,
• aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-d-C6-alkoxy, aryl-d-Cβ-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyI, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-d- Ce-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl, wherein the cyclic moieties optionally may be substituted with one or more substituents selected from R44,
R41 and R42 are independently selected from hydrogen, -OH, d-Ce-alkyl, d-Ce-alkenyl, aryl- d-Ce-alkyl or aryl, wherein the alkyl moieties may optionally be substituted with one or more substituents independently selected from R45, and the aryl moieties may optionally be substituted with one or more substituents independently selected from R46; R41 and R42 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R43 is independently selected from halogen, -CN, -CF3, -OCF3, -OR41, and -NR41R42 R44 is independently selected from halogen, -C(O)OR41, -CH2C(O)OR41, -CH2OR41, -CN, -
CF3, -OCF3, -N02, -OR41, -NR41R42 and d-Cβ-alkyl,
R45 is independently selected from halogen, -CN, -CF3, -OGF3, -O-C Ce-alkyl, -C(0)-O-C
Cβ-alkyl, -COOH and -NH2,
R46 is independently selected from halogen, -G(0)Od-G6-alkyl, -COOH, -CN, -CF3, -OCF3, -
N02, -OH, -Od-Ce-alkyl, -NH2, C(=O) or d-Ce-alkyl,
Q is a valence bond, d-C6-alkytene, -d-G6-alkyl-O-, -d-C6-alkyl-NH-, -NH-C G6-alkyl, -NH-C(=0)-, -C(=O)-NH-, -O-Ci-Ce-alkyl, -C(=O)-, or -d-C6-alkyl-G(=0)-N(R47)- wherein the alkyl moieties are optionally substituted with one or more substituents independently selected from R48,
R47 and R48 are independently selected from hydrogen, d-C6-alkyl, aryl optionally substituted with one or more R49,
R49 is independently selected from halogen and -COOH,
T is
• hydrogen,
• d-d-alkyl, C2-C6-alkenyl , C2-C6-alkynyl, Cι-C6-alkyloxy-carbonyl, wherein the alkyl, alkenyl and alkynyl moieties are optionally substituted with one or more substituents independently selected from R50,
• aryl, aryloxy, aryloxy-carbonyl, aryl-C -C6-alkyl, aroyl, aryl-Cι-C6-alkoxy, aryI-C2- Ce-alkenyl, aryl-C2-C6-alkyny-, heteroaryl, heteroaryl-Cι-C6-alkyl, heteroaryl-C2- Cβ-alkenyl, heteroaryl-C2-C6-alkynyl,
wherein any alkyl, alkenyl , alkynyl, aryl and heteroaryl moiety is optionally substituted with one or more substituents independently selected from Rso,
R50 is CrCe-alkyl, C C6-alkoxy, aryl, aryloxy, aryl-C dralkoxy, -C(=O)-NH-C1-C6-alkyl-aryl, heteroaryl, heteroaryl-d-Ce-alkoxy, -CrCe-alkyl-COOH, -O-Gι-C6-alkyl-COOH, -S(O)2R51, -C2-C6-alkenyl-COOH, -OR51, -N02, halogen, -COOH, -CF3, -CN, =O, -N(R51R52), wherein the aryl or heteroaryl moieties are optionally substituted with one or more R53, R51 and R52 are independently selected from hydrogen and d-Cβ-alkyl,
R53 is independently selected from d-Ce-alkyl, d-C6-alkoxy, -d-Ce-alkyl-COOH, -C2-
Ce-alkenyl-COOH, -OR51, -NO2, halogen, -COOH, -CF3, -CN, or -N(R51R52),
or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
Embodiment 146. A pharmaceutical composition according to embodiment 148 wherein K is a valence bond, C C6-alkylene, -NH-C(=0)-U-, -GrC6-alkyl-S-, -C C6-alkyl-0-, or -C(=0)-, wherein any Cι-C6-alkyl moiety is optionally substituted with R38.
Embodiment 147. A pharmaceutical composition according to embodiment 149 wherein K is a valence bond, d-C6-alkyIene, -NH-C(=O)-U-, -d-C6-alkyl-S-, or -C C6-alkyl-O, wherein any CrC6-alkyl moiety is optionally substituted with R38.
Embodiment 148. A pharmaceutical composition according to embodiment 150 wherein K is a valence bond, C^Ce-alkylene, or -NH-C(=O)-U, wherein any Ci-Ge-alkyl moiety is optionally substituted with R38.
Embodiment 149. A pharmaceutical composition according to embodiment 151 wherein K is a valence bond or C C6-alkylene, wherein any d-C6-alkyl moiety is optionally substituted with R38.
Embodiment 150. A pharmaceutical composition according to embodiment 151 wherein K is a valence bond or -NH-C(=O)-U.
Embodiment 151. A pharmaceutical composition according to embodiment 152 wherein K is a valence bond.
Embodiment 152. A pharmaceutical composition according to any one of the embodiments
148 to 154 wherein U is a valence bond or -C C6-alkyl-O-.
Embodiment 153. A pharmaceutical composition according to embodiment 155 wherein U is a valence bond.
Embodiment 154. A pharmaceutical composition according to any one of the embodiments
148 to 156 wherein M is arylene or heteroarylene, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from
R40.
Embodiment 155. A pharmaceutical composition according to embodiment 157 wherein is
ArG1 or Het1, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40. Embodiment 156. A pharmaceutical composition according to embodiment 158 wherein M is
ArG1 or Het2, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
Embodiment 157. A pharmaceutical composition according to embodiment 159 wherein M is
ArG1 or Het3, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R4Q.
Embodiment 158. A pharmaceutical composition according to embodiment 160 wherein M is phenylene optionally substituted with one or more substituents independently selected from
R40.
Embodiment 159. A pharmaceutical composition according to embodiment 160 wherein M is indolylene optionally substituted with one or more substituents independently selected from
R40.
Embodiment 160. A pharmaceutical composition according to embodiment 162 wherein M is
Figure imgf000041_0001
Embodiment 161. A pharmaceutical composition according to embodiment 160 wherein M is carbazolylene optionally substituted with one or more substituents independently selected from R40. Embodiment 162. A pharmaceutical composition according to embodiment 164 wherein M is
Figure imgf000041_0002
Embodiment 163. A pharmaceutical composition according to any one of the embodiments
148 to 165 wherein R40 is selected from
• hydrogen, halogen, -CN, -CF3, -OCF3l -NO2, -OR41, -NR41R42, -SR41, -S(O)2R41, -NR41C(O)R42, -Od-C6-alkyl-C(O)NR41R42, -C2-C6-alkenyl-C(=O)OR41, -C(O)OR41, =O, -NH-C(=O)-O-Ci-C6-alkyl, or -NH-C(=O)-C(=O)-O-Cι-C6-aIkyl,
CrCe-alkyl or G2-C6- alkenyl which may each optionally be substituted with one or more substituents independently selected from R43, • aryl, aryloxy, aryl-d-C6-alkoxy, aryl-Cι-C6-alkyl, aryl-C2-C6-alkenyl, heteroaryl, het- eroaryl-d-Ce-alkyl, or heteroaryl-C2-C6-alkenyl, wherein the cyclic moieties optionally may be substituted with one or more substituents selected from R44. Embodiment 164. A pharmaceutical composition according to embodiment 166 wherein R40 is selected from o hydrogen, halogen, -CN, -CF3, -OCF3, -N02, -OR41, -NR41R42, -SR41, -S(0)2R41, -NR41C(0)R42, -Od-C6-alkyl-C(0)NR41R42, -C2-C6-alkenyl-C(=O)OR41 ! -C(O)OR41, =O, -NH-C(=O)-O-d-C6-alkyl, or -NH-C(=O)-C(=O)-O-CrC6-alkyl,
Ci-Cβ-alkyl or C2-C6- alkenyl which may each optionally be substituted with one or more substituents independently selected from R43,
• ArG1, ArG1-O-, ArG1-d-C6-alkoxy, ArG1 -CrCe-alkyl, ArG1-C2-C6-alkenyI, Het3, Het3-CrC6-alkyl, or Het3-C2-C6-alkenyl, wherein the cyclic moieties optionally may be substituted with one or more substituents selected from R44.
Embodiment 165. A pharmaceutical composition according to embodiment 167 wherein R40 is selected from
• hydrogen, halogen, -CF3, -NO2, -OR41, -NR41R42, -C(O)OR41, =O, or -NR41C(O)R42,
• CrCe-alkyl,
• ArG1.
Embodiment 166. A pharmaceutical composition according to embodiment 168 wherein R40 is selected from
• Halogen, -NO2, -OR41, -NR41R42, -C(O)OR41, or -NR41C(O)R42,
• Methyl,
• Phenyl.
Embodiment 167. A pharmaceutical composition according to any one of the embodiments 148 to 170 wherein R41 and R42 are independently selected from hydrogen, d-Ce-alkyl, or aryl, wherein the aryl moieties may optionally be substituted with halogen or -COOH. Embodiment 168. A pharmaceutical composition according to embodiment 171 wherein R41 and R42 are independently selected from hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties may optionally be substituted with halogen or -COOH. Embodiment 169. A pharmaceutical composition according to any one of the embodiments 148 to 172 wherein Q is a valence bond, Cι-C6-alkylene, -d-Ce-alkyl-O-, -d-Ce-alkyl-NH-, -NH-CrC6-alkyl, -NH-C(=O)-, -C(=0)-NH-, -O-d-Ce-alkyl, -G(=O)-, or -d- C6-alkyl-C(=O)-N(R47)- wherein the alkyl moieties are optionally substituted with one or more substituents independently selected from R48.
Embodiment 170. A pharmaceutical composition according to embodiment 173 wherein Q is a valence bond, -CH2-, -CH2-CH2-, -CH2-O-, -CH2-CH2-O-, -CH2-NH-, -CH2-CH2-NH-, -NH-CHjr, -NH-CH2-CH2-, -NH-C(=O)-, -C(=0)-NH-, -0-CH2-, -0-CH2-CH2-, or -C(=0)-. Embodiment 171. A pharmaceutical composition according to any one of the embodiments 148 to 174 wherein R4' and R48 are independently selected from hydrogen, methyl and phenyl.
Embodiment 172. A pharmaceutical composition according to any one of the embodiments 148 to 178 wherein T is o Hydrogen,
• d-C6-alkyl optionally substituted with one or more substituents independently selected from R50,
• aryl, aryl-CrC6-alkyl, heteroaryl, wherein the alkyl, aryl and heteroaryl moieties are optionally substituted with one or more substituents independently selected from R50.
Embodiment 173. A pharmaceutical composition according to embodiment 179 wherein T is
• hydrogen,
• d-Ce-alkyl optionally substituted with one or more substituents independently selected from R50,
• ArG1, ArG1-CrC6-alkyl, Het3, wherein the alkyl, aryl and heteroaryl moieties are optionally substituted with one or more substituents independently selected from R50.
Embodiment 174. A pharmaceutical composition according to embodiment 180 wherein T is
• hydrogen,
• d-Ce-alkyl, optionally substituted with one or more substituents independently selected from R50,
• phenyl, phenyl-CrC6-alkyl, wherein the alkyl and phenyl moieties are optionally substituted with one or more substituents independently selected from R50.
Embodiment 175. A pharmaceutical composition according to any one of the embodiments 148 to 181 wherein R50 is d-Ce-alkyl, d-Ce-alkoxy, aryl, aryloxy, aryl-d-C6-alkoxy, -C(=O)-NH-GrC6-alkyl-aryl, heteroaryl, -C C6-alkyl-COOH, -O-C C6-alkyl-COOH, -S(O)2R51, -C2-C6-alkenyl-COOH, -OR51, -NO2, halogen, -COOH, -CF3, -CN, =O, -N(R51R52), wherein the aryl or heteroaryl moieties are optionally substituted with one or more R53. Embodiment 176. A pharmaceutical composition according to embodiment 183 wherein R50 is CrCe-alkyl, GrC6-alkoxy, aryl, aryloxy, aryl-d-Ce-alkoxy , -OR51, -NO2, halogen, -COOH, -CF3, wherein any aryl moiety is optionally substituted with one or more R53. Embodiment 177. A pharmaceutical composition according to embodiment 184 wherein R50 is CrCe-alkyl, aryloxy, aryl-CrCe-alkoxy , -OR51, halogen, -COOH, -CF3, wherein any aryl moiety is optionally substituted with one or more R53.
Embodiment 178. A pharmaceutical composition according to embodiment 185 wherein R50 is d-Cβ-alkyl, ArG1-0-, ArG1-C Cβ-alkoxy , -OR51, halogen, -COOH, -CF3, wherein any aryl moiety is optionally substituted with one or more R53.
Embodiment 179. A pharmaceutical composition according to embodiment 186 wherein R50 is phenyl, methyl or ethyl.
Embodiment 180. A pharmaceutical composition according to embodiment 188 wherein R50 is methyl or ethyl.
Embodiment 181. A pharmaceutical composition according to any one of the embodiments
148 to 189 wherein R51 is methyl.
Embodiment 182. A pharmaceutical composition according to any one of the embodiments
148 to 192 wherein R53 is CrC6-alkyl, CrC6-alkoxy, -OR51, halogen.or -CF3.
Embodiment 183. A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn2+ site of an R-state insulin hexamer is
Figure imgf000044_0001
wherein V is CrCe-alkyl, aryl, heteroaryl, aryl-C -6-alkyl- or aryl-C2.6-alkenyl-, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R54, and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R55,
R54 is independently selected from halogen, -CN, -CF3, -OCF3, aryl, -COOH and -NH2)
R55 is independently selected from
• hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(O)2CF3, -OS(O)2CF3, -SCF3, -NO2, -OR56, -NR56R57, -SR56, -NR56S(O)2R57, -S(O)2NR56R57, -S(O)NR56R57, -S(O)R56, -S(O)2R56, -OS(0)2 R56, -C(0)NR56R57, -OC(O)NR56R57, -NR56C(O)R57, -CH2C(O)NR56R57, -OC Cβ- alkyl-C(O)NR56R57, -CH2OR56, -CH2OC(O)R56, -CH2NR56R57, -OC(O)R56, -OC C8- alkyl-C(O)OR56, -OCrC6-alkyl-OR56, -SCrC6-alkyl-C(O)OR56, -C2-C6-alkenyl- C(=O)OR56, -NR56-C(=O)-CrC6-alkyl-C(=O)OR56, -NR56-C(=O)-CrC6- alkenyl-C(=O)OR56 , -C(O)OR56, or -C2-C6-alkenyl-C(=O)R58,
o CrCe-alkyl, C2-C6-alkenyl or G2-C6-alkynyl,
which may optionally be substituted with one or more substituents selected from R58,
oaryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-CrC6-alkoxy, aryl-GrCe-alkyl, aryl-C2-C6-aIkenyl, aroyl-G2-C6-alkenyI, aryl-G2-C6-alkynyl, heteroaryl, heteroaryl-Gr Ce-alkyl, heteroaryl-G2-C6-alkenyl or heteroaryl-G2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R59,
R56 and R57 are independently selected from hydrogen, OH, CF3, CrCι2-alkyl, aryl-d-Ce- alkyl, -C(=O)-CrC6-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R60, and the aryl groups may optionally be substituted with one or more substituents independently selected from R61; R56 and R57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R58 is independently selected from halogen, -CN, -CF3, -OCF3, -OR56, and -NR56R57,
R59 is independently selected from halogen, -C(O)OR56, -CH2C(O)OR56, -CH2OR56, -CN, - CF3, -OCF3, -NO2, -OR56, -NR56R57 and d-C6-alkyl,
R60 is independently selected from halogen, -CN, -CF3, -OCF3, -OCrC6-alkyl, -C(O)OCrC6- alkyl, -C(=O)-R62, -COOH and -NH2,
R61 is independently selected from halogen, -C(O)OGrCe-alkyl, -COOH, -CN, -CF3, -OCF3, - NO2, -OH, -OCrCe-alkyl, -NH2, C(=O) or d-C6-alkyl, R62 is d-Ce-alkyl, aryl optionally substituted with one or more substituents independently selected from halogen, or heteroaryl optionally substituted with one or more d-C6-alkyl independently, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
Embodiment 184. A pharmaceutical composition according to embodiment 196 wherein V is aryl, heteroaryl, or aryl-d-6-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected R54, and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R55.
Embodiment 185. A pharmaceutical composition according to embodiment 197 wherein V is aryl, Het1 , or aryl-Cι.6-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from R54, and the aryl or heteroaryl moiety is optionally substituted with one or more substituents independently selected from R55. Embodiment 186. A pharmaceutical composition according to embodiment 198 wherein V is aryl, Het2, or aryl-d.6-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from R54, and the aryl or heteroaryl moiety is optionally substituted with one or more substituents independently selected from R55. Embodiment 187. A pharmaceutical composition according to embodiment 199 wherein V is aryl, Het3, or aryl-Cι.6-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from R54, and the aryl or heteroaryl moiety is optionally substituted with one or more substituents independently selected from R55. Embodiment 188. A pharmaceutical composition according to embodiment 200 wherein V is aryl optionally substituted with one or more substituents independently selected from R55. Embodiment 189. A pharmaceutical composition according to embodiment 201 wherein V is ArG1 optionally substituted with one or more substituents independently selected from R55. Embodiment 190. A pharmaceutical composition according to embodiment 202 wherein V is phenyl, naphthyl or anthranyl optionally substituted with one or more substituents independently selected from R55.
Embodiment 191. A pharmaceutical composition according to embodiment 203 wherein V is phenyl optionally substituted with one or more substituents independently selected from R55. Embodiment 192. A pharmaceutical composition according to any one of the embodiments 196 to 204 wherein R55 is independently selected from o halogen, d-Ce-alkyl, -CN, -OCF3 ,-CF3, -NO2, -OR56, -NR56R57, -NR56G(O)R57
-SR56, -QCrC8-alkyl-G(0)OR56, or -C(O)OR56, • CrCe-alkyl optionally substituted with one or more substituents independently selected from R58 βaryl, aryl-CrC6-alkyl, heteroaryl, or heteroaryl-GrC6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents independently selected from R59. Embodiment 193. A pharmaceutical composition according to embodiment 205 wherein R55 is independently selected from o halogen, d-Cβ-alkyl, -CN, -OCF3 ,-CF3, -NO2, -OR58, -NR56R57, -NR56C(0)R57 -SR56, -OCrC8-alkyl-G(O)OR56, or -C(0)OR56
®C C6-aIkyl optionally substituted with one or more substituents independently selected from R58
•ArG1, ArG1-d-C6-alkyl, Het3, or Het3-C C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents independently selected from R59. Embodiment 194. A pharmaceutical composition according to embodiment 206 wherein R55 is independently selected from halogen, -OR56, -NR56R57, -C(O)OR56, -OC Cβ- alkyl-C(O)OR56, -NR56C(O)R57 or d-C6-alkyl.
Embodiment 195. A pharmaceutical composition according to embodiment 207 wherein R55 is independently selected from halogen, -OR56, -NR56R57, -C(O)OR56, -OC C8- alkyl-C(O)OR56, -NR56C(O)R57, methyl or ethyl.
Embodiment 196. A pharmaceutical composition according to any one of the embodiments 196 to 208 wherein R56 and R57 are independently selected from hydrogen, CF3, d-C12-alkyl, or -C(=O)-CrC6-alkyl; R56 and R57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom.
Embodiment 197. A pharmaceutical composition according to embodiment 209 wherein R56 and R57 are independently selected from hydrogen or d-C 2-alkyl, R56 and R57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom.
Embodiment 198. A pharmaceutical composition according to embodiment 210 wherein R56 and R57 are independently selected from hydrogen or methyl, ethyl, propyl butyl, R56 and R57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom.
Embodiment 199. A pharmaceutical composition according to embodiment 1 wherein the ligand that binds reversibly to a HisBlO Zn2+ site of an R-state insulin hexamer is
Figure imgf000048_0001
wherein AA is d-Ce-alkyl, aryl, heteroaryl, aryl-G -6-alkyl- or aryl-G2-6-alkenyl-, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R63, and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R64,
R63 is independently selected from halogen, -CN, -CF3, -OCF3, aryl, -COOH and -NH2,
R64 is independently selected from
• hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(O)2CF3, -OS(O)2CF3, -SCF3, -NO2, -OR65, -NR65R66, -SR65, -NR65S(O)2R66, -S(O)2NR65R66, -S(O)NR65R66, -S(O)R65, -S(O)2R65, -OS(O)2 R65, -C(O)NR65R66, -OC(O)NR65R66, -NR65C(O)R66, -CH2C(O)NR65R66, -OCι-C6- alkyl-C(O)NR65R66, -CH2OR65, -CH2OC(O)R65, -CH2NR65R66, -OC(O)R65, -OCrC6- alkyl-C(O)OR65, -OCrC6-alkyl-OR65, -SC C6-alkyl-C(O)OR65, -C2-C6-alkenyl- C(=0)OR65, -NR65-C(=O)-C C6-alkyl-C(=O)OR65, _NR65-C(=O)-C C6- alkenyl-C(=O)OR65 , -C(O)OR65, or -C2-C6-alkenyl-C(=O)R65,
• CrCe-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, each of which may optionally be substituted with one or more substituents selected from R67,
• aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-CrC6-alkoxy, aryl-CrC6-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-Cβ-alkenyl, aryl-C2-Ce-alkynyl, heteroaryl, heteroaryl-d- C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-d-Ce-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R68,
R65 and R66 are independently selected from hydrogen, OH, CF3, Crd2-alkyl, aryl-CrC6- alkyl, -C(=O)-R69, aryl or heteroaryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from R70, and the aryl and heteroaryl groups may optionally be substituted with one or more substituents independently selected from R71; R65 and R66 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R67 is independently selected from halogen, -CN, -CF3, -OCF3, -OR65, and -NR65R66,
R68 is independently selected from halogen, -G(O)OR65, -CH2C(O)OR65, -CH2OR65, -CN, - CF3, -OCF3, -N02, -OR65, -NR65R66 and d-Cβ-ε
R69 is independently selected from d-Cβ-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl optionally substituted with one or more d-Cβ-alkyl,
R70 is independently selected from halogen, -CN, -CF3, -OCF3, -OCrC6-alkyl, -C(O)Od-C6- alkyl, -COOH and -NH2,
R71 is independently selected from halogen, -C(O)OCrC6-alkyl, -COOH, -CN, -CF3, -OCF3, - NO2, -OH, -OCrCe-alkyl, -NH2, C(=O) or CrC6-alkyl,
or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
Embodiment 200. A pharmaceutical composition according to embodiment 212 wherein AA is aryl, heteroaryl or aryl-Ci-β-alkyl-, wherein the alkyl is optionally substituted with one or more R63, and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R64.
Embodiment 201. A pharmaceutical composition according to embodiment 213 wherein AA is aryl or heteroaryl optionally substituted with one or more substituents independently selected from R64.
Embodiment 202. A pharmaceutical composition according to embodiment 214 wherein AA is ArG1 or Het1 optionally substituted with one or more substituents independently selected from R64. Embodiment 203. A pharmaceutical composition according to embodiment 215 wherein AA is ArG1 or Het2 optionally substituted with one or more substituents independently selected from R64.
Embodiment 204. A pharmaceutical composition according to embodiment 216 wherein AA is ArG1 or Het3 optionally substituted with one or more substituents independently selected from R64.
Embodiment 205. A pharmaceutical composition according to embodiment 217 wherein AA is phenyl, naphtyl, anthryl, carbazolyl, thienyl, pyridyl, or benzodioxyl optionally substituted with one or more substituents independently selected from R64.
Embodiment 206. A pharmaceutical composition according to embodiment 218 wherein AA is phenyl or naphtyl optionally substituted with one or more substituents independently selected from R64.
Embodiment 207. A pharmaceutical composition according to any one of the embodiments 212 to 219 wherein R64 is independently selected from hydrogen, halogen, -CF3, -OCF3, -OR65, -NR65R66, d-Ce-alkyl , -OC(O)R65, -OCrC6-alkyl-C(O)OR65, aryl-C2-C6-alkenyl, aryloxy or aryl, wherein C C6-alkyl is optionally substituted with one or more substituents independently selected from R67, and the cyclic moieties optionally are substituted with one or more substituents independently selected from R68.
Embodiment 208. A pharmaceutical composition according to embodiment 220 wherein R64 is independently selected from halogen, -CF3, -OCF3, -OR65, -NR65R66, methyl, ethyl, propyl, -OC(O)R65, -OCH2-C(O)OR65, -OCH2-CH2-C(O)OR65, phenoxy optionally substituted with one or more substituents independently selected from R68.
Embodiment 209. A pharmaceutical composition according to any one of the embodiments 212 to 221 wherein R65 and R66 are independently selected from hydrogen, CF3, C Cι2-alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R71.
Embodiment 210. A pharmaceutical composition according to embodiment 222 wherein R65 and R66 are independently hydrogen, d-Cι2-alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R71.
Embodiment 211. A pharmaceutical composition according to embodiment 223 wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, ArG1 or Het1 optionally substituted with one or more substituents independently selected from R71. Embodiment 212. A pharmaceutical composition according to embodiment 224 wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, ArG1 or Het2 optionally substituted with one or more substituents independently selected from R71. Embodiment 213. A pharmaceutical composition according to embodiment 225 wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, ArG1 or Het3 optionally substituted with one or more substituents independently selected from R71. Embodiment 214. A pharmaceutical composition according to embodiment 226 wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, phenyl, naphtyl, thiadia∑olyl optionally substituted with one or more R71 independently; or isoxa∑olyl optionally substituted with one or more substituents independently selected from R71. Embodiment 215. A pharmaceutical composition according to any one of the embodiments 212 to 227 wherein R71 is halogen or d-Ce-alkyl.
Embodiment 216. A pharmaceutical composition according to embodiment 228 wherein R71 is halogen or methyl.
Embodiment 217. Method of prolonging the action of an insulin preparation comprising insulin, protamine and zinc ions wherein said method comprises adding a zinc-binding ligand according to any of embodiments 21 to 216 to the insulin preparation. Embodiment 218. A method of treating type 1 or type 2 diabetes comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical preparation according to any one of the embodiments 1 to216.
Embodiment 219. Use of a preparation according to any one of the embodiments 1 to 216for the preparation of a medicament for treatment of type 1 or type 2 diabetes. Embodiment 220. A method of preparing a pharmaceutical preparation comprising the steps of mixing
• insulin
• a ligand for the His810 Zn2+ site of the insulin hexamer according to any of embodiments 21 to 216
• zinc ions
• protamine
• optionally further ingredients selected from the group consisting of phenolic preservative, buffer, isotonicity agent, viscosity increasing agent, and a non-ionic surfactant, and allowing the mixture to stand until crystals are formed.
Embodiment 221. A method according to embodiment 233 wherein the ligand for the His810
Zn2+ site is added to the mixture before crystal growth.
Embodiment 222. A method according to embodiment 233 wherein the ligand for the His810
Zn2+ site is added to the mixture after completion of crystal growth. The novel NPH-insulin preparations disclosed here can be used for parenteral or pulmonal administration.
In another embodiment the NPH preparations of the present invention are used in connection with pen-like injection devices, which may be prefilled and disposable, or the insulin preparations may be supplied from a reservoir which is removable. Non-limiting examples of pen-like injection devices are FlexPen®, InnoLet®, InDuo™, Innovo®.
In a further embodiment NPH preparations of the present invention may be used in connection with devices for pulmonary administration of aqueous insulin preparations. In one embodiment hereof, the NPH preparation of the invention is dried to form a powder. In that embodiment, suitable devices used in pulmonary administration of a NPH preparation according to the present invention may be the dry powder formulation and delivery devices being developed by Inhale Therapeutic Systems, Inc., and the Spiros® dry powder inhaler system being developed by Dura Pharmaceuticals, Inc.
In one aspect of the invention the zinc-binding ligand for the His810 Zn2+ site is present in the preparation in a smaller concentration than that of Zn +- In such an embodiment not all of the insulin hexamers will have zinc-binding ligand for the His810 Zn2+ site present, and thus insulin from these hexamers will be released rapidly. Such a preparation will therefore have a dual-release profile after administration, i.e. the administration will result in a both a rapid release of insulin and a protracted release.
PHARMACEUTICAL COMPOSITIONS
Insulin formulations of the invention are usually administered from multi-dose containers where a preservative effect is desired. Since phenolic preservatives also stabilize the R-state hexamer the formulations may contain up to 50 mM of phenolic molecules. The phenolic molecules in the insulin formulation may be selected from the group consisting of phenol, m- cresol, chloro-cresol, thymol, m-chlor-phenol, resorcinole, 7-hydroxyindole or any mixture thereof.
In one embodiment of the invention 0.5 to 5.0 mg/ml of phenolic compound may be employed. In another embodiment of the invention 0.6 to 5.0 mg/ml of m-cresol may be employed. In another embodiment of the invention 0.5 to 5.0 mg/ml of phenol may be employed. In another embodiment of the invention 1.4 to 5.0 mg/ml of phenol may be employed. In another embodiment of the invention 0.5 to 5.0 mg/ml of a mixture of m-cresol or phenol may be employed.
In another embodiment of the invention 1.4 to 5.0 mg/ml of a mixture of m-cresol or phenol may be employed.
The pharmaceutical preparation may further comprises a buffer substance, such as a TRIS, phosphate, glycine or glycylglycine (or another ∑witterionic substance) buffer, an isotonicity agent, such as NaCl, glycerol, mannitol and/or lactose. Chloride would be used at moderate concentrations (e.g. up to 50 mM) to avoid competition with the zinc-site ligands of the present invention.
The action of insulin may further be slowed down in vivo by the addition of physiologically acceptable agents that increase the viscosity of the pharmaceutical preparation. Thus, the pharmaceutical preparation according to the invention may furthermore comprise an agent which increases the viscosity, such as polyethylene glycol, polypropylene glycol, copolymers thereof, dextrans and/or polylactides.
In a particular embodiment the insulin preparation of the invention comprises between 0.001 % by weight and 1 % by weight of a non-ionic surfactant, for example tween 20 or Poloxamer 188.
The insulin preparation of the present invention may have a pH value in the range of 3.5 to 8.5, more preferably 7.1 to 7.9.
COMBINATION TREATMENT
The invention furthermore relates to treatment of a patient in which the pharmaceutical preparation of the invention, i.e. comprising zinc ions, acid-stabilised insulin analogue and a ligand for the R-state His810 Zn2+ site, is combined with another form of treatment.
In one aspect of the invention, treatment of a patient with the pharmaceutical preparation of the invention is combined with diet and/or exercise.
In another aspect of the invention the pharmaceutical preparation of the invention is administered in combination with one or more further active substances in any suitable ratios. Such further active substances may e.g. be selected from antiobesity agents, antidiabetics, antihypertensive agents, agents for the treatment of complications resulting from or associated with diabetes and agents for the treatment of complications and disorders resulting from or associated with obesity. Thus, in a further aspect of the invention the pharmaceutical preparation of the invention may be administered in combination with one or more antiobesity agents or appetite regulating agents.
Such agents may be selected from the group consisting of CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, ΪV1C4 (rnelano- cortin 4) agonists, MC3 (melanocortin 3) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corlicotropin releasing factor binding protein) antagonists, urocortin agonists, β3 adrenergic agonists such as CL-316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140, MSH (melanoeyte- stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CGK (cholecystokinin) agonists, serotonin re-uptake inhibitors such as fluoxetine, seroxat or cita- lopram, serotonin and noradrenaline re-uptake inhibitors, mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists, bombesin agonists, galanin antagonists, growth hormone, growth factors such as prolactin or placental lactogen, growth hormone releasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptin, doprexin), lipase/amylase inhibitors, PPAR (peroxisome proliferator-activated receptor) modulators, RXR (retinoid X receptor) modulators, TR β agonists, AGRP (Agouti related protein) inhibitors, H3 histamine antagonists, opioid antagonists (such as naltrexone), exendin-4, GLP-1 and ciliary neurotro- phic factor.
In one embodiment of the invention the antiobesity agent is leptin.
In another embodiment the antiobesity agent is dexamphetamine or amphetamine.
In another embodiment the antiobesity agent is fenfluramine or dexfenfluramine.
In still another embodiment the antiobesity agent is sibutramine.
In a further embodiment the antiobesity agent is orlistat.
In another embodiment the antiobesity agent is mazindol or phentermine.
In still another embodiment the antiobesity agent is phendimetrazine, diethylpropion, fluoxetine, bupropion, topiramate or ecopipam.
The orally active hypoglycemic agents comprise imidazolines, sulphonylureas, biguanides, meglitinides, oxadiazolidinediones, thiazolidinediones, insulin sensitizers, insulin secretagogues such as glimepride, α-glucosidase inhibitors, agents acting on the ATP- dependent potassium channel of the β-cells eg potassium channel openers such as those disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (Novo Nordisk A/S) which are incorporated herein by reference, or mitiglinide, or a potassium channel blocker, such as BTS-67582, nateglinide, glucagon antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference, GLP-1 agonists such as those disclosed in WO 00/42026 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference, DPP-IV (dipeptidyl pepfidase-IV) inhibitors, PTPase (protein tyrosine phosphatase) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis, glucose uptake modulators, GSK-3 (glycogen synthase kinase-3) inhibitors, compounds modifying the lipid metabolism such as antilipidemic agents, compounds lowering food intake, PPAR (peroxisome proliferator-activated receptor) and RXR (retinoid X receptor) agonists, such as ALRT-268, LG-1268 or LG-1069.
In a further embodiment of the invention the pharmaceutical preparation of the invention is administered in combination with a sulphonylurea e.g. tolbutamide, chlorpropa- mide, tolazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide.
In another embodiment of the invention the pharmaceutical preparation of the invention is administered in combination with a biguanide, e.g. metformin.
In yet another embodiment of the invention the pharmaceutical preparation of the invention is administered in combination with a meglitinide eg repaglinide or nateglinide.
In still another embodiment of the invention the pharmaceutical preparation of the invention is administered in combination with a thiazolidinedione insulin sensitizer, e.g. trogli- tazone, ciglitazone, pioglitazone, rosiglitazone, isaglitazone, darglitazone, englitazone, CS- 011/CI-1037 or T 174 or the compounds disclosed in WO 97/41097, WO 97/41119, WO 97/41120, WO 00/41121 and WO 98/45292 (Dr. Reddy's Research Foundation), which are incorporated herein by reference.
In still another embodiment of the invention the pharmaceutical preparation of the invention may be administered in combination with an insulin sensitizer, e.g. such as GI 262570, YM-440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW-409544, CRE-16336, AR-H049020, LY510929, MBX- 02, CLX-0940, GW-501516 or the compounds disclosed in WO 99/19313, WO 00/50414, WO 00/63191, WO 00/63192, WO 00/63193 (Dr. Reddy's Research Foundation) and WO 00/23425, WO 00/23415, WO 00/23451, WO 00/23445, WO 00/23417, WO 00/23416, WO 00/63153, WO 00/63196, WO 00/63209, WO 00/63190 and WO 00/63189 (Novo Nordisk A/S), which are incorporated herein by reference.
In a further embodiment of the invention the pharmaceutical preparation of the invention is administered in combination with an α-glucosidase inhibitor, e.g. voglibose, emigli- tate, miglitol or acarbose. In another embodiment of the invention the pharmaceutical preparation of the invention is administered in combination with an agent acting on the ATP-dependent potassium channel of the β-cells, e.g. tolbutamide, glibenclamide, glipizide, glicazide, BTS-67582 or re- paglinide.
In yet another embodiment of the invention the pharmaceutical preparation of the invention may be administered in combination with nateglinide.
In still another embodiment of the invention the pharmaceutical preparation of the invention is administered in combination with an antilipidemic agent, e.g. cholestyramine, coleslipol, clofibrate, gemfibro∑il, lovastatin, pravaslatin, simvastatin, probucol or dextrothy- roxine.
In another aspect of the invention, the pharmaceutical preparation of the invention is administered in combination with more than one of the above-mentioned compounds, e.g. in combination with metformin and a sulphonylurea such as glyburide; a sulphonylurea and acarbose; nateglinide and metformin; acarbose and metformin; a sulphonylurea, metformin and troglitazone; metformin and a sulphonylurea; etc.
Furthermore, the pharmaceutical preparation of the invention may be administered in combination with one or more antihypertensive agents. Examples of antihypertensive agents are β-blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and α- blockers such as doxazosin, urapidil, prazosin and terazosin. The pharmaceutical preparation of the invention may also be combined with NEP inhibitors such as candoxatril.
Further reference can be made to Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. It should be understood that any suitable combination of the compounds according to the invention with diet and/or exercise, one or more of the above-mentioned compounds and optionally one or more other active substances are considered to be within the scope of the present invention.
EXAMPLES
The following examples and general procedures refer to intermediate compounds and final products identified in the specification and in the synthesis schemes. The preparation of the compounds of the present invention is described in detail using the following examples, but the chemical reactions described are disclosed in terms of their general applicability to the preparation of compounds of the invention. Occasionally, the reaction may not be applicable as described to each compound included within the disclosed scope of the invention. The compounds for which this occurs will be readily recognised by those skilled in the art. In these cases the reactions can be successfully performed by conventional modifications known to those skilled in the art, that is, by appropriate protection of interfering groups, by changing to other conventional reagents, or by routine modification of reaction conditions. Alternatively, other reactions disclosed herein or otherwise conventional will be applicable to the preparation of the corresponding compounds of the invention. In all preparative methods, all starting materials are known or may easily be prepared from known starting materials. All temperatures are set forth in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight when referring to yields and all parts are by volume when referring to solvents and eluents.
HPLC-MS (Method A)
The following instrumentation was used:
• Hewlett Packard series 1100 G1312A Bin Pump
• Hewlett Packard series 1 00 Column compartment
• Hewlett Packard series 1100 G13 15A DAD diode array detector
• Hewlett Packard series 1100 MSD
The instrument was controlled by HP Chemstation software.
The HPLC pump was connected to two eluent reservoirs containing:
A: 0.01% TFA in water
B: 0.01 % TFA in acetonitrile
The analysis was performed at 40 °C by injecting an appropriate volume of the sample (preferably 1 μL) onto the column, which was eluted with a gradient of acetonitrile. The HPLC conditions, detector settings and mass spectrometer settings used are given in the following table.
Column Waters Xterra MS C-18 X 3 mm id
Gradient 10% - 100% acetonitrile lineary during 7.5 min at 1.0 mL/min
Detection UV: 210 nm (analog output from DAD)
MS lonisation mode: API-ES
Scan 100-1000 amu step 0.1 amu
HPLC-MS (Method B)
The following instrumentation was used:
Sciex AP1 100 Single quadropole mass spectrometer
Perkin Elmer Series 200 Quard pump
Perkin Elmer Series 200 autosampler
Applied Biosystems 785A UV detector
Sedex 55 evaporative light scattering detector
A Valco column switch with a Valco actuator controlled by timed events from the pump.
The Sciex Sample control software running on a Macintosh PowerPC 7200 computer was used for the instrument control and data acquisition.
The HPLC pump was connected to four eluent reservoirs containing:
A Acetonitrile B Water C 0.5% TFA in water D 0.02 M ammonium acetate
The requirements for samples are that they contain approximately 500 μg/mL of the compound to be analysed in an acceptable solvent such as methanol, ethanol, acetonitrile, THF, water and mixtures thereof. (High concentrations of strongly eluting solvents will interfere with the chromatography at low acetonitrile concentrations.)
The analysis was performed at room temperature by injecting 20 μL of the sample solution on the column, which was eluted with a gradient of acetonitrile in either 0.05% TFA or 0.002 M ammonium acetate. Depending on the analysis method varying elution conditions were used.
The eluate from the column was passed through a flow splitting T-connector, which passed approximately 20 μL/min through approx. 1 m. 75 μ fused silica capillary to the API interface of AP1 100 spectrometer.
The remaining 1.48 mL/min was passed through the UV detector and to the ELS detector.
During the LC-analysis the detection data were acquired concurrently from the mass spectrometer, the UV detector and the ELS detector.
The LC conditions, detector settings and mass spectrometer settings used for the different methods are given in the following table.
Figure imgf000059_0001
HPLC-MS (Method C) The following instrumentation is used:
• Hewlett Packard series 1100 G1312A Bin Pump
• Hewlett Packard series 1100 Column compartment
• Hewlett Packard series 1100 G1315A DAD diode array detector
• Hewlett Packard series 1100 MSD
• Sedere 75 Evaporative Light Scattering detector The instrument is controlled by HP Ghemstation software.
The HPLC pump is connected to two eluent reservoirs containing:
A 0.01% TFA In water
B 0.01 % TFA in acetonitrile The analysis is performed at 40 °C by injecting an appropriate volume of the sample (preferably 1 μ\) onto the column which is eluted with a gradient of acetonitrile. The HPLC conditions, detector settings and mass spectrometer settings used are given in the following table.
Column Waters 4terra MS C-1843 mm id μm
Gradient 5% - 100% acetoni ril© linear during 7.5 min at 1. ml/min
Detection 210 nm (analogue output from DAD)
ELS (analogue output from ELS)
MS ionisation mode API-ES
Scan 100-1000 amu step 0.1 amu
After the DAD the flow is divided yielding approximately 1 ml/min to the ELS and 0.5 ml/min to the MS.
HPLC-MS (Method D)
The following instrumentation was used:
Sciex API 150 Single Quadropole mass spectrometer
Hewlett Packard Series 1100 G1312A Bin pump
Gilson 215 micro injector
Hewlett Packard Series 1100 G1315A DAD diode array detector
Sedex 55 evaporative light scattering detector
A Valco column switch with a Valco actuator controlled by timed events from the pump.
The Sciex Sample control software running on a Macintosh Power G3 computer was used for the instrument control and data acquisition.
The HPLC pump was connected to two eluent reservoirs containing:
A: Acetonitrile containing 0.05% TFA
B: Water containing 0.05% TFA The requirements for the samples are that they contain approximately 500 μg/ml of the compound to be analysed in an acceptable solvent such as methanol, ethanol, acetonitrile, THF, water and mixtures thereof. (High concentrations of strongly eluting solvents will interfere with the chromatography at low acetonitrile concentrations.)
The analysis was performed at room temperature by injecting 20 μl of the sample solution on the column, which was eluted with a gradient of acetonitrile in 0.05% TFA The eluate from the column was passed through a flow splitting T-connector, which passed approximately 20 μl/min through approx. 1 m 75 μ fused silica capillary to the API interface of AP1 150 spectrometer.
The remaining 1.48 ml/min was passed through the UV detector and to the ELS detector. During the LC-analysis the detection data were acquired concurrently from the mass spectrometer, the UV detector and the ELS detector.
The LG conditions, detector settings and mass spectrometer settings used for the different methods are given in the following table.
Figure imgf000061_0001
EXAMPLES
Example 1
1 -/-Benzotriazole
Figure imgf000062_0001
Example 2
5,6-Dimethyl-1 H-benzotriazole
Figure imgf000062_0002
Example 3
1 H-Benzotriazole-5-carboxylic acid
Figure imgf000062_0003
Example 4
4-Nitro-1 --benzotriazole
Figure imgf000062_0004
Example 5
5-Amino-1 H-benzotria∑ole
Figure imgf000062_0005
Example 6
5-Chloro-1 H-benzotriazole
Figure imgf000063_0001
Example 7
5-Nitro- 1 W-benzotriazole
Figure imgf000063_0002
Example 8
4-[(1 H-Benzotriazole-5-carbonyl)amino]benzoic acid
Figure imgf000063_0003
4-[(1f -Benzotriazole-5-carbonyl)amino]benzoic acid methyl ester (5.2 g, 17.6 mmol) was dissolved in THF (60 mL) and methanol (10 mL) was added followed by 1N sodium hydroxide (35 mL). The mixture was stirred at room temperature for 16 hours and then 1 N hydrochloric acid (45 mL) was added. The mixture was added water (200 mL) and extracted with ethyl acetate (2 x 500 mL). The combined organic phases were evaporated in vacuo to afford 0.44 g of 4-[(1H-benzotriazole-5-carbonyl)amino]benzoic acid. By filtration of the aqueous phase a further crop of 4-[(1 H-benzotriazole-5-carbonyl)amino]benzoic acid was isolated (0.52 g).
1H-NMR (DMSO-dβ): δ 7.97 (4H, s), 8.03 (2H, m), 8.66 (1H, bs), 10.7 (1H, s), 12.6 (1H, bs); HPLC-MS (Method A): m/z: 283 (M+1); Rt = 1.85 min. General procedure (A) for preparation of compounds of general formula :
Figure imgf000064_0001
wherein D, E and R19 are as defined above, and E is optionally substituted with up to three substituents R21, R22 and R23 independently as defined above.
The carboxylic acid of 1 H-ben∑otriazoIe-5-carboxylic acid is activated, ie the OH functionality is converted into a leaving group L (selected from eg fluorine, chlorine, bromine, iodine, 1- imida∑olyl, 1,2,4-tria∑oIyl, 1-ben∑otriazolyloxy, 1-(4-aza benzotriazolyl)oxy, pentafluoro- phenoxy, N-succinyloxy 3,4-dihydro-4-oxo-3-(1,2,3-benzotriazinyl)oxy, benzotriazole 5-COO, or any other leaving group known to act as a leaving group in acylation reactions. The activated benzotriazole-5-carboxylic acid is then reacted with R2-(CH2)n-B' in the presence of a base. The base can be either absent (i.e. R2-(CH2)n-B' acts as a base) or triethylamine, N- ethyl-N,N.-diisopropylamine, N-methylmorpholine, 2,6-lutidine, 2,2,6,6-tetramethylpiperidine, potassium carbonate, sodium carbonate, caesium carbonate or any other base known to be useful in acylation reactions. The reaction is performed in a solvent solvent such as THF, di- oxane, toluene, dichloromethane, DMF, NMP or a mixture of two or more of these. The reaction is performed between 0 °C and 80 °C, preferably between 20 °C and 40 °C. When the acylation is complete, the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art.
The general procedure (A) is further illustrated in the following example:
Example 9 (General Procedure (A))
1 /-/-Benzotriazole-5-carboxylic acid phenylamide
Figure imgf000064_0002
Benzotria∑ole-5-carboxylic acid (856 mg), HOAt (715 mg) and EDAC (1.00 g) were dissolved in DMF (17.5 mL) and the mixture was stirred at room temperature 1 hour. A 0.5 mL aliqot of this mixture was added to aniline (13.7 L, 0.15 mmol) and the resulting mixture was vigorously shaken at room temperature for 16 hours. 1N hydrochloric acid (2 mL) and ethyl ace- tate (1 mL) were added and the mixture was vigorously shaken at room temperature for 2 hours. The organic phase was isolated and concentrated in vacuo to afford the title compound.
HPLG-MS (Method B): m/∑: 239 (M+1); Rf = 3.93 min.
The compounds in the following examples were similarly made. Optionally, the compounds may be isolated by filtration or by chromatography.
Example 10 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid (4-methoxyphenyl)amide
Figure imgf000065_0001
HPLC-MS (Method A): m/z: 269 (M+1) & 291 (M+23); Rt = 2.41 min HPLC-MS (Method B): m/z: 239 (M+1); Rt = 3.93 min.
Example 11 (General Procedure (A)) {4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}carbamic acid ferf-butyl ester
Figure imgf000065_0002
HPLC-MS (Method B): m/z: 354 (M+1); Rt = 4.58 min.
Example 12 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid (4-acetylaminophenyl)amide
Figure imgf000065_0003
HPLC-MS (Method B): m/z: 296 (M+1); Rt = 3.32 min.
Example 13 (General Procedure (A)) 1H-Benzotria∑ole-5-carboxylic acid (3-fluorophenyl)amidβ
Figure imgf000066_0001
HPLC-MS (Method B): m/z: 257 (M+1); Rt = 4.33 min.
Example 14 (General Procedure (A)) 1H-Ben∑otria∑ole-5-carboxylic acid (2-chlorophenyl)amide
Figure imgf000066_0002
HPLC-MS (Method B): m/z: 273 (M+1); Rt = 4.18 min.
Example 15 (General Procedure (A)) 4-[(1/Y-Benzotriazole-5-carbonyl)amino]benzoic acid methyl ester
Figure imgf000066_0003
HPLC-MS (Method A):m/z: 297 (M+1); Rt : 2,60 min. HPLC-MS (Method B): m/z: 297 (M+1);
Rt = 4.30 min.
Example 16 (General Procedure (A))
1H-Benzotriazole-5-carboxylic acid (4-butylphenyl)amide
Figure imgf000066_0004
HPLC-MS (Method B): m/z: 295 (M+1); Rt = 5.80 min.
Example 17 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid (l-phenylethyl)amide
Figure imgf000066_0005
HPLC-MS (Method B): m/z: 267 (M+1); Rt = 4.08 min. Example 18 (General Procedure (A)) IH-Benzotriazole-S-carboxylic acid benzylamide
Figure imgf000067_0001
HPLC-MS (Method B): m/z: 253 (M+1); Rf = 3.88 min.
Example 19 (General Procedure (A)) 1H-Benzotria∑ole-5-carboxylic acid 4-chlorobenzylamid.
Figure imgf000067_0002
HPLC-MS (Method B): m/z: 287 (M+1); Rt = 4.40 min.
Example 20 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid 2-chlorobenzylamide
Figure imgf000067_0003
HPLC-MS (Method B): m/z: 287 (M+1); Rt = 4.25 min.
Example 21 (General Procedure (A)) Ir -Benzotriazole-δ-carboxylic acid 4-methoxybenzylamide
Figure imgf000067_0004
HPLC-MS (Method B): m/z: 283 (M+1); Rt = 3.93 min.
Example 22 (General Procedure (A)) 1ry-Benzotriazole-5-carboxylic acid 3-methoxybenzylamide
Figure imgf000067_0005
HPLC-MS (Method B): m/z: 283 (M+1); Rt = 3.97 min. Example 23 (General Procedure (A)) IH-Benzotriazole-δ-carboxylic acid (1,2-diphenylethyl)amide
Figure imgf000068_0001
HPLC-MS (Method B): m/z: 343 (M+1); Rt = 5.05 min.
Example 24 (General Procedure (A))
1 H-Benzotriazole-S-carboxylic acid 3-bromobβnzylamide
Figure imgf000068_0002
HPLC-MS (Method B): m/z: 331 (M+1); Rt = 4.45 min.
Example 25 (General Procedure (A))
4-{[(1 H-Benzotriazole-5-carbonyl)amino]methyl}benzoic acid
Figure imgf000068_0003
HPLC-MS (Method B): m/z: 297 (M+1); Rt = 3.35 min.
Example 26 (General Procedure (A)) l/V-Benzotriazole-S-carboxylic acid phenethylamide
Figure imgf000068_0004
HPLC-MS (Method B): m/z: 267 (M+1); Rt = 4.08 min.
Example 27 (General Procedure (A))
1 H-Benzotriazole-5-carboxylic acid [2-(4-chlorophenyl)ethyl]amide
Figure imgf000068_0005
HPLC-MS (Method B): m/z: 301 (M+1); Rt = 4.50 min. Example 28 (General Procedure (A))
1 H-Benzotriazole-5-carboxylic acid [2-(4-methoxyphenyl)ethyl]amide
Figure imgf000069_0001
HPLC-MS (Method B): m/z: 297 (M+1); Rt = 4.15 min.
Example 29 (General Procedure (A))
1 H-Benzotriazole-5-carboxylic acid [2-(3-methoxyphenyI)ethyl]amide
Figure imgf000069_0002
HPLC-MS (Method B): m/z: 297 (M+1); Rt = 4.13 min.
Example 30 (General Procedure (A))
1 H-Benzotriazole-5-carboxylic acid [2-(3-chlorophenyl)ethyl]amide
Figure imgf000069_0003
HPLC-MS (Method B): m/z: 301 (M+1); Rt = 4.55 min.
Example 31 (General Procedure (A)) 1 --Benzotriazole-5-carboxylic acid (2,2-diphenylethyl)amide
Figure imgf000069_0004
HPLC-MS (Method B): m/z: 343 (M+1); Rt = 5.00 min.
Example 32 (General Procedure (A))
1 H-Benzotriazole-5-carboxylic acid (3,4-dichlorophenyI)methylamide
Figure imgf000069_0005
HPLC-MS (Method B): m/z: 321 (M+1); Rt = 4.67 min.
Example 33 (General Procedure (A)) 1H-Benzotria∑ole-5-carboxylic acid methylphenylamide
Figure imgf000070_0001
HPLC-MS (Method B): m/z: 253 (M+1); Rt = 3.82 min.
Example 34 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid benzylmethylamide
Figure imgf000070_0002
HPLC-MS (Method B): m/z: 267 (M+1); Rt = 4.05 min.
Example 35 (General Procedure (A))
1 H-Benzotriazole-5-carboxylic acid [2-(3-chloro-4-methoxyphenyl)ethyl]methyl-amide
Figure imgf000070_0003
HPLC-MS (Method B): m/z: 345 (M+1); Rt = 4.37 min.
Example 36 (General Procedure (A)) 1 -/-Benzotriazole-5-carboxylic acid methylphenethylamide
Figure imgf000070_0004
HPLC-MS (Method B): m/z: 281 (M+1); Rt = 4.15 min.
Example 37 (General Procedure (A))
1 H-Benzotria∑ole-5-carboxylic acid [2-(3,4-dimethoxyphenyl)ethyl]methylamide
Figure imgf000071_0001
HPLC-MS (Method B): m/z: 341 (M+1); Rt = 3.78 min;
Example 38 (General Procedure (A))
1 H-Benzotriazole-5-carboxylic acid (2-hydroxy-2-phenylethyl)methylamid€
Figure imgf000071_0002
HPLC-MS (Method B): m/z: 297 (M+1); Rt = 3.48 min.
Example 39 (General procedure (A))
1 H-Benzotriazole-5-carboxylic acid (3-bromopheny!)amide
Figure imgf000071_0003
HPLC-MS (Method A): m/z: 317 (M+1); Rt = 3.19 min.
Example 40 (General procedure (A)) 1 H-Benzotriazole-5-carboxylic acid (4-bromophenyl)amide
Figure imgf000071_0004
HPLC-MS (Method A): m/z: 317 (M+1); Rt = 3.18 min.
Example 41 (General procedure (A))
{4-[(1H-Benzotriazole-5-carbonyl)amino]benzoylamino}acetic acid
Figure imgf000071_0005
HPLC-MS (Method A): m/z: 340 (M+1); Rt = 1.71 min. Example 42 (General procedure (A))
{4-[(1 H-Benzotriazole-5-carbonyl)amino]phenyl}acetic acid
Figure imgf000072_0001
HPLC-MS (Method A): m/z: 297 (M+1); Rt = 2.02 min.
Example 43 (General procedure (A)) 3-{4-[(1 H-Ben∑otriazole-5-carbonyl)amino]phenyl}acrylic acid
Figure imgf000072_0002
HPLC-MS (Method A): m/z: 309 (M+1); Rt = 3.19 min.
Example 44 (General procedure (A))
{3-[(1 H-Benzotriazole-5-carbonyl)amino]phenyl}acetic acid
Figure imgf000072_0003
HPLC-MS (Method A): m/z: 297 (M+1); Rt = 2.10 min.
Example 45 (General procedure (A))
2-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenoxy}-2-methylpropionic acid
Figure imgf000072_0004
HPLC-MS (Method A): m/z: 341 (M+1); Rt = 2.42 min.
Example 46 (General procedure (A)) 3-{4-[(1 H-Benzotriazole-5-carbonyl)amino]benzoylamino}propionic acid
Figure imgf000073_0001
HPLC-MS (Method A): m/z: 354 (M+1); Rt = 1.78 min.
Example 47 (General procedure (A))
3-{4-[(1 H-Benzotriazole-5-carbonyl)amino]phenyl}propionic acid
Figure imgf000073_0002
HPLC-MS (Method A): m/z: 311 (M+1); Rt = 2.20 min.
Example 48 (General procedure (A)) 1H-Benzotriazole-5-carboxylic acid (4-benzyloxyphenyl)amide
Figure imgf000073_0003
HPLC-MS (Method A): m/z: 345 (M+1); Rt = 3.60 min.
Example 49 (General procedure (A))
1 H-Benzotriazole-5-carboxylic acid (3-chloro-4-methoxyphenyi)amide
Figure imgf000073_0004
HPLC-MS (Method A): m/z: 303 (M+1); Rt = 2.88 min.
Example 50 (General procedure (A))
1 H-Benzotriazole-5-carboxylic acid (4-phenoxyphenyl)amide
Figure imgf000074_0001
HPLC-MS (Method A): m/z: 331 (M+1); Rt = 3.62 min.
Example 51 (General procedure (A))
1 H-Benzotriazole-5-carboxylic acid (4-butoxyphenyl)amid_
Figure imgf000074_0002
HPLC-MS (Method A): m/z: 311 (M+1); Rt = 3.59 min.
Example 52 (General procedure (A))
1 H-Benzotriazole-5-carboxylic acid (3-bromo-4-trifluoromethoxyphenyl)amide
Figure imgf000074_0003
HPLC-MS (Method A): m/z: 402 (M+1); Rt = 3.93 min.
Example 53 (General procedure (A))
1 H-Benzotriazole-5-carboxylic acid (3,5-dichloro-4-hydroxyphenyl)amide
Figure imgf000074_0004
HPLC-MS (Method A): m/z: 323 (M+1); Rt = 2.57 min.
Example 54 (General procedure (A))
4-{[(1 H-Ben∑otriazole-5-carbonyl)amino]methyl}benzoic acid
Figure imgf000075_0001
HPLG-MS (Method A): m/z: 297 (M+1); Rt = 1.86 min.
Example 55 (General procedure (A)) {4-[(1H-Benzotriazole-5-carbonyl)amino]phenylsulfanyl}acetic acid
Figure imgf000075_0002
HPLC-MS (Method A): m/z: 329 (M+1); Rt = 2.34 min.
Example 56
N-(1H-Benzotriazol-5-yl)acetamide
Figure imgf000075_0003
HPLC-MS (Method A): m/z: 177 (M+1); Rt = 0.84 min.
Example 57 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid 4-nitrobenzylamide
Figure imgf000075_0004
The following compound is prepared according to general procedure (N) as described below: Example 58 (General procedure (N)) 1H-Benzotriazole-5-carboxylic acid 4-chlorobenzylamide
Figure imgf000075_0005
HPLC-MS (Method B): m/z: 287 (M+1); Rt = 4.40 min. Example 59 2-[(1 H-Benzotriazol-5-ylimino)methyl]-4,6-dichlorophenol
Figure imgf000076_0001
Example 60 Diethyl 2-[(1H-benzotriazol-6-ylamino)methylidene]malonate
Figure imgf000076_0002
Example 61 N1-(1H-Benzotriazol-5-yl)-3-chlorobenzamide
Figure imgf000076_0003
Example 62 N1-(1H-Benzotriazol-5-yl)-3,4,5-trimethoxybenzamide
Figure imgf000076_0004
Example 63 N2-(1 H-Benzotriazol-5-yl)-3-chlorobenzo[b]thiophene-2-carboxamide
Figure imgf000077_0001
Example 64 6-Bromo-1 H-benzotriazole
Figure imgf000077_0002
Example 65 2-[(1 H-Benzotriazol-5-ylimino)methyl]-4-bromophenol
Figure imgf000077_0003
General procedure (B) for preparation of compounds of general formula
Figure imgf000077_0004
I, wherein X, Y, A and R3 are as defined above and A is optionally substituted with up to four substituents R7, R8, R9, and R10 as defined above.
The chemistry is well known (eg Lohray et al., J. Med. Chem., 1SS8, 42, 2569-81) and is generally performed by reacting a carbonyl compound (aldehyde or ketone) with the heterocyclic ring (eg thiazolidine-2,4-dione (X = O; Y = S), rhodanine (X = Y = S) and hydantoin (X = O; Y = NH) in the presence of a base, such as sodium acetate, potassium acetate, ammonium acetate, piperidinium benzoate or an amine (eg piperidine, triethylamine and the like) in a solvent (eg acetic acid, ethanol, methanol, DMSO, DMF, NMP, toluene, benzene) or in a mixture of two or more of these solvents. The reaction is performed at room temperature or at elevated temperature, most often at or near the boiling point of the mixture. Optionally, azeotropic removal of the formed water can be done.
This general procedure (B) is further illustrated in the following example: Example 66 (General procedure (B)) 5-(3-Phenoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000078_0001
A solution of thiazolidine-2,4-dione (90%, 78 mg, 0.6 mmol) and ammonium acetate (92 mg, 1.2 mmol) in acetic acid (1 mL) was added to 3-phenoxybenzaldehyde (52 μL, 0.6 mmol) and the resulting mixture was shaken at 115 °C for 16 hours. After cooling, the mixture was concentrated in vacuo to afford the title compound.
HPLC-MS (Method A): m/z: 298 (M+1); Rt = 4.54 min.
The compounds in the following examples were similarly prepared. Optionally, the compounds can be further purified by filtration and washing with water, ethanol and / or heptane instead of concentration in vacuo. Also optionally the compounds can be purified by washing with ethanol, water and/or heptane, or by chromatography, such as preparative HPLC. Example 67 (General procedure (B)) 5-(4-Dimethylaminobenzylidene)thiazolidine-2,4-dione
Figure imgf000078_0002
HPLC-MS (Method C): m/z: 249 (M+1); Rt = 4.90 min
Example 68 (General procedure (B)) 5-Naphthalen-1-ylmethylenethia∑olidine-2,4-dione
Figure imgf000079_0001
HPLC-MS (Method A): m/∑: 256 (M+1); Rt = 4,16 min.
Example 69 (General procedure (B)) 5-Ben∑ylidene-thia∑olidine-2,4-dione
Figure imgf000079_0002
HPLC-MS (Method A): m/z: 206 (M+1); Rt = 4,87 min.
Example 70 (General procedure (B)) 5-(4-Diethylaminobenzylidene)thiazolidine-2,4-dione
Figure imgf000079_0003
HPLC-MS (Method A): m/z: 277 (M+1); Rt = 4.73 min.
Example 71 (General procedure (B)) 5-(4-Methoxy-benzylidene)-thiazolidine-2,4-dione
Figure imgf000079_0004
HPLC-MS (Method A): m/z: 263 (M+1); Rt = 4,90 min.
Example 72 (General procedure (B)) 5-(4-Chloro-benzylidene)-thia∑olidine-2,4-dione
Figure imgf000080_0001
HPLC-MS (Method A): m/z: 240 (M+1); Rt = 5,53 min.
Example 73 (General procedure (B)) 5-(4-Nitro-benzylidene)-thia∑olidine-2,4-dione
Figure imgf000080_0002
HPLC-MS (Method A): m/z: 251 (M+1); Rt = 4,87 min.
Example 74 (General procedure (B)) 5-(4-Hydroxy-3-methoxy-benzylidene)-thiazolidine-2,4-dione
Figure imgf000080_0003
HPLC-MS (Method A): m/z: 252 (M+1); Rt = 4,07 min.
Example 75 (General procedure (B)) 5-(4-Methylsulfanylbenzylidene)thiazolidine-2,4-dione
Figure imgf000080_0004
HPLC-MS (Method A): m/z: 252 (M+1); Rt = 5,43 min.
Example 76 (General procedure (B)) 5-(2-Pentyloxybenzylidene)thiazolidine-2,4-dione
Figure imgf000081_0001
HPLC-MS (Method C): m/z: 292 (M+1); Rt = 4.75 min.
1H NMR (DMSO-cf6): 6 = 0.90 (3H, t), 1.39 (4H, m), 1.77 (2H, p), 4.08 (2H, t), 7.08 (1H, t),
7.14 (1H, d), 7.43 (2H, m), 8.03 (1H, s), 12.6 (1H, bs).
Example 77 (General procedure (B)) 5-(3-Fluoro-4-methoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000081_0002
HPLC-MS (Method A): m/z: 354 (M+1); Rt = 4,97 min.
Example 78 (General procedure (B)) 5-(4-tert-Butylbenzylidene)thiazolidine-2,4-dione
Figure imgf000081_0003
HPLC-MS (Method A): m/z: 262 (M+1); Rt = 6,70 min.
Example 79 (General procedure (B)) Λ/-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acetamide
Figure imgf000081_0004
HPLC-MS (Method A): m/z: 263 (M+1); Rt = 3,90 min. Example 80 (General procedure (B)) 5-Biphenyl-4-ylmethylene-thiazolidine-2,4-dione
Figure imgf000082_0001
HPLC-MS (Method A): m/z: 282 (M+1); Rt = 4,52 min.
Example 81 (General procedure (B)) 5-(4-Phenoxy-benzylidene)-thiazolidine-2,4-dione
Figure imgf000082_0002
HPLC-MS (Method A): m/z: 298 (M+1); Rt = 6,50 min.
Example 82 (General procedure (B)) 5-(3-Benzyloxybenzylidene)thiazolidine-2,4-dione
Figure imgf000082_0003
HPLC-MS (Method A): m/z: 312 (M+1); Rt = 6,37 min.
Example 83 (General procedure (B)) 5-(3-p-Tolyloxybenzylidene)thiazolidine-2,4-dione
Figure imgf000082_0004
HPLC-MS (Method A): m/z: 312 (M+1); Rt = 6,87 min.
Example 84 (General procedure (B)) 5-Naphthalen-2-ylmethylene-thiazolidine-2,4-dione
Figure imgf000082_0005
HPLC-MS (Method A): m/z: 256 (M+1); Rt = 4.15 min.
Example 85 (General procedure (B)) 5-Ben∑o[1 ,3]dioxol-5-ylmethylenethia∑olidine-2,4-dion.
Figure imgf000083_0001
HPLC-MS (Method A): m/∑: 250 (M+1), Rt = 3.18 min.
Example 86 (General procedure (B)) 5-(4-Chlorobenzylidene)-2-thioxothiazolidin-4-one
Figure imgf000083_0002
HPLC-MS (Method A): m/z: 256 (M+1); Rt = 4,51 min.
Example 87 (General procedure (B)) 5-(4-Dimethylaminobenzylidene)-2-thioxothiazolidin-4-one
Figure imgf000083_0003
HPLC-MS (Method A): m/z: 265 (M+1); Rt = 5,66 min.
Example 88 (General procedure (B)) 5-(4-Nitrobenzylidene)-2-thioxothiazolidin-4-one
Figure imgf000083_0004
HPLC-MS (Method A): m/z: 267 (M+1); Rt = 3,94 min. Example 89 (General procedure (B)) 5-(4-Methylsulfanylbenzylidene)-2-thioxothiazolidin-4-one
Figure imgf000084_0001
HPLC-MS (Method A): m/z: 268 (M+1); Rl = 6,39 min.
Example 90 (General procedure (B)) 5-(3-Fluoro-4-melhoxybenzylidene)-2-thioxothia∑olidin-4-ons
Figure imgf000084_0002
HPLC-MS (Method A): m/z: 270 (M+1); Rt = 5,52 min.
Example 91 (General procedure (B)) 5-Naphthalen-2-ylmethylene-2-thioxothiazolidin-4-one
Figure imgf000084_0003
HPLC-MS (Method A): m/z: 272 (M+1); Rt = 6,75 min.
Example 92 (General procedure (B)) 5-(4-Diethylaminobenzylidene)-2-thioxothiazolidin-4-one
Figure imgf000084_0004
HPLC-MS (Method A): m/z: 293 (M+1); Rt = 5,99 min.
Example 93 (General procedure (B)) 5-Biphenyl-4-ylmethylene-2-lhioxothiazolidin-4-one
Figure imgf000085_0001
HPLC-MS (Method A): m/z: 298 (M+1); Rt = 7,03 min.
Example 94 (General procedure (B)) 5-(3-Phenoxyben∑ylidene)-2-thioxothia∑olidin-4-one
Figure imgf000085_0002
HPLC-MS (Method A): m/z: 314 (M+1); Rt = 6,89 min.
Example 95 (General procedure (B)) 5-(3-Benzyloxybenzylidene)-2-thioxothiazolidin-4-one
Figure imgf000085_0003
HPLC-MS (Method A): m/z: 328 (M+1); Rt = 6,95 min.
Example 96 (General procedure (B)) 5-(4-Benzyloxybenzylidene)-2-thioxothiazolidin-4-one
Figure imgf000085_0004
HPLC-MS (Method A): m/z: 328 (M+1); RT = 6,89 min.
Example 97 (General procedure (B))
5-Naphthalen-1-ylmethylene-2-thioxothiazolidin-4-one
Figure imgf000085_0005
HPLC-MS (Method A): m/z: 272 (M+1); Rt = 6,43 min. Example 98 (General procedure (B))
5-(3-Methoxybenzyl)thiazolidine-2,4-dione
Figure imgf000086_0001
HPLC-MS (Method A): m/z: 236 (M+1); Rt = 3,05 min.
Example 99 (General procedure (D)) 4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid ethyl ester
Figure imgf000086_0002
HPLC-MS (Method A): m/z: 392 (M+23), Rt = 4.32 min.
Example 100 (General procedure (D)) 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenoxy]-butyric acid
Figure imgf000086_0003
HPLC-MS (Method A): m/z: 410 (M+23); Rt = 3,35 min.
Example 101 (General procedure (B)) 5-(3-Bromobenzylidene)thiazolidine-2,4-dione
Figure imgf000086_0004
HPLC-MS (Method A): m/z: 285 (M+1); Rt = 4.01 min.
Example 102 (General procedure (B)) 5-(4-Bromobenzylidene)thiazolidine-2,4-dione
Figure imgf000087_0001
HPLC-MS (Method A): m/z: 285 (M+1); Rf = 4.05 min.
Example 103 (General procedure (B)) 5-(3-Ghloroben∑ylidene)thia∑olidine-2,4-dione
Figure imgf000087_0002
HPLC-MS (Method A): m/z: 240 (M+1); Rt = 3.91 min.
Example 104 (General procedure (B)) 5-Thiophen-2-ylmethylenethiazolidine-2,4-dione
Figure imgf000087_0003
HPLC-MS (Method A): m/z: 212 (M+1); Rt = 3.09 min.
Example 105 (General procedure (B)) 5-(4-Bromothiophen-2-ylmethylene)thiazolidine-2,4-dione
Figure imgf000087_0004
HPLC-MS (Method A): m/z: 291 (M+1); Rt = 3.85 min.
Example 106 (General procedure (B)) 5-(3,5-Dichlorobenzylidene)thiazolidine-2,4-dione
Figure imgf000088_0001
HPLC-MS (fl lethod A): m/z: 274 (M+1); Rt = 4.52 min.
Example 107 (General procedure (B))
5-(1 -Methyl-1 H-indol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000088_0002
HPLC-MS (Method A): m/z: 259 (M+1); Rt = 3.55 min.
Example 108 (General procedure (B)) 5-(1 H-lndol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000088_0003
HPLC-MS (Method A): m/z: 245 (M+1); Rt = 2.73 min.
Example 109 (General procedure (B)) 5-Fluoren-9-ylidenethiazolidine-2,4-dione
Figure imgf000088_0004
HPLC-MS (Method A): m/z: 280 (M+1); Rt = 4.34 min. Example 110 (General procedure (B)) 5-(1-Phenylethylidene)thiazolidine-2,4-dione
Figure imgf000089_0001
HPLC-MS (Method A): m/z: 220 (M+1); Rt = 3,38 min.
Example 111 (General procedure (B)) 5-[1-(4-Methoxyphenyl)-ethylidene]-thiazolidine-2,4-dione
Figure imgf000089_0002
HPLC-MS (Method A): m/z: 250 (M+1); Rt = 3.55 min.
Example 112 (General procedure (B)) 5-(1-Naphthalen-2-yl-ethylidene)-thiazolidine-2,4-dione
Figure imgf000089_0003
HPLC-MS (Method A): m/z: 270 (M+1); Rt = 4,30 min.
Example 113 (General procedure (B)) 5-[1-(4-Bromophenyl)-ethylidene]-thiazolidine-2,4-dione
Figure imgf000089_0004
HPLC-MS (Method A): m/z: 300 (M+1); Rt = 4,18 min. Example 114 (General procedure (B)) 5-(2,2-Diphenylethylidene)-thiazolidine-2,4-dione
Figure imgf000090_0001
HPLC-MS (Method A): m/z: 296 (M+1); Rt = 4,49 min.
Example 115 (General procedure (B)) 5-[1-(3-Methoxyphenyl)-ethylidene]-thiazolidine-2,4-dione
Figure imgf000090_0002
HPLC-MS (Method A): m/z: 250 (M+1); Rt = 3,60 min.
Example 116 (General procedure (B)) 5-[1-(6-Methoxynaphthalen-2-yl)-ethylidene]-thiazolidine-2,4-dione
Figure imgf000090_0003
HPLC-MS (Method A): m/z: 300 (M+1); Rt = 4,26 min.
Example 117 (General procedure (B)) 5-[1-(4-Phenoxyphenyl)-ethylidene]-thiazolidine-2,4-dione
Figure imgf000090_0004
HPLC-MS (Method A): m/z: 312 (M+1); Rt = 4,68 min.
Example 118 (General procedure (B)) 5-[1-(3-Fluoro-4-methoxyphenyl)ethylidene]thiazolidine-2,4-dione
Figure imgf000091_0001
HPLC-MS (Method A): m/z: 268 (M+1); Rt = 3,58 min.
Example 119 (General procedure (B)) 5-[1-(3-Bromophenyl)-ethylidene]-thiazolidine-2,4-dione
Figure imgf000091_0002
HPLC-MS (Method A): m/z: 300 (M+1); Rt = 4,13 min.
Example 120 (General procedure (B)) 5-Anthracen-9-ylmethylenethiazolidine-2,4-dione
Figure imgf000091_0003
HPLC-MS (Method A): m/z: 306 (M+1); Rt = 4,64 min.
Example 121 (General procedure (B)) 5-(2-Methoxynaphthalen-1-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000091_0004
HPLC-MS (Method A): m/z: 286 (M+1); Rt = 4,02 min.
Example 122 (General procedure (B)) 5-(4-Mefhoxynaphthalen-1-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000092_0001
HPLC-MS (Method A): m/z: 286 (M+1); Rt = 4,31 min.
Example 123 (General procedure (B)) 5-(4-Dimethylaminonaphthalen-1-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000092_0002
HPLC-MS (Method A): m/z: 299 (M+1); Rt = 4,22 min.
Example 124 (General procedure (B)) 5-(4-Methylnaphthalen-1-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000092_0003
HPLC-MS (Method A): m/z: 270 (M+1); Rt = 4,47 min.
Example 125 (General procedure (B)) 5-Pyridin-2-ylmethylene-thiazolidine-2,4-dione
Figure imgf000092_0004
Example 126 5-Pyridin-2-ylmethyl-thiazolidine-2,4-dione
Figure imgf000093_0001
5-Pyridin-2-ylmethylene-thia∑olidine-2,4-dione (5 g) in tetrahydrofuran (300 ml) was added 10% Pd/C (1 g) and the mixture was hydrogenated at ambient pressure for 1 hours. More 10% Pd/C (5 g) was added and the mixture was hydrogenated at 50 psi for 16 hours. After filtration and evaporation in vacuo, the residue was purified by column chromatography eluting with a mixture of ethyl acetate and heptane (1:1). This afforded the title compound (0.8 g, 16%) as a solid.
TLC: Rf = 0.30 (SiO2; EtOAc: heptane 1:1)
Example 127 (General procedure (B)) 5-(1H-lmidazol-4-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000093_0002
Example 128 (General procedure (B)) 5-(4-Benzyloxy-benzylidene)-thiazolidine-2,4-dione
Figure imgf000093_0003
HPLC-MS (Method A): m/z: 6,43 min ; 99 % (2A)
Example 129 (General procedure (B))
5-[4-(4-Fluoroben∑yloxy)benzylidene]-2-thioxothiazolidin-4-on.
Figure imgf000093_0004
Example 130 (General procedure (B)) 5-(4-Butoxybenzylidene)-2-thioxothiazolidin-4-one
Figure imgf000094_0001
Example 131 (General procedure (B)) 5-(3-Methoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000094_0002
HPLC-MS (Method A): m/z: 236 (M+1); Rt = 4,97 min
Example 132 (General procedure (B)) 5-(3-Methoxybenzylidene)imidazolidine-2,4-dione
Figure imgf000094_0003
HPLC-MS (Method A): m/z: 219 (M+1); Rt = 2.43 min.
Example 133 (General procedure (B)) 5-(4-Methoxybenzylidene)imidazolidine-2,4-dione
Figure imgf000094_0004
HPLC-MS (Method A): m/∑: 219 (M+1); Rt = 2.38 min. Example 134 (General procedure (B)) 5-(2,3-Dichlorobenzylidene)thiazolidine-2,4-dione
Figure imgf000095_0001
Example 135 (General procedure (B)) 5-Benzofuran-7-ylmethylenethia∑olidine-2,4-dione
Figure imgf000095_0002
HPLC-MS (Method C): m/z: 247 (M+1); Rt = 4,57 min.
Example 136 (General procedure (B)) 5-Benzo[1 ,3]dioxol-4-ylmethylenethiazolidine-2,4-dione
Figure imgf000095_0003
HPLC-MS (Method C): m/z: 250 (M+1); Rt = 4,00 min.
Example 137 (General procedure (B)) 5-(4-Methoxy-2,3-dimethylbenzylidene)thiazolidine-2,4-dione
Figure imgf000095_0004
HPLC-MS (Method C): m/z: 264 (M+1); Rt = 5,05 min.
Example 138 (General procedure (B)) 5-(2-Benzyloxy-3-methoxybenzylidene)thia∑olidine-2,4-dione
Figure imgf000096_0001
HPLC-MS (Method C): m/z: 342 (M+1); Rt = 5,14 min.
Example 139 (General procedure (B))
5-(2-Hydro) yben∑ylidene)thia∑olidine-2,4-dione
Figure imgf000096_0002
HPLC-MS (Method C): m/z: 222 (M+1); Rt = 3,67 min.
Example 140 (General procedure (B)) 5-(2,4-Dichlorobenzylidene)thiazolidine-2,4-dione
HN J ό ci
1H-NMR (DMSO-d6): 7.60 (2H, "s"), 7.78 (1H, s), 7.82 (1H, s).
Example 141 (General procedure (B)) 5-(2-Chlorobenzylidene)thiazolidine-2,4-dione
HN 5^ ) & Cl
1H-NMR (DMSO-cY6): 7.40 (1 H, t), 7.46 (1H, t), 7.57 (1 H, d), 7.62 (1 H, d), 7.74 (1H, s).
Example 142 (General procedure (B)) 5-(2-Bromobenzylidene)thiazolidine-2,4-dione
Figure imgf000096_0003
1H-NMR (DMSO-dβ): 7.33 (1H, t), 7.52 (1H, t), 7.60 (1H, d), 7.71 (1H, s), 7.77 (1H, d). Example 143 (General procedure (B)) 5-(2,4-Dimethoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000097_0001
HPLC-MS (Method C): m/z: 266 (M+1) Rt = 4,40 min.
Example 144 (General procedure (B)) 5-(2-Methoxybenzylidene)thia∑olidine-2,4-dione
Figure imgf000097_0002
HPLC-MS (Method C): m/z: 236 (M+1); Rt = 4,17 min.
Example 145 (General procedure (B)) 5-(2,6-Difluorobenzylidene)thiazolidine-2,4-dione
Figure imgf000097_0003
HPLC-MS (Method C): m/z: 242 (M+1); Rt = 4,30 min.
Example 146 (General procedure (B)) 5-(2,4-Dimethylbenzylidene)thiazolidine-2,4-dione
Figure imgf000097_0004
HPLC-MS (Method C): m/z: 234 (M+1); Rt = 5,00 min.
Example 147 (General procedure (B)) 5-(2,4,6-Trimethoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000098_0001
HPLC-MS (Method C): m/∑: 296 (M+1); Rt = 4,27 min.
Example 148 (General procedure (B)) 5-(4-Hydroxy-2-methoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000098_0002
HPLC-MS (Method C): m/z: 252 (M+1); Rt = 3,64 min.
Example 149 (General procedure (B)) 5-(4-Hydroxynaphthalen-1-ylmethylene)thiazolidine-2,4-dione
Figure imgf000098_0003
1H-NMR (DMSO-d6): δ = 7.04 (1H, d), 7.57 (2H, m), 7.67 (1H, t), 8.11 (1H, d), 8.25 (1H, d), 8.39 (1H, s) 11.1 (1H, s), 12.5 (1H, bs). HPLC-MS (Method C): m/z: 272 (M+1); Rt = 3.44 min.
Example 150 (General procedure (B)) 5-(2-Trifluoromethoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000098_0004
HPLC-MS (Method C): m/z: 290 (M+1); Rt = 4,94 min.
Example 151 (General procedure (B)) 5-Biphenyl-2-ylmethylenethiazolidine-2,4-dione
Figure imgf000099_0001
HPLC-MS (Method C): m/z: 282 (M+1); Rt = 5,17 min.
Example 152 (General procedure (B)) 5-(2-Benzyloxybenzylidene)thia∑olidine-2,4-dione
Figure imgf000099_0002
HPLC-MS (Method C): m/z: 312 (M+1); Rt = 5,40 min.
Example 153 (General procedure (B)) 5-Adamantan-2-ylidenethiazolidine-2,4-dione
Figure imgf000099_0003
HPLC-MS (Method A): m/z: 250 (M+1); Rt = 4,30 min.
Example 154 (General Procedure (B)) 5-[3-(4-Nitrophenyl)allylidene]thiazolidine-2,4-dione
Figure imgf000099_0004
HPLC-MS (Method C): m/z: 277 (M+1); Rt = 3.63 min.
Example 155 (General Procedure (B)) 5-[3-(2-Methoxyphenyl)allylidene]thia∑olidine-2,4-dione
Figure imgf000100_0001
m/∑: 262 (M+1); Rt = 3.81 min.
Example 156 (General Procedure (B)) 5-[3-(4-Methoxyphenyl)allylidene]thia∑olidine-2,4-dion.
Figure imgf000100_0002
HPLC-MS (Method C): m/z: 262 (M+1); Rt = 3.67 min.
Example 157 (General procedure (B))
5-(4-Hydroxybenzylidene)thiazolidine-2,4-dione
Figure imgf000100_0003
Example 158 (General procedure (B)) 5-(4-Dimethylaminobenzylidene)pyrimidine-2,4,6-trione
Figure imgf000100_0004
HPLC-MS (Method C): m/z = 260 (M+1) Rt = 2,16 min.
Example 159 (General procedure (B)) 5-(9-Ethyl-9H-carbazol-2-ylmethylene)-pyrimidine-2,4,6-trione
Figure imgf000101_0001
HPLC-MS (Method C): m/z = 334 (M+1); Rt = 3,55 min.
Example 160 (General procedure (B)) 5-(4-Hexyloxynaphthalen-1-ylmethylene)thiazolidine-2,4-dione
Figure imgf000101_0002
HPLC-MS (Method C): m/z = 356 (M+1); Rt = 5.75 min.
Example 161 (General procedure (B)) 5-(4-Decyloxynaphthalen-1-ylmethylene)thiazolidine-2,4-dione
Figure imgf000101_0003
HPLC-MS (Method C): m/z = 412 (M+1); Rt = 6.44 min.
Example 162 (General procedure (B)) 5-[4-(2-Aminoethoxy)-naphthalen-1-ylmethylene]-thiazolidine-2,4-dione
Figure imgf000102_0001
HPLC-MS (Method C): m/z = 315 (M+1); Rt = 3,24 min.
Example 163 (General procedure 5-(2,4-Dimethyl-9H-carba∑ol-3-ylmethylene)-pyrimidine-2,4,6-trione
Figure imgf000102_0002
HPLC-MS (Method C): m/z = 334 (M+1); Rt = 3,14 min.
Example 164 (General procedure (B)) 4-(4-Hydroxy-3-methoxybenzylidine)hydantoin
Figure imgf000102_0003
Example 165 (General procedure (B)) 5-Benzylidenehydantoin
Figure imgf000102_0004
General procedure (C) for preparation of compounds of general formula l2:
Figure imgf000103_0001
>2 wherein X, Y, A, and R3 are as defined above and A is optionally substituted with up to four substituents R7, R8, R9, and R10 as defined above.
This general procedure (C) is quite similar to general procedure (B) and is further illustrated in the following example:
Example 166 (General procedure (C)) 5-(3,4-Dibromobenzylidene)thiazolidine-2,4-dione
Figure imgf000103_0002
A mixture of thiazolidine-2,4-dione (90%, 65 mg, 0.5 mmol), 3,4-dibromobenzaldehyde (132 mg, 0.5 mmol), and piperidine (247 μL, 2.5 mmol) was shaken in acetic acid (2 mL) at 110 °C for 16 hours. After cooling, the mixture was concentrated to dryness in vacuo . The resulting crude product was shaken with water, centrifuged, and the supernatant was discarded. Subsequently the residue was shaken with ethanol, centrifuged, the supernatant was discarded and the residue was further evaporated to dryness to afford the title compound.
1H NMR (Acetone-dβ): δH 7.99 (d,1H), 7.90 (d,1H), 7.70 (s,1H), 7.54 (d,1H); HPLC-MS (Method A): m/z: 364 (M+1); Rt = 4.31 min.
The compounds in the following examples were similarly prepared. Optionally, the compounds can be further purified by filtration and washing with water instead of concentration in vacuo. Also optionally the compounds can be purified by washing with ethanol, water and/or heptane, or by preparative HPLC.
Example 167 (General procedure (G)) 5-(4-Hydroxy-3-iodo-5-methoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000104_0001
Mp = 256 °G; 1H NMR (DMSQ-cf6) δ = 12.5 (s,broad,1H), 10.5 (s,broad,1H), 7.69 (s,1H), 7.51 (d,1H), 7.19 (d,1H)3.88 (s,3H), 13C NMR (DMSO-ri6) δc = 168.0, 167.7 , 149.0, 147.4, 133.0, 131.2, 126.7, 121.2, 113.5, 85.5, 56.5; HPLC-MS (Method A): m/z: 378 (M+1); Rt = 3.21 min.
Example 168 (General procedure (C)) 5-(4-Hydroxy-2,6-dimelhylbenzylidene)thiazolidine-2,4-dione
Figure imgf000104_0002
HPLC-MS (Method C): m/z: 250 (M+1); Rt.= 2.45 min.
Example 169 (General procedure (C)) 4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-2-yloxymethyl]-benzoic acid
Figure imgf000104_0003
HPLC-MS (Method C): m/z: 506 (M+23); Rt.= 4.27 min.
Example 170 (General procedure (C)) 5-(4-Bromo-2,6-dichlorobenzylidene)thiazolidine-2,4-dione
Figure imgf000104_0004
HPLC-MS (Method G): m/z: 354 (M+1); Rt = 4.36 min. Example 171 (General procedure (C)) 5-(6-Hydroxy-2-naphthylmethylene) thiazolidine-2,4-dione
Figure imgf000105_0001
Mp 310-314 °C, 1H NMR (DMSO-cfe): δH = 12.5 (s,broad,1H), 8.06(d,1H), 7.90- 7.78(m,2H),7.86 (s,1H), 7.58 (dd,1H),7.20 7.12 (m,2H). 13C NMR (DMSO-of6): ^c = 166.2, 165.8 , 155.4, 133.3, 130.1, 129.1, 128.6, 125.4, 125.3, 125.1 , 124.3, 120.0, 117.8, 106.8; HPLC-MS (Method A): m/z: 272 (M+1); Rf = 3.12 min.
Preparation of the starting material, 6-hydroxy-2-naphtalenecarbaldehyde:
6-Cyano-2-naphthalenecarbaldehyde (1.0 g, 5.9 mmol) was dissolved in dry hexane (15 mL) under nitrogen. The solution was cooled to -60 °C and a solution of diisobutyl aluminium hydride (DIBAH) (15 mL, 1M in hexane) was added dropwise. After the addition, the solution was left at room temperature overnight. Saturated ammonium chloride solution (20 mL) was added and the mixture was stirred at room temperature for 20 min, subsequently aqueous H2S0 (10% solution, 15 mL) was added followed by water until all salt was dissolved. The resulting solution was extracted with ethyl acetate (3x), the combined organic phases were dried with MgS0 , evaporated to dryness to afford 0.89 g of 6-hydroxy-2- naphtalenecarbaldehyde.
Mp.: 153.5-156.5 °G; HPLC-MS (Method A): m/z: 173 (M+1); Rt = 2.67 min; 1 H NMR (DMSO- dβ): δH = 10.32(s,1H), 8.95 (d,1H), 10.02 (s,1H), 8.42 (s,broad,1H), 8.01 (d,1H), 7.82-7.78 (m,2H), 7.23-7.18 (m,2H).
Alternative preparation of 6-hydroxy-2-naphtalenecarbaldehyde: To a stirred cooled mixture of 6-bromo-2-hydroxynaphthalene (25.3 g, 0.113 mol) in THF (600 mL) at -78 °C was added n-Buϋ (2.5 M, 100 mL, 0.250 mol) dropwise. The mixture turned yellow and the temperature rose to -64 °G. After ca 5 min a suspension appeared. After addition, the mixture was maintained at -78 °C. After 20 minutes, a solution of DMF (28.9 mL, 0.373 mol) in THF (100 mL) was added over 20 minutes. After addition, the mixture was allowed to warm slowly to room temperature. After 1 hour, the mixture was poured in ice/water (200 mL). To the mixture citric acid was added to a pH of 5. The mixture was stirred for 0.5 hour. Ethyl acetate (200 mL) was added and the organic layer was separated and washed with brine (100 mL), dried over Na2S0 and concentrated. To the residue was added heptane with 20% ethyl acetate (ca 50 mL) and the mixture was stirred for 1 hour. The mixture was filtered and the solid was washed with ethyl acetate and dried in vacuo to afford
16 g of the title compound.
Example 172 (General procedure (C)) 5-(3-lodo-4-methoxyben∑ylidene)thiazolidiene-2,4-dione
Figure imgf000106_0001
1H NMR (DMSO-d6): δH 12.55 (s,broad,1H), 8.02 (d,1H), 7.72 (s,1H), 7.61 (d,1H)7.18(d,1H), 3.88 (s,3H); 3C NMR (DMSO-dβ): δc 168.1 , 167.7 , 159.8, 141.5, 132.0, 130.8, 128.0, 122.1, 112.5, 87.5, 57.3. HPLC-MS (Method A): m/z: 362 (M+1); Rt = 4.08 min.
Preparation of the starting material, 3-iodo-4-methoxybenzaldehyde:
4-Methoxybenzaldehyde (0.5 g, 3.67 mmol) and silver trifluoroacetate (0.92 g, 4.19 mmol) were mixed in dichloromethane (25 mL). Iodine (1.19 g, 4.7 mmol) was added in small portions and the mixture was stirred overnight at room temperature under nitrogen. The mixture was subsequently filtered and the residue washed with DCM. The combined filtrates were treated with an acqueous sodium thiosulfate solution (1 M) until the colour disappeared. Subsequent extraction with dichloromethane (3 x 20 mL) followed by drying with MgS04 and evaporation in vacuo afforded 0.94 g of 3-iodo-4-methoxybenzaldehyde.
Mp 104-107 °C; HPLC-MS (Method A): m/z:263 (M+1); Rt = 3.56 min.;1H NMR (CDCI3): δH = 8.80 (s,1H), 8.31 (d,1 H), 7.85 (dd,1H) 6.92 (d,1H), 3.99 (s, 3H).
Example 173 (General procedure (C)) 5-(1-Bromonaphthalen-2-ylmethylene)thiazolidine-2,4-dione
Figure imgf000106_0002
HPLC-MS (Method A): m/z: =336 (M+1); Rt = 4.46 min. Example 174 (General procedure (C))
1 -[5-(2,4-Dioxothiazolidin-5-ylidenemethyl)thiazol-2-yl]piperidine-4-carboxylic acid ethyl ester
Figure imgf000107_0001
H NMR (DMSO-cfβ): δH = 7.88 (s,1H), 7.78 (s,1H), 4.10 (q,2H), 4.0-3.8 (m,2H), 3.40-3.1. (m,2H), 2.75-2.60 (m,1H), 2.04-1.88 (m,2H), 1.73-1.49 (m,2H) , 1.08 (t,3H); HPLC-MS (Method A): m/z: 368 (M+1); Rt = 3.41 min.
Example 175 (General procedure (C)) 5-(2-Phenyl-[1,2,3]triazol-4-ylmethylene) thiazolidine-2,4-dione
Figure imgf000107_0002
1H NMR (DMSO-dβ): δ = 12.6 (s,broad,1 H), 8.46 (s,1H), 8.08 (dd,2H), 7.82 (s,1H), 7.70-7.45 (m, 3H). HPLC-MS (Method A): m/z: 273 (M+1); Rt = 3.76 min.
Example 176 (General procedure (C)) 5-(Quinolin-4-ylmethylene)thiazolidine-2,4-dione
Figure imgf000107_0003
HPLC-MS (Method A): m/z: 257 (M+1); Rt = 2.40 min.
Example 177 (General procedure (C)) 5-(6-Methylpyridin-2-ylmethylene)thiazolidine-2,4-dione
Figure imgf000108_0001
1H NMR (DMSO-de): H = 12.35 (s,broad,1H), 7.82 (t,1H), 7.78 (s,1H), 7.65 (d,1H), 7.1? (d,1H), 2.52 (s,3 H); HPLC-MS (Method A): m/∑: 221 (M+1); Rt = 3.03 min.
Example 178 (General procedure (C)) 5-(2,4-dioxothiazolidin-5-ylidenemethyl)-furan-2-ylmethylacetate
Figure imgf000108_0002
H NMR (DMSO-dβ): δH = 12.46 (s,broad,1H), 7.58 (s,1H), 7.05 (d,1H), 6.74 (s,1H), 5.13 (s,2H), 2.10 (s,3H). HPLC-MS (Method A): m/z: 208 (M-CH3COO); Rt = 2.67 min.
Example 179 (General procedure (C)) 5-(2,4-Dioxothiazolidin-5-ylidenemethyl)furan-2-sulfonicacid
Figure imgf000108_0003
HPLC-MS (Method A): m/z:276 (M+1 ); Rt = 0.98 min.
Example 180 (General procedure (C)) 5-(5-Benzyloxy-1H-pyrrolo[2,3-c]pyridin-3-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000108_0004
HPLC-MS (Method A): m/z: 352 (M+1); Rt = 3.01 min.
Example 181 (General procedure (C)) 5-(Quinolin-2-ylmethylene)thiazolidine-2,4-dione
Figure imgf000109_0001
HPLC-MS (Method A): m/z: 257 (M+1); Rt = 3.40 min.
Example 182 (General procedure (G))
5-(2,4-Dioxothia∑olidin-5-ylidenemethyl)thiophene-2-carboxylic acid
Figure imgf000109_0002
HPLC-MS (Method A): m/z: 256 (M+1); Rt = 1.96 min.
Example 183 (General procedure (C)) 5-(2-Phenyl-1H-imidazol-4-ylmethylene)thiazolidine-2,4-dione
Figure imgf000109_0003
HPLC-MS (Method A): m/z: 272 (M+1); Rt = 2.89 min.
Example 184 (General procedure (C)) 5-(4-lmidazol-1-yl-benzylidene)thiazolidine-2,4-dione
Figure imgf000109_0004
HPLC-MS (Method A): m/z: 272 (M+1); Rt = 1.38 min.
Example 185 (General procedure (C)) 5-(9-Ethyl-9H-carbazol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000109_0005
HPLC-MS (Method A): m/z: 323 (M+1); Rt = 4.52 min.
Example 186 (General procedure (C)) 5-(1 ,4-Dimethyl-9H-carbazol-3-ylmethylene)thia∑olidine-2,4-dione
Figure imgf000110_0001
HPLC-MS (Method A): m/z: 323 (M+1); Rt = 4.35 min.
Example 187 (General procedure (C)) 5-(2-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000110_0002
HPLC-MS (Method A): m/z: 259 (M+1); Rt = 3.24 min.
Example 188 (General procedure (C)) 5-(2-Ethylindol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000110_0003
2-Methylindole (1.0 g, 7.6mmol) dissolved in diethyl ether (100 mL) under nitrogen was treated with n-Butyl lithium (2 M in pentane, 22.8 mmol) and potassium ferf-butoxide (15.2 mmol) with stirring at RT for 30 min. The temperature was lowered to -70 C and methyl Iodide (15.2 mmol) was added and the resulting mixture was stirred at -70 for 2 h. Then 5 drops of water was added and the mixture allowed to warm up to RT. Subsequently, the mixture was poured into water (300 mL), pH was adjusted to 6 by means of 1 N hydrochloric acid and the mixture was extracted with diethyl ether. The organic phase was dried with Na2S04 and evaporated to dryness. The residue was purified by column chromatography on silica gel using heptane/elher( 4/1) as eluent. This afforded 720 mg (69 %) of 2-ethylindole. 1H NMR (DMSO- 6 ): δ = 10.85 (1 H,s); 7.39 (1H,d); 7.25 (1 H,d); 6.98(1H,t); 6.90(1 H,t); 6.10 (1 H,s); 2.71 (2H,q); 1.28 (3H,t).
2-Ethylindole (0.5 g, 3.4mmol) dissolved in DMF (2 mL) was added to a cold (0 °C) premixed (30 minutes) mixture of DMF (1.15 mL) and phosphorous oxychloride (0.64 g, 4.16 mmol). After addition of 2-ethylindole, the mixture was heated to 40 °C for 1 h, water (5 mL) was added and the pH adjusted to 5 by means of 1 N sodium hydroxide.The mixture was subsequently extracted with diethyl ether, the organic phase isolated, dried with MgS04 and evaporated to dryness affording 2-ethylindole-3-carbaldehyde (300 mg ).
HPLC-MS (Method C): m/z:174 (M+1); Rt. =2.47 min.
2-Ethylindole-3-carbaldehyde (170 mg) was treated with thiazolidine-2,4-dione using the general procedure (C) to afford the title compound (50 mg).
HPLC-MS (Method C):m/z: 273 (M+1); Rt.= 3.26 min.
Example 189 (General procedure (C))
5-[2-(4-Bromophenylsulfanyl)-1-methyl-1 H-indol-3-ylmethylene]thiazolidine-2,4-dione
Figure imgf000111_0001
HPLC-MS (Method A): m/z: 447 (M+1); Rt = 5.25 min.
Example 190 (General procedure (C)) 5-[2-(2,4-Dichlorobenzyloxy)-naphthalen-1-ylmethylene]thiazolidine-2,4-dione
Figure imgf000112_0001
HPLC-MS (Method A): (anyone 1) m/z: 430 (M+1); Rt = 5.47 min.
Example 191 (General procedure (C))
5-{4-[3-(4-Bromophenyl)-3-oxopropenyl]-benzylidene}thiazolidine-2,4-dionε
Figure imgf000112_0002
HPLC-MS (Method A): m/z: 416 (M+1); Rt = 5.02 min.
Example 192 (General procedure (C)) 5-(4-Pyridin-2-ylbenzylidene)thiazolidine-2,4-dione
HPLC-MS (Method A): m/z: 283 (M+1), Rt = 2.97 min.
Example 193 (General procedure (C)) 5-(3,4-Bisbenzyloxybenzylidene)thiazolidine-2,4-dione
Figure imgf000112_0004
HPLC-MS (Method A): m/z: 418 (M+1); Rt = 5.13 min.
Example 194 (General procedure (C)) 5-[4-(4-Nitrobenzyloxy)-benzylidene]thiazolidine-2,4-dione
Figure imgf000113_0001
HPLC-MS (Method A): m/z: 357 (M+1); Rt = 4.45 min.
Example 195 (General procedure (C)) 5-(2-Phenyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dions
Figure imgf000113_0002
HPLC-MS (Method A): m/z: 321 (M+1); Rt = 3.93 min.
Example 196 (General procedure (C)) 5-(5-Benzyloxy-1H-indol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000113_0003
HPLC-MS (Method A): m/z: 351 (M+1); Rt = 4.18 min.
Example 197 (General procedure (C)) 5-(4-Hydroxybenzylidene)thiazolidine-2,4-dione
Figure imgf000113_0004
HPLC-MS (Method A): m/z: 222 (M+1); Rt = 2.42 min.
Example 198 (General procedure (C))
5-(1 -Methyl-1 H-indol-2-ylmethylene)thiazolidine-2,4-dionc
Figure imgf000114_0001
1H NMR (DMSO-d6): δH = 12.60 (s,broad,1H), 7.85 (s,1H), 7.68 (dd, 1H), 7.55 (dd,1H), 7.38 (dt,1H), 7.11 (dt,1H) 6.84 (s,1H), 3.88 (s,3H); HPLG-MS (Method A): m/z: 259 (M+1); Rt = 4.00 min.
Example 199 (General procedure (G)) 5-(5-Nitro-1H-indol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000114_0002
.Mp 330-333 °C, 1H NMR (DMSO-cfe): δH = 12.62 (s,broad,1H), 8.95 (d,1H), 8.20 (s,1H), 8.12 (dd,1H), 7.98 (s,broad,1H), 7.68 (d,1H); HPLC-MS (Method A): m/z: 290 (M+1); Rt = 3.18 min.
Example 200 (General procedure (C)) 5-(6-Methoxynaphthalen-2-ylmethylene)thiazolidine-2,4-dione
Figure imgf000114_0003
HPLC-MS (Method A): m/z: 286 (M+1); Rt = 4.27 min.
Example 201 (General procedure (C)) 5-(3-Bromo-4-methoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000114_0004
HPLC-MS (Method A): m/z: 314 (M+1), Rt = 3.96 min.
Example 202 (General procedure (C)) 3-{(2-Cyanoethyl)-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]amino}propionitrile
Figure imgf000115_0001
HPLC-MS (Method A): m/z: 327 (M+1); Rt = 2.90 min.
Example 203 (General procedure (C)) 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methyl ester
Figure imgf000115_0002
HPLC-MS (Method A): m/z: 303 (M+1); Rt = 3.22-3-90 min.
Example 204
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid pentyl ester.
Figure imgf000115_0003
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methyl ester (example 203, 59 mg; 0.195mmol) was stirred in pentanol (20 mL) at 145 °C for 16 hours. The mixture was evaporated to dryness affording the title compound (69 mg).
HPLC-MS (Method C): m/z: 359 (M+1); Rt.= 4.25 min.
Example 205 (General procedure (C)) 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carboxylic acid
Figure imgf000115_0004
HPLC-MS (Method A): m/z: 289 (M+1); Rt = 2.67 min.
Example 206 (General procedure (C)) 5-(1-Benzylindol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000116_0001
HPLC-MS (Method A): m/∑: 335 (M+1); Rt = 4.55 min.
Example 207 (General procedure (C)) 5-(1-Ben∑enesulfonylindol-3-ylmethylene)thia∑olidine-2,4-dionξ
Figure imgf000116_0002
HPLC-MS (Method A): m/z: = 385 (M+1); Rt = 4.59 min.
Example 208 (General procedure (C)) 5-(4-[1 ,2,3]Thiadiazol-4-ylbenzylidene)thiazolidine-2,4-dione
Figure imgf000116_0003
HPLC-MS (Method A): m/z: 290 (M+1); Rt = 3.45 min.
Example 209 (General procedure (C)) 5-[4-(4-Nitrobenzyloxy)-benzylidene]thiazolidine-2,4-dione
Figure imgf000116_0004
HPLC-MS (Method A): m/z: 357 (M+1); Rt = 4.42 min. Example 210 (General procedure (C))
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-1-carboxylic acid ethyl ester
Figure imgf000117_0001
HPLC-MS (Method A): m/z: 317 (M+1); Rt = 4.35 min.
Example 211 (General procedure (C)) 5-[2-(4-Pentylbenzoyl)-benzofuran-5-ylmethylene]thiazolidine-2,4-dione
Figure imgf000117_0002
HPLC-MS (Method A): m/z: 420 (M+1); Rt = 5.92 min.
Example 212 (General procedure (C)) 5-[1-(2-Fluorobenzyl)-4-nitroindol-3-ylmethylene]thiazolidine-2,4-dione
Figure imgf000117_0003
HPLC-MS (Method A): (Anyone 1) m/z: 398 (M+1); Rt = 4.42 min.
Example 213 (General procedure (C)) 5-(4-Benzyloxyindol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000117_0004
HPLC-MS (Method A): m/z: 351 (M+1); Rt = 3.95 min. Example 214 (General procedure (C)) 5-(4-lsobutylbenzylidene)-thiazolidine-2,4-dione
Figure imgf000118_0001
HPLC-MS (Method A): m/z: 262 (M+1); Rt = 4.97 min.
Example 215 (General procedure (C))
Trifluoromethanesulfonic acid 4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yl ester
Figure imgf000118_0002
HPLC-MS (Method A): m/z: 404 (M+1); Rt = 4.96 min.
Preparation of starting material:
4-Hydroxy-1-naphthaldehyde (10 g, 58 mmol) was dissolved in pyridin (50 ml) and the mixture was cooled to 0-5 °C. With stirring, trifluoromethanesulfonic acid anhydride (11.7 ml, 70 mmol) was added drop-wise. After addition was complete, the mixture was allowed to warm up to room temperature, and diethyl ether (200 ml) was added. The mixture was washed with water (2 x 250 ml), hydrochloric acid (3N, 200 ml), and saturated aqueous sodium chloride (100 ml). After drying (MgSO4), filtration and concentration in vacuo, the residue was purified by column chromatography on silica gel eluting with a mixture of ethyl acetate and heptane (1:4). This afforded 8.35 g (47%) trifluoromethanesulfonic acid 4-formylnaphthalen-1-yl ester, mp 44-46.6 °C.
Example 216 (General procedure (C)) 5-(4-Nitroindol-3-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000118_0003
HPLC-MS (Method A): m/z: 290 (M+1); Rt = 3.14 min. Example 217 (General procedure (C)) 5-(3,5-Dibromo-4-hydroxy-benzylidene)thiazolidine-2,4-dione
Figure imgf000119_0001
1H NMR (DMSQ-de): H = 12.65 (broad,1H), 10.85 (broad,1H), 7.78 (s,2H), 7.70 (s,1H); HPLC-MS (Method A): m/z: 380 (M+1); Rt = 3.56 min.
Example 218 (General procedure (C))
Figure imgf000119_0002
HPLC-MS (Method A): m/z: 385 (M+1); Rt = 5.08 min.
General procedure for preparation of starting materials for examples 218 - 221 : lndole-3-carbaldehyde (3.8 g, 26 mmol) was stirred with potassium hydroxide (1.7 g) in acetone (200 mL) at RT until a solution was obtained indicating full conversion to the indole potassium salt. Subsequently the solution was evaporated to dryness in vacuo. The residue was dissolved in acetone to give a solution containing 2.6 mmol/20 mL.
20 mL portions of this solution were mixed with equimolar amounts of arylmethylbromides in acetone (10 mL). The mixtures were stirred at RT for 4 days and subsequently evaporated to dryness and checked by HPLC-MS. The crude products, 1-benzylated indole-3- carbaldehydes, were used for the reaction with thiazolidine-2,4-dione using the general procedure C.
Example 219 (General procedure (C)) 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-ylmethyl]ben∑oic acid methyl ester
Figure imgf000120_0001
HPLC-MS (Method A): m/z: 393 (M+1); Rt = 4.60 min.
Example 220 (General procedure (C))
5-[1 -(9, 10-Dioxo-9, 10-dihydroanthracen-2-ylmethyl)-1 H-indol-3-ylmethylene]thia∑olidine-2,4- dione
Figure imgf000120_0002
HPLC-MS (Method A): m/z: 465 (M+1); Rt = 5.02 min.
Example 221 (General procedure (C)) 4,-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-ylmethyl]biphenyl-2-carbonitrile
Figure imgf000120_0003
HPLC-MS (Method A): m/z: 458 (M+23); Rt = 4.81 min.
Example 222 (General procedure (C)) 3-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-methylindol-1-ylmethyl]benzonitrile.
Figure imgf000120_0004
2-Methylindole-3-carbaldehyde (200 mg, 1.26 mmol) was added to a slurry of 3- bromomethylbenzenecarbonitrile (1.26 mmol) followed by sodium hydride, 60%, (1.26 mmol) in DMF (2 mL). The mixture was shaken for 16 hours, evaporated to dryness and washed with water and ethanol. The residue was treated with thiazolidine-2,4-dione following the general procedure C to afford the title compound (100 mg).
HPLC-MS (Method C): m/z: 374 (M+1); Rt. = 3.95 min.
Example 223 (General procedure (C)) 5-(1-Benzyl-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione.
Figure imgf000121_0001
This compound was prepared in analogy with the compound described in example 222 from benzyl bromide and 2-methylindole-3-carbaldehyde, followed by reaction with thiazolidine- 2,4-dione resulting in 50 mg of the title compound.
HPLC-MS (Method C): m/z: 349 (M+1); Rt. = 4.19 min.
Example 224
4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-methylindol-1-ylmethyl]benzoic acid methyl ester
Figure imgf000121_0002
This compound was prepared in analogy with the compound described in example 222 from 4-(bromomethyl)benzoic acid methyl ester and 2-methylindole-3-carbaldehyde, followed by reaction with thiazolidine-2,4-dione.
HPLC-MS (Method C): m/z: 407 (M+1); Rt.= 4.19 min. Example 225 (General procedure (C))
5-(2-Chloro-1-methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000122_0001
HPLC-MS (Method A): m/z: 293 (M+1); Rt = 4,10 min.
Example 226 (General procedure (C)) 5-(4-Hydroxy-3,5-diiodo-benzylidene)-thiazolidine-2,4-dion€
Figure imgf000122_0002
HPLC-MS (Method A): m/z: 474 (M+1); Rt = 6.61 min.
Example 227 (General procedure (C)) 5-(4-Hydroxy-3-iodobenzylidene)thiazolidine-2,4-dione
Figure imgf000122_0003
HPLC-MS (Method C): m/z: 348 (M+1); Rt. = 3.13 min
1H-NMR: (DMSO-dβ): 11.5 (IH.broad); 7.95(1 H.d); 7.65(1 H,s); 7.45 (1H,dd); 7.01(1H,dd);
3.4 (1H,broad).
Example 228 (General procedure (C))
5-(2,3,6-Trichlorobenzylidene)thiazolidine-2,4-dione
Figure imgf000123_0001
H PLC-MS (Method C): m/z: 309 (M+1); Rt.= 4.07 min
Example 229 (General procedure (C)) 5-(2,6-Dichlorobenzylidene)thiazolidine-2,4-dione
Figure imgf000123_0002
Mp. 152-154°C.
HPLC-MS (Method C): m/z: 274 (M+1), Rt .= 3.70 min
1H-NMR: (DMSO-d6): 12.8 (1 H, broad); 7.72 (1H,s); 7.60 (2H,d); 7.50 (1H,t).
Example 230 (General procedure (C))
5-[1-(2,6-Dichloro-4-trifluoromethylphenyl)-2,5-dimethyl-1r7,-pyrroI-3-yImethylene]thiazolidine-
2,4-dione
Figure imgf000123_0003
HPLC-MS (Method C): m/z: 436 (M+1); Rt. 4.81 min
Example 231 (General procedure (C))
5-[1-(3,5-Dichlorophenyl)-5-(4-methanesulfonylphenyl)-2-methyl-1H-pyrrol-3-ylmethylene]- thia∑olidine-2,4-dione
Figure imgf000124_0001
HPLC-MS (Method C): m/z: 508 (M+1); Rt. = 4.31 min
Example 232 (General procedure (C))
5-[1-(2,5-Dimethoxyphenyl)-5-(4-methanesulfonylphenyl)-2-methyl-1H- ■3-ylm. thia∑olidine-2,4-dione
Figure imgf000124_0002
HPLC-MS (Method C): m/z: 499 (M+1); Rt. = 3.70 min
Example 233 (General procedure (C)) 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2,5-dimethylpyrrol-1-yl]benzoic acid
Figure imgf000124_0003
HPLC-MS (Method C): m/z:342 (M+1); Rt.= 3.19 min
Example 234 (General procedure (C)) 5-(4-Hydroxy-2,6-dimethoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000124_0004
HPLC-MS (Method C): m/z:282( M+1); Rt .= 2.56, mp=331-333 °C Example 235 (General procedure (C)) 5-(2,6-Dimethylbenzylidene)thiazolidine-2,4-dione
Figure imgf000125_0001
M.p: 104-105 °C
HPLC-MS (Method C): m/z: 234 (M+1); Rt .= 3.58 min,
Example 236 (General procedure (C)) 5-(2,6-Dimethoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000125_0002
Mp: 241-242 °C
HPLC-MS (Method C): m/z: 266 (M+1); Rt .= 3.25 min;
Example 237 (General procedure (C)) 5-[4-(2-Fluoro-6-nitrobenzyloxy)-2,6-dimethoxybenzylidene]thiazolidine-2,4-dione
Figure imgf000125_0003
Mp: 255-256 °C
HPLC-MS (Method C): m/z: 435 (M+1), Rt 4.13 min,
Example 238 (General procedure (C)) 5-Benzofuran-2-ylmethylenethiazolidine-2,4-dione
Figure imgf000125_0004
HPLC-MS (Method C): m/z:246 (M+1); Rt.= 3.65 min, mp = 265-266 °C Example 239 (General procedure (C)) 5-[3-(4-Dimethylaminophenyl)allylidene]thiazolidine-2,4-dione
Figure imgf000126_0001
HPLC-MS (Method C): m/z:276(M+1); Rt.= 3.63, mp = 259-263 °C
1H-NMR: (DMSO-d6) δ= 12.3 (IH.broad); 7.46 (2H,d); 7.39 (1H,d); 7.11 (1H,d); 6.69 (2H,d);
6.59 (1H, dd); 2.98 (3H,s).
Example 240 (General procedure (C)) 5-(2-Methyl-3-phenylallylidene)thiazoIidine-2,4-dione
Figure imgf000126_0002
Mp: 203-210 °C
HPLC-MS (Method C): m/z: 246 (M+1); Rt = 3.79 min.
Example 241 (General procedure (C)) 5-(2-Chloro-3-phenylallylidene)thiazolidine-2,4-dione
Figure imgf000126_0003
Mp: 251-254 °C
HPLC-MS (Method C): m/z: 266 (M+1; Rt = 3.90 min
Example 242 (General procedure (C)) 5-(2-Oxo-1,2-dihydroquinolin-3-yImethylene)thiazolidine-2,4-dione
Figure imgf000126_0004
Mp: 338-347 °C HPLC-MS (Method C): m/z: 273 (M+1); Rt. = 2.59 min.
Example 243 (General procedure (C)) 5-(2,4,6-Tribromo-3-hydroxyben∑ylidene)thiazolidine-2,4-dione.
Figure imgf000127_0001
HPLC-MS (Method C): m/∑: 459 (M+1);Rt.= 3.65 min.
Example 244 (General procedure (C)) 5-(5-Bromo-2-methylindσl-3-ylmethylene)thiazolidine-2,4-dione.
Figure imgf000127_0002
HPLC-MS (Method C): m/z: 339 (M+1); Rt = 3.37min.
Example 245 (General procedure (C)) 5-(7-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione.
Figure imgf000127_0003
HPLC-MS (Method C): m/z: 319 (M+1); Rt = 3.48min.
Example 246 (General procedure (C)) 5-(6-Bromoindol-3-ylmethylene)thiazolidine-2,4-dione.
Figure imgf000127_0004
HPLC-MS (Method C): m/z: 325 (M+1); Rt = 3.54 min.
Example 247 (General procedure (C)) 5-(8-Methyl-2-oxo-1 ,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione.
Figure imgf000128_0001
HPLC-MS (Method C): m/z: 287 (M+1); Rt = 2.86 min.
Example 248 (General procedure (C)) 5-(6-Methoxy-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dions
Figure imgf000128_0002
HPLC-MS (Method C): m/z: 303 (M+1); Rt = 2.65 min.
Example 249 (General procedure (C)) 5-Quinolin-3-ylmethylenethiazolidine-2,4-dione.
Figure imgf000128_0003
HPLC-MS (Method C): m/z: 257 (M+1); Rt = 2.77 min.
Example 250 (General procedure (C)) 5-(8-Hydroxyquinolin-2-ylmethylene)thiazolidine-2,4-dione.
Figure imgf000128_0004
HPLC-MS (Method C): m/z: 273 (M+1); Rt = 3.44 min.
Example 251 (General procedure (C)) 5-Quinolin-8-ylmethylenethiazolidine-2,4-dione.
Figure imgf000128_0005
HPLC-MS (Method C): m/z: 257 (M+1); Rt = 3.15 min. Example 252 (General procedure (C)) 5-(1-Bromo-6-methoxynaphthalen-2-ylmethylene)thiazolidine-2,4-dione.
Figure imgf000129_0001
HPLC-MS (Method C): m/z: 366 (M+1); Rt = 4.44 min.
Example 253 (General procedure (C)) 5-(6-Methyl-2-oxo-1 ,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione.
Figure imgf000129_0002
HPLC-MS (Method C): m/z: 287 (M+1); Rt. = 2.89 min.
Example 254 (General procedure (D)) 5-(2,6-Dichloro-4-dibenzylaminobenzylidene)thiazolidine-2,4-dione.
Figure imgf000129_0003
HPLC-MS (Method C): m/z: 469 (M+1); Rt = 5.35 min.
Example 255 (General Procedure (C))
7-(2,4-Dioxothiazolidin-5-ylidenemethyl)-4-methoxybenzofuran-2-carboxylic acid
Figure imgf000129_0004
HPLC-MS (Method C): m/z: 320 (M+1); Rt = 2.71 min.
Preparation of the intermediate, 7-formyl-4-ιmethoxybenzofuran-2-carboxylic acid: A mixture of 2-hydroxy-6-methoxybenzaldehyde (6.4 g, 42 mmol), ethyl bromoacetate (14.2 mL, 128 mmol) and potassium carbonate (26 g, 185 mmol) was heated to 130 °C. After 3 h the mixture was cooled to room temperature and acetone (100 mL) was added, the mixture was subsequently filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with a mixture of ethyl acetate and heptane (1 :4). This afforded 7.5 g (55%) of ethyl 4-methoxybenzofuran-2-carboxylate.
A solution of ethyl 4-methoxyben∑ofuran-2-carboxylate (6.9 g, 31.3 mmol) in dichloromethane (70 ml) was cooled to 0 °C and a solution of titanium tetrachloride (13.08 g, 69 mmol) was added drop wise. After 10 minutes dichloromethoxymethane (3.958 g, 34 mmol) was added over 10 minutes. After addition, the mixture was warmed to room temperature for 18 hours and the mixture poured into hydrochloric acid (2N, 100 L). The mixture was stirred for 0.5 hour and then extracted with a mixture of ethyl acetate and toluene (1:1). The organic phase was dried over Na2S0 and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with a mixture of ethyl acetate and heptane (1:4). This afforded 5.8 g (80%) of ethyl 7-formyl-4-methoxybenzofuran-2-carboxylate.
7-formyl-4-methoxybenzofuran-2-carboxylate (5.0 g, 21.5 mmol) and sodium carbonate (43 mmol) in water (100 mL) was refluxed until a clear solution appeared (about 0.5 hour). The solution was filtered and acidified to pH =1 with hydrochloric acid (2 N), the resulting product was filtered off and washed with ethyl acetate and ethanol and dried to afford 3.5 g (74%) of 7-formyl-4-methoxybenzofuran-2-carboxylic acid as a solid.
1H NMR (DMSO-d6): δ = 10.20 (s, 1H) ; 8.07 (d, 1H) ; 7.70 (s, 1 H) ; 7.17 (d, 1H) ; 4.08 (s, 3H).
Example 256 (General Procedure (C)) 5-(4-Methoxybenzofuran-7-ylmethylene)thiazolidine-2,4-dione
Figure imgf000130_0001
HPLC-MS (Method C): m/z: 267 (M+1); Rt = 3.30 min. Preparation of the intermediate, 4-methoxybenzofuran-7-carbaldehyde:
A mixture of 7-formyl-4-methoxybenzofuran-2-carboxylic acid (3.0 g, 13.6 mmol) and Cu (0.6 g, 9.44 mmol) in quinoline (6 mL) was refluxed. After 0.5 h the mixture was cooled to room temperature and water (100 mL) and hydrochloric acid (10 N, 20 mL) were added. The mixture was extracted with a mixture of ethyl acetate and toluene (1:1), filtered through celite and the organic layer separated and washed with a sodium carbonate solution, dried over Na2S04 and concentrated in vacuo to afford 1.5 g crude product. Column chromatography Si02, EtOAc/heptanes=1/4 gave 1.1 g (46%) of 4-methoxyben∑ofuran-7-carbaldβhyde as a solid.
1H NMR (CDCI3): δ: 10.30 (s,1H) ; 7.85 (d,1H) ; 7.75 (d,1H) ; 6.98 (d,1H) ; 6.87 (d,1H) ; 4.10 (s,3H). HPLC-MS (Method C) :m/z: 177 (M+1); Rt. = 7.65 min.
Example 257 (General Procedure (C)) 5-(4-Hydroxybenzofuran-7-ylmethylene)thiazolidine-2,4-dione
Figure imgf000131_0001
HPLC-MS (Method C): m/z: = 262 (M+1); Rt 2.45 min.
Preparation of the intermediate, 4-hydroxybenzofuran-7-carbaldehyde
A mixture of 4-methoxybenzofuran-7-carbaldehyde (1.6 g, 9.1 mmol) and pyridine hydrochlo- ride (4.8 g, 41.7mmol) in quinoline (8 mL) was refluxed. After 8 h the mixture was cooled to room temperature and poured into water (100 mL) and hydrochloric acid (2 N) was added to pH = 2. The mixture was extracted with a mixture of ethyl acetate and toluene (1:1), washed with a sodium carbonate solution, dried with Na2SO and concentrated in vacuo to afford 0.8 g crude product. This was purified by column chromatography on silica gel, eluting with a mixture of ethyl acetate and heptane (1:3). This afforded 250 mg of 4-hydroxyben∑ofuran-7- carbaldehyde as a solid. 1H NMR (DMSO-d6): δ = 11.35 (s, broad, 1H) ; 10.15 (s, 1H) ; 8.05 (d, 1H) ; 7.75 (d, 1H) ; 7.10 (d, 1H); 6.83 (d, 1H). HPLC-MS (Method C): m/z: 163 (M+1); Rt. = 6.36 min.
Example 258 (General Procedure (C)) 5-(5-Bromo-2,3-dihydrobenzofuran-7-ylmethylene)thia∑olidine-2,4-dione
Figure imgf000132_0001
HPLC-MS (Method C): m/z: 328 (M+1); Rt = 3.66 min.
Preparation of the intermediate, 5-bromo-2,3-dihydrobenzofuran-7-carbaldehyde: To a cooled (15 °C) stirred mixture dihydrobenzofuran (50.9 g, 0.424 mol) in acetic acid (500 mL), a solution of bromine (65.5 mL, 1.27 mol) in acetic acid (200 mL) was added drop wise over 1 hour. After stirring for 18 hours, a mixture of Na2S2O5 (150 g) in water (250 mL) was added carefully, and the mixture was concentrated in vacuo. Water (200 mL) was added and the mixture was extracted with ethyl acetate containing 10% heptane, dried over Na2SO4 and concentrated in vacuo to give crude 5,7-dibromo-2,3-dihydrobenzofuran which was used as such for the following reaction steps. To a cooled solution (-78 °C) of crude 5,7-dibromo-2,3- dihydrobenzofuran (50.7 g, 0.182 mol) in THF (375 mL) a solution of n-BuLi (2.5 M, 80 mL, 0.200 mol) in hexane was added. After addition, the mixture was stirred for 20 min. DMF (16 mL) was then added drop wise at -78 °C. After addition, the mixture was stirred at room temperature for 3 h and then the mixture was poured into a mixture of ice water, (500 mL) and hydrochloric acid (10 N, 40 mL) and extracted with toluene, dried over Na2SO4 and concentrated in vacuo. Column chromatography on silica gel eluting with a mixture of ethyl acetate and heptane (1 :4) afforede 23 g of 5-bromo-2,3-dihydrobenzofuran-7-carbaldehyde as a solid.
1H NMR (CDCI3): Λ10.18 (s,1H) ; 7.75 (d,1H) ;7.55 (d,1H) ; 4.80 (t,2H) ; 3.28 (t,2H).
Example 259 (General Procedure (C)) 5-(4-Cyclohexylbenzylidene)thiazolidine-2,4-dione
Figure imgf000133_0001
HPLC-MS (Method C): m/z: 288 (M+1); Rt = 5.03 min.
Preparation of the intermediate, 4-cyclohexylbenzaldehyde:
This compound was synthesized according to a modified literature procedure (J. Org. Chem., 37, No.24, (1972), 3972-3973).
Cyclohexylbenzene (112.5 g, 0.702 mol) and hexamethylenetetramine (99.3 g, 0.708 mol) were mixed in TFA (375 mL). The mixture was stirred under nitrogen at 90 °C for 3 days. After cooling to room temperature the red-brown mixture was poured into ice-water (3600 ml) and stirred for 1 hour. The solution was neutralized with Na2CO3 (2 M solution in water) and extracted with dichloromethane (2.5 L). The organic phase was dried (Na2SO4) and the solvent was removed in vacuo. The remaining red-brown oil was purified by fractional distillation to afford the title compound (51 g, 39%). H NMR (CDCI3): £9.96 (s, 1H), 7.80 (d, 2H), 7.35 (d, 2H), 2.58 (m, 1H), 1.94-1.70 (m, 5 H), 1.51-1.17 (m, 5H)
Other ligands of the invention include
3,,5'-Dichloro-4'-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-carboxylic acid:
Figure imgf000133_0002
Example 260 (General procedure (C)) 5-(1-Bromo-6-hydroxynaphthalen-2-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000134_0001
HPLC-MS (Method C): m/z = 350 (M+1); Rt. = 3.45 min.
Example 261 (General procedure (C))
5-[4-(2-Bromoethoxy)-naphthalen-1-ylmethylene]-thiazolidine-2,4-dion€
Figure imgf000134_0002
HPLC-MS (Method C): m/z = 380 (M+1); Rt = 3.52 min.
Example 262 (General procedure (C)) 5-(2-Methyl-5-nitro-1H-indol-3-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000134_0003
HPLC-MS (Method C): m/z = 304 (M+1); Rt = 2.95 min.
Example 263 (General procedure (C)) 5-(4-Naphthalen-2-yl-thiazol-2-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000135_0001
HPLC-MS (Method C): m/z = 339 (M+1); Rt.= 4.498 min.
Example 264 (General procedure (C)) 5-[4-(4-Methoxy-naphthalen-1-yl)-thiazol-2-ylmethylene]-thiazolidine-2,4-dione
Figure imgf000135_0002
HPLC-MS (Method C): m/z = 369 (M+1); Rt.= 4.456 min.
Example 265 (General procedure (C)) 5-(2-Pyridin-4-yl-1H-indol-3-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000135_0003
HPLC-MS (Method C): m/z = 322 (M+1); Rt. = 2.307 min. Example 266 (General procedure (C)) 5-[5-(4-Chlorophenyl)-1H-pyrazol-4-ylmethylene]-thiazolidine-2,4-dione
Figure imgf000136_0001
HPLC-MS (Method C): m/z = 306 (M+1); Rt.= 3.60 min.
Example 267 (General procedure (C))
5-[5-(2,5-Dimethylphenyl)-1H-pyrazol-4-ylmethylene]-thiazolidine-2,4-dione
Figure imgf000136_0002
HPLC-MS (Method C): m/z = 300 (M+1); Rt. = 3.063 min.
Example 268 (General procedure (C)) 5-(2-Phenyl-benzo[d]imidazo[2,1-b]thiazol-3-ylmethyIene)-thiazolidine-2,4-dione
Figure imgf000136_0003
HPLC-MS (Method C): m/z = 378 (M+1); Rt = 3.90 min. Example 269 (General procedure (C)) N-{4-[2-(2,4-Dioxothiazolidin-5-ylidenemethyl)-phenoxy]-phenyl}-acetamide
Figure imgf000137_0001
HPLC-MS (Method C): m/z = 355 (M+1); Rt 3.33 min.
Example 270 (General procedure (C)) 5-(2-Phenyl-imidazo[1,2-a]pyridin-3-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000137_0002
HPLC-MS (Method C): m/z = 322 (M+1); Rt. = 2.78 min.
Example 271 (General procedure (C)) 5-(2-Naphthalen-2-yl-imidazo[1,2-a]pyridin-3-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000137_0003
HPLC-MS (Method C): m/z = 372 (M+1); Rl. = 2.78 min.
Example 272 (General procedure (C)) 5-[6-Bromo-2-(3-methoxyphenyl)-imidazo[1,2-a]pyridin-3-ylmethylene]-thiazolidine-2,4-dione
Figure imgf000138_0001
HPLC-MS (Method C): m/z = 431 (M+1); Rt.= 3.30 min.
Example 273 (General procedure (C)) 5-(1,2,3,4-Tetrahydrophenanthren-9-ylmethylene)thiazolidine-2,4-dione
Figure imgf000138_0002
HPLC-MS (Method C): m/z = 310 (M+1); Rt .= 4.97 min.
Example 274 (General procedure (C)) 5-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydro-naphthalen-2-ylmethylene)thiazolidine-2,4-dione
Figure imgf000138_0003
HPLC-MS (Method C): m/z = 330 (M+1); Rt .= 5.33 min. Example 275 (General procedure (C)) 5-[6-(2,4-Dichloro-phenyl)-imidazo[2,1-b]thiazol-5-ylmethylene]-thiazolidine-2,4-dione
Figure imgf000139_0001
HPLC-MS (Method C): m/z = 396 (M+1); Rt. = 3.82 min.
Example 276 (General procedure (C)) 5-(5-Bromobenzofuran-7-ylmethylene)-thiazolidine-2,4-dione
Figure imgf000139_0002
HPLC-MS (Method C): m/z = 324 (M+1); Rt. = 3.82 min.
Example 277 (General procedure (C)) 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-1,4-dimethylcarbazol-9-ylmethyl]-benzoic acid
Figure imgf000139_0003
HPLC-MS (Method C): m/z = 457 (M+1); Rt = 4,23 min. Preparation of intermediary aldehyde:
1,4 Dimethylcarbazol-3-carbaldehyde (0.68 g, 3.08 mmol) was dissolved in dry DMF (15 mL), NaH (diethyl ether washed) (0.162 g, 6.7 mol) was slowly added under nitrogen and the mixture was stirred for 1 hour at room temperature. 4-Bromomethylbenzoic acid (0.73 g, 3.4 mmol) was slowly added and the resulting slurry was heated to 40 °C for 16 hours. Water (5 mL) and hydrochloric acid (6N, 3 mL) were added. After stirring for 20 min at room temperature, the precipitate was filtered off and washed twice with acetone to afford after drying 0.38 g (34%) of 4-(3-formyl-1 ,4-dimethylcarbazol-9-ylmethyl)ben∑oic acid.
HPLC-MS (Method C) : m/z = 358 (M+1), RT. = 4,15 min.
Example 278 (General procedure (C)) 4-[7-(2,4-Dioxothiazolidin-5-ylidenemethyl)-benzofuran-5-yl]-benzoic acid
Figure imgf000140_0001
Starting aldehyde commercially available (Syncom BV, NL) HPLC-MS (Method C): m/z = 366 (M+1); Rt. = 3.37 min.
Example 279 (General procedure (C)) 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-nitrophenoxy]-benzoic acid methyl ester
Figure imgf000140_0002
HPLC-MS (Method C): m/z = 401 (M+1); Rt. = 4.08 min. Example 280 (General procedure (C))
3',5'-Dichloro-4,-(2,4-dioxothiazolidin-5-ylidenemethyl)-biphenyl-4-carboxylic acid
Figure imgf000141_0001
Starting aldehyde commercially available (Syncom BV, NL)
HPLC-MS (Method C): m/z = 394 (M+1); Rt. = 3.71 min.
Example 281 (General procedure (C))
Figure imgf000141_0002
HPLC-MS (Method C): m/z = 232( M+1); Rt .= 3.6 min.
Example 282 5-(2-Methyl-1H-indol-3-ylmethyl)-thiazolidine-2,4-dione
Figure imgf000141_0003
5-(2-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione (prepared as described in exampl. 187, 1.5 g, 5.8 mmol) was dissolved in pyridine (20 mL) and THF (50 mL), LiBH4 (2 M in THF, 23.2 mmol) was slowly added with a syringe under cooling on ice. The mixture was heated to 85 °C for 2 days. After cooling, the mixture was acidified with concentrated hydro- chloric acid to pH 1. The aquous layer was extracted 3 times with ethyl acetate, dried with MgS04 treated with activated carbon, filtered and the resulting filtrate was evaporated in vacuo to give 1.3 g (88%) of the title compound.
HPLC-MS (Method C): m/z = 261 (M+1); Rt. = 3.00 min.
Example 283 4-[4-(2,4-Dioxothia∑olidin-5-ylmethyl)naphthalen-1-yloxy]butyric acid
Figure imgf000142_0001
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid (4.98 g, 13.9 mmol, prepared as described in example 469) was dissolved in dry THF (50 mL) and added dry pyridine (50 mL) and, in portions, lithium borohydride (2.0 M, in THF, 14 mL). The resulting slurry was refluxed under nitrogen for 16 hours, added (after cooling) more lithium borohydride (2.0 M, in THF, 7 mL). The resulting mixture was refluxed under nitrogen for 16 hours. The mixture was cooled and added more lithium borohydride (2.0 M, in THF, 5 mL). The resulting mixture was refluxed under nitrogen for 16 hours. After cooling to 5 °C, the mixture was added water (300 mL) and hydrochloric acid (150 mL). The solid was isolated by filtration, washed with water (3 x 500 mL) and dried. Recrystallization from acetonitrile (500 mL) afforded2.5 g of the title compound.
1H-NMR (DMSO-d6, selected peaks): δ = 3.42 (1H, dd), 3.90 (1H, dd), 4.16 (2H, T), 4.95 (1H, dd), 6.92 (1H, d), 7.31 (1H, d), 7.54 (1H, t), 7.62 (1H, t), 8.02 (1H, d), 8.23 (1H, d), 12.1 (1H, bs), 12.2 (1 H, bs). HPLC-MS (Method C): m/z = 382 (M+23); Rt = 3,23 min.
Example 284 5-Naphthalen-1-ylmethylthiazolidine-2,4-dione
Figure imgf000143_0001
5-Naphihalen-1-ylmethylenethiazolidine-2,4-dione (1.08 g, 4.2 mmol, prepared as described in example 68) was dissolved in dry THF (15 mL) and added dry pyridine (15 mL) and, in portions, lithium borohydride (2.0 M, in THF, 4.6 mL). The resulting mixture was refluxed under nitrogen for 16 hours. After cooling to 5 °C, the mixture was added water (100 mL), and, in portions, concentrated hydrochloric acid (40 mL). More water (100 mL) was added, and the mixture was extracted with ethyl acetate (200 mL). The organic phase was washed with water (3 x 100 mL), dried and concentrated in vacuo. The residue was dissolved in ethyl acetate (50 mL) added activated carbon, filtered and concentrated in vacuo and dried to afford 0.82 g (75%) of the title compound. H-NMR (DMSO-dβ): δ = 3.54 (1H, dd), 3.98 (1 H, dd), 5.00 (1 H, dd), 7.4-7.6 (4H, m), 7.87 (1H, d), 7.96 (1H, d), 8.11 (1H, d), 12.2 (1H, bs). HPLC-MS (Method C): m/z = 258 (M+1); Rt = 3,638 min.
The following preferred compounds of the invention may be prepared according to procedures similar to those described in the three examples above:
Figure imgf000143_0002
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
The following compounds are commercially available and may be prepared using general procedures (B) and / or (C). Example 380 5-(5-Bromo-1H-indol-3-ylmethylene)thiazolidine-2,4-dione
Figure imgf000155_0002
Example 381
5-Pyridin-4-ylmethylenethiazolidine-2,4-dione
Figure imgf000156_0001
Example 382 5-(3-Bromo-4-methoxybenzylidene)thiazolidine-2,4-dione
Figure imgf000156_0002
Example 383 5-(3-Nitrobenzylidene)thiazolidine-2,4-dione
Figure imgf000156_0003
Example 384 5-Cyclohexylidene-1,3-thiazolidine-2,4-dione
Figure imgf000156_0004
Example 385 5-(3,4-Dihydroxybenzylidene)thiazolidine-2,4-dione
Figure imgf000156_0005
Example 386 5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione
Figure imgf000157_0001
Example 387 5-(4-Hydroxy-3-methoxy-5-nitrobenzylidene)thiazolidine-2,4-dione
Figure imgf000157_0002
Example 388 5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione
Figure imgf000157_0003
Example 389 5-(4-Hydroxy-3,5-dimethoxybenzylidene)thiazoIidine-2,4-dione
Figure imgf000157_0004
Example 390 5-(3-Bromo-5-ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione
Figure imgf000157_0005
Example 391 5-(3-Ethoxy-4-hydroxy-5-nitrobenzyIidene)thiazolidine-2,4-dione
Figure imgf000158_0001
Example 392
Figure imgf000158_0002
Example 393
Figure imgf000158_0003
Example 394
Figure imgf000158_0004
Example 395
Figure imgf000158_0005
Example 396
Figure imgf000159_0001
Example 397
Figure imgf000159_0002
Example 398
Figure imgf000159_0003
Example 399
Figure imgf000159_0004
Example 400
Figure imgf000159_0005
Example 401
Figure imgf000160_0001
Example 402
Figure imgf000160_0002
Example 403
Figure imgf000160_0003
Example 404
Figure imgf000160_0004
Example 405 5-(3-Hydroxy-5-methyl-phenylamino)-thiazolidine-2,4-dione
Figure imgf000161_0001
Example 406
Figure imgf000161_0002
Example 407
Figure imgf000161_0003
Example 408
Figure imgf000161_0004
Example 409
Figure imgf000162_0001
Example 410
Figure imgf000162_0002
Example 411
Figure imgf000162_0003
Example 412
Figure imgf000162_0004
Example 413
Figure imgf000163_0001
Example 414
Figure imgf000163_0002
Example 415
Figure imgf000163_0003
Example 416
Figure imgf000163_0004
Example 417
Figure imgf000164_0001
Example 418
Figure imgf000164_0002
Example 419
Figure imgf000164_0003
Example 420
Figure imgf000164_0004
Example 421
Figure imgf000164_0005
Example 422
Figure imgf000165_0001
Example 423
Figure imgf000165_0002
Example 424
Figure imgf000165_0003
Example 425
Figure imgf000165_0004
Example 426
Figure imgf000165_0005
Example 427
Figure imgf000166_0001
Example 428
Figure imgf000166_0002
Example 429
Figure imgf000166_0003
Figure imgf000166_0004
Example 431 5-(4-Diethylamino-2-methoxy-benzylidene)-imidazolidine-2,4-dione
Figure imgf000167_0001
Example 432
Figure imgf000167_0002
Example 433
Figure imgf000167_0003
Example 434
Figure imgf000167_0004
Example 435
Figure imgf000168_0001
Example 436
Figure imgf000168_0002
Example 437
Figure imgf000168_0003
Example 438
Figure imgf000168_0004
Example 439
Figure imgf000169_0001
Example 440
Figure imgf000169_0002
Example 441
Figure imgf000169_0003
Figure imgf000169_0004
Example 443
Figure imgf000170_0001
Example 444
Figure imgf000170_0002
Example 445
Figure imgf000170_0003
Example 446
Figure imgf000170_0004
Example 447
Figure imgf000171_0001
Example 448
Figure imgf000171_0002
Example 449
Figure imgf000171_0003
Example 450
Figure imgf000171_0004
Example 451
Figure imgf000172_0001
Example 452
Figure imgf000172_0002
Example 453
Figure imgf000172_0003
Example 454 5-(4-Diethylamino-benzylidene)-2-imino-thiazolidin-4-one
Figure imgf000172_0004
Example 455
Figure imgf000172_0005
Example 456
Figure imgf000173_0001
Example 457
Figure imgf000173_0002
Example 458
Figure imgf000173_0003
Example 459
Figure imgf000173_0004
General procedure (D) for preparation of compounds of general formula l3:
Figure imgf000174_0001
^ι©p ?
Figure imgf000174_0002
wherein X, Y, and R3 are as defined above, n is 1 or 3-20,
E is arylene or heterarylene (including up to four optional substituents, R13, R14, R15, and R15A as defined above),
R' is a standard carboxylic acid protecting group, such as d-Cβ-alkyl or benzyl and Lea is a leaving group, such as chloro, bromo, iodo, methanesulfonyloxy, toluenesulfonyloxy or the like.
Step 1 is an alkylation of a phenol moiety. The reaction is preformed by reacting R10-C(=O)- E-OH with an ω-bromo-alkane-carboxylic acid ester (or a synthetic equivalent) in the presence of a base such as sodium or potassium carbonate, sodium or potassium hydroxide, sodium hydride, sodium or potassium alkoxide in a solvent, such as DMF, NMP, DMSO, acetone, acetonitrile, ethyl acetate or isopropyl acetate. The reaction is performed at 20 - 160 °C, usually at room temperature, but when the phenol moiety has one or more substituents heating to 50 °C or more can be beneficial, especially when the substituents are in the ortho position relatively to the phenol. This will readily be recognised by those skilled in the art.
Step 2 is a hydrolysis of the product from step 1. Step 3 is similar to general procedure (B) and (C).
This general procedure (D) is further illustrated in the following examples:
Example 460 (General procedure (D)) 4-[4-(2,4-Dioxothia∑olidin-5-ylidenemethyl)phenoxy]butyric cid
Figure imgf000175_0001
Step l:
A mixture of 4-hydroxybenzaldehyde (9.21 g, 75 mmol), potassium carbonate (56 g, 410 mmol) and 4-bromobutyric acid ethyl ester (12.9 mL, 90 mmol) in Λ/,Λ/-dimethylformamide (250 mL) was stirred vigorously for 16 hours at room temperature. The mixture was filtered and concentrated in vacuo to afford 19.6 g (100%) of 4-(4-formylphenoxy)butyric acid ethyl ester as an oil. 1H-NMR (DMSO-d6): δ 1.21 (3H, t), 2.05 (2H, p), 2.49 (2H, t), 4.12 (4H, m), 7.13 (2H, d), 7.87 (2H, d), 9.90 (1 H, s). HPLC-MS (Method A): m/z = 237 (M+1); Rt = 3.46 min.
Step 2:
4-(4-Formylphenoxy)butyric acid ethyl ester (19.6 g, 75 mmol) was dissolved in methanol (250 mL) and 1 N sodium hydroxide (100 mL) was added and the resulting mixture was stirred at room temperature for 16 hours. The organic solvent was evaporated in vacuo (40 °C, 120 mBar) and the residue was acidified with 1 N hydrochloric acid (110 mL). The mixture was filtered and washed with water and dried in vacuo to afford 14.3 g (91%) 4-(4- formylphenoxy)butyric acid as a solid. 1H-NMR (DMSO-d6): δ 1.99 (2H, p), 2.42 (2H, t), 4.13 (2H, t), 7.14 (2H, d), 7.88 (2H, d), 9.90 (1 H, s), 12.2 (1 H, bs). HPLC-MS (Method A): m/z = 209 (M+1); Rt = 2.19 min.
Step 3:
Thiazolidine-2,4-dione (3.55 g, 27.6 mmol), 4-(4-formylphenoxy)butyric acid (5.74 g, 27.6 mmol), anhydrous sodium acetate (1 .3 g, 138 mmol) and acetic acid (100 mL) was refluxed for 16 h. After cooling, the mixture was filtered and washed with acetic acid and water. Drying in vacuo afforded 2.74 g (32%) of 4-[4-(2,4-dioxothia∑olidin-5-ylidenemethyl)phenoxy]butyric acid as a solid. 1H-NMR (DMSO-d6): 51.97 (2H, p), 2.40 (2H, t), 4.07 (2H, t), 7.08 (2H, d), 7.56 (2H, d), 7.77 (1 H, s), 12.2 (1 H, bs), 12.5 (1 H, bs); HPLC-MS (Method A): m/z: 308 (M+1 ); Rt = 2.89 min.
Example 461 (General procedure (D)) [3-(2,4-Dioxothia∑olidin-5-ylidenemethyl)phenoxy]acetic acid
Figure imgf000176_0001
Thiazolidine-2,4-dione (3.9 g, 33 mmol), 3-formylphenoxyacetic acid (6.0 g, 33 mmol), anhydrous sodium acetate (13.6 g, 165 mmol) and acetic acid (100 mL) was refluxed for 16 h. After cooling, the mixture was filtered and washed with acetic acid and water. Drying in vacuo afforded 5.13 g (56%) of [3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid as a solid.
1H-NMR (DMSO-d6): δ 4.69 (2H, s), 6.95 (1H, dd), 7.09 (1H, t), 7.15 (1H, d), 7.39 (1H, t),7.53 (1H, s); HPLC-MS (Method A): m/z = 280 (M+1) (poor ionisation); Rt = 2.49 min.
The compounds in the following examples were similarly prepared.
Example 462 (General procedure (D)) 3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acrylic acid
Figure imgf000176_0002
H-NMR (DMSO-d6): 56.63 (1H, d), 7.59-7.64 (3H, m), 7.77 (1H, s), 7.83 (2H, m).
Example 463 (General procedure (D)) [4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid
Figure imgf000177_0001
Triethylamine salt: 1H-NMR (DMSO-o*6): δ 4.27 (2H, s), 6.90 (2H, d), 7.26 (1H, s), 7.40 (2H, d).
Example 464 (General procedure (D)) 4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid
Figure imgf000177_0002
Example 465 (General procedure (D)) 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid
Figure imgf000177_0003
1 H-NMR (DMSO-d6): δ 7.57 (1H, s), 7.60 (1 H, t), 7.79 (1H, dt), 7.92 (1 H, dt), 8.14 (1H, t).
Example 466 (General procedure (D))
4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid
Figure imgf000177_0004
1H-NMR (DMSO-dβ): δ 2.00 (2H, p), 2.45 (2H, t), 4.17 (2H, t), 7.31 (1 H, d), 7.54 (1 H, 7.69 (1H, d), 7.74 (1 H, s), 12.2 (1 H, bs), 12.6 (1 H, bs). HPLC-MS (Method A): m/z: 364 (M+23); Rt = 3.19 min.
Example 467 (General procedure (D)) 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid
Figure imgf000178_0001
1 H-NMR (DMSO-d5): δ 1.99 (2H, p), 2.46 (2H, t), 4.17 (2H, t), 7.28 (1 H, d), 7.57 (1H, dd), 7.25 (1 H, s), 7.85 (1 H, d), 12.2 (1 H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 410 (M+23); Rt = 3.35 min.
Example 468 (General procedure (D))
4-[2-Bromo-4-(4-oxo-2-thioxothia∑olidin-5-ylidenemethyl)phenoxy]butyric acid
Figure imgf000178_0002
1H-NMR (DMSO-d6): δ 1.99 (2H, p), 2.45 (2H, t), 4.18 (2H, t), 7.28 (1 H, d), 7.55 (1 H, dd), 7.60 (1 H, s), 7.86 (1 H, d), 12.2 (1H, bs), 13.8 (1H, bs). HPLC-MS (Method A): m/z: 424 (M+23); Rt = 3.84 min. HPLC-MS (Method A): m/z: 424 (M+23); Rt = 3,84 min
Example 469 (General procedure (D)) 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid
Figure imgf000178_0003
1H-NMR (DMSO-d6): δ 2.12 (2H, p), 2.5 (below DMSO), 4.28 (2H, t), 7.12 (1H, d), 7.6-7.7 (3H, m), 8.12 (1H, d), 8.31 (1H, d), 8.39 (1H, s), 12.2 (1H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 380 (M+23); Rt = 3.76 min.
Example 470 (General procedure (D)) 5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoic acid
Figure imgf000179_0001
HPLC-MS (Method A): m/z: 394 (M+23); Rt = 3.62 min.
'H-NMR (DMSO-d6): δ 1.78 (2H, m), 1.90 (2H, m), 2.38 (2H, t), 4.27 (2H, t), 7.16 (1H, d),
7.6-7.75 (3H, m), 8.13 (1H, d), 8.28 (1H, d), 8.39 (1H, s), 12.1 (1H, bs), 12.6 (1 H, bs).
Example 471 5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoic acid.
Figure imgf000179_0002
5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]pentanoic acid (example 470, 185 mg, 0.5 mmol) was treated with an equimolar amount of bromine in acetic acid (10 mL). Stirring at RT for 14 days followed by evaporation to dryness afforded a mixture of the bro- minated compound and unchanged starting material. Purification by preparative HPLC on a C18 column using acetonitrile and water as eluent afforded 8 mg of the title compound.
HPLC-MS (Method C): m/z: 473 (M+23), Rt. = 3.77 min
Example 472 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid.
Figure imgf000179_0003
Starting with 4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyric acid (example 469, 0.5 mmol) using the same method as in example 471 afforded 66 mg of the title compound.
HPLC-MS (Method C): m/z: 459 (M+23) ; Rt. = 3.59 min. Example 473 (General procedure (D)) [2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid
Figure imgf000180_0001
1H-NMR (DMSO-de): δ 4.90 (2H, s), 7.12 (1H, d), 7.52 (1H, dd), 7.65 (1H, s) 7.84 (1H, d).HPLC-MS (Method A): m/z: not observed; Rt = 2.89 min.
Example 474 (General procedure (D)) 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid
Figure imgf000180_0002
1H-NMR (DMSO-d6): δ 1.98 (2H, p), 2.42 (2H, t), 4.04 (2H, t), 7.05 (1H, dd), 7.15 (2H, m), 7.45 (1H, t), 7.77 (1H, s), 12.1 (1H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 330 (M+23); Rt = 3.05 min.
Example 475 (General procedure (D)) [4-(2,4-Dioxothiazolidin-5-ylidenemethyI)-3-methoxyphenoxy]acetic acid
Figure imgf000180_0003
HPLC-MS (Method B): m/z: 310 (M+1); Rt = 3,43 min.
Example 476 (General procedure (D)) [4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetic acid
Figure imgf000181_0001
HPLC-MS (Method A): m/z: 330 (M+1); Rt = 3.25 min.
Example 477 (General procedure (D)) 8-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalene-1-carboxylic acid
Figure imgf000181_0002
HPLC-MS (Method A): m/z: 299 (M+1); Rt = 2,49 min.
Example 478 (General procedure (D)) [3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]acetic acid
Figure imgf000181_0003
HPLC-MS (Method A): m/z: 303 (M+1); Rt = 2.90 min.
Preparation of starting material:
3-Formylindol (10 g, 69 mmol) was dissolved in N,Λ/-dimethylformamide (100 mL) and under an atmosphere of nitrogenand with external cooling, keeping the temperature below 15 °C, sodium hydride (60% in mineral oil, 3.0 g, 76 mmol) was added in portions. Then a solution of ethyl bromoacetate (8.4 mL, 76 mmol) in Λ ,Λ-dimethylformamide (15 mL) was added dropwise over 30 minutes and the resulting mixture was stirred at room temperature for 16 hours. The mixture was concentrated in vacuo and the residue was partitioned between water (300 mL) and ethyl acetate (2 x 150 mL). The combined organic extracts were washed with a saturated aqueous solution of ammonium chloride (100 mL), dried (MgS04) and con- centrated in vacuo to afford 15.9 g (quant.) of (3-formylindol-1-yl)acetic acid ethyl ester as an oil.
1H-NMR (CDCI3): 4. = 130 (3H, t), 4.23 (2H, q), 4.90 (2H, s), 7.3 (3H, m), 7.77 (1H, s), 8.32 (1 H, d), 10.0 (1 H, s).
(3-Formylindol-1-yl)acetic acid ethyl ester (15.9 g 69 mmol) was dissolved in 1 ,4-dioxane (100 mL) and 1 sodium hydroxide (10 mL) was added and the resulting mixture was stirred at room temperature for 4 days. Water (500 mL) was added and the mixture was washed with diethyl ether (150 mL). The aqueous phase was acidified with 5N hydrochloric acid and extracted with ethyl acetate (250 + 150 mL). The combined organic extracts were dried (MgS04) and concentrated in vacuo to afford 10.3 g (73%) of (3-formylindol-1-yl)acetic acid as a solid.
1H-NMR (DMSO-d6): δH = 5.20 (2H, s), 7.3 (2H, m), 7.55 (1H, d), 8.12 (1H, d), 8.30 (1H, s), 9.95 (1H, s), 13.3 (1H, bs).
Example 479 (General procedure (D))
3-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]propionic acid
Figure imgf000182_0001
HPLC-MS (Method A): m/z: 317 (M+1); Rt = 3.08 min.
Preparation of starting material:
A mixture of 3-formylindol (10 g, 69 mmol), ethyl 3-bromopropionate (10.5 mL, 83 mmol) and potassium carbonate (28.5 g, 207 mmol) and acetonitrile (100 mL) was stirred vigorously at refux temperature for 2 days. After cooling, the mixture was filtered and the filtrate was concentrated in vacuo to afford 17.5 g (quant.) of 3-(3-formylindol-1-yl)propionic acid ethyl ester as a solid. 1H-NMR (DMSO-dβ): δH = 1.10 (3H, t), 2.94 (2H, t), 4.02 (2H, q), 4.55 (2H, t), 7.3 (2H, m), 7.67 (1 H, d), 8.12 (1 H, d), 8.30 (1 H, s), 9.90 (1 H, s).
3-(3-Formylindol-1-yl)propionic acid ethyl ester (17.5 g 69 mmol) was hydrolysed as described above to afford 12.5 g (83%) of 3-(3-formylindol-1-yl)propionic acid as a solid.
1H-NMR (DMSO-dβ): δH = 2.87 (2H, t), 4.50 (2H, t), 7.3 (2H, m), 7.68 (1H, d), 8.12 (1H, d), 8.31 (1H, s), 9.95 (1 H, s), 12.5 (1H, bs).
Example 480 (General procedure (D))
{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)ben∑ylidene]-4-oxo-2-thioxothia∑olidin-3-yl}acetic acid
Figure imgf000183_0001
HPLC-MS (Method A): m/z: 429 (M+23); Rt = 3.89 min.
Example 481 (General procedure (D)) 6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxyoctanoic acid
Figure imgf000183_0002
HPLC-MS (Method C): m/z: 436 (M+23); Rt.= 4.36 min
The intermediate aldehyde for this compound was prepared by a slightly modified procedure: 6-Hydroxynaphthalene-2-carbaldehyde (1.0 g, 5.8 mmol) was dissolved in DMF (10 mL) and sodium hydride 60% (278 mg) was added and the mixture stirred at RT for 15 min. 8- Bromooctanoic acid (0.37 g, 1.7 mmol) was converted to the sodium salt by addition of sodium hydride 60% and added to an aliquot (2.5 mL) of the above naphtholate solution and the resulting mixture was stirred at RT for 16 hours. Aqueous acetic acid (10 %) was added and the mixture was extracted 3 times with diethyl ether. The combined organic phases were dried with MgSO4 and evaporated to dryness affording 300 mg of 8-(6-formylnaphthalen-2- yloxy)octanoic acid.
HPLC-MS (Method C): m/z 315 (M+1); Rt. = 4.24 min.
Example 482 (General procedure (D)) 12-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]dodecanoic acid.
Figure imgf000184_0001
HPLC-MS (Method C): m/z: 492 (M+23); Rt.= 5.3 min.
The intermediate aldehyde was prepared similarly as described in example 481.
Example 483 (General procedure (D))
11 -[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoic acid.
Figure imgf000184_0002
HPLC-MS (Method C): m/z:478 (M+23); Rt .= 5.17 min.
The intermediate aldehyde was prepared similarly as described in example 481.
Example 484 (General procedure (D)) 15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoic acid.
Figure imgf000184_0003
HPLC-MS (Method C): m/z: 534 (M+23); Rt.= 6.07 min.
The intermediate aldehyde was prepared similarly as described in example 481.
Example 485 (General procedure (D)) 6-[6-(2,4-Dioxothia∑olidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoic acid.
Figure imgf000185_0001
HPLC-MS (Method C): m/z: 408 (M+23); Rt .= 3.71 min.
Example 486 (General procedure (D)) 4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyric acid.
Figure imgf000185_0002
HPLC-MS (Method C): m/z: 380 (M+23); Rt.= 3.23 min.
Example 487 (General procedure (D)) 6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoic acid ethyl ester.
Figure imgf000185_0003
HPLC-MS (Method C): m/z: 436 (M+23); Rt.= 4.64 min.
Example 488 (General procedure (D)) 4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyric acid ethyl ester.
Figure imgf000185_0004
HPLC-MS (Method C): m/z: 408 (M+23); Rt.= 4.28 min.
Example 489 (General procedure (D)) 2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentyl}malonic acid
Figure imgf000186_0001
HPLC-MS (Method C): m/∑ = 444 (M+1); Rt = 3,84 min.
Example 490 (General procedure (D)
2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1 -yloxy]pentyl}malonic acid diethyl ester
Figure imgf000186_0002
HPLC-MS (Method C): m/z = 500 (M+1); Rt = 5.18 min.
Example 491 (General procedure (D)) 4-[4-(2,4,6-Trioxotetrahydropyrimidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid
Figure imgf000186_0003
HPLC-MS (Method C): m/z = 369 (M+1); Rt = 2,68 min.
Example 492
N-(3-Aminopropyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]- butyramide
Figure imgf000187_0001
To a mixture of 4-[4-(2,4-dioxothia∑olidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid (example 469, 5.9 g, 16.5 mmol) and 1-hydroxybenzotriazole (3.35 g, 24.8 mmol) in DMF (60 mL) was added 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (4.75 g, 24.8 mmol) and the resulting mixture was stirred at room temperature for 2 hours. /V-(3-amino- propylcarbamic acid fe/ -butyl ester (3.45 g, 19.8 mmol) was added and the resulting mixture was stirred al room temperature for 16 hours. The mixture was concentrated in vacuo and ethyl acetate and dichloromethane were added to the residue. The mixture was filtered, washed with water and dried in vacuo to afford 4.98 g (59%) of (3-{4-[4-(2,4-dioxothiazolidin- 5-ylidenemethyl)naphthalen-1-yloxy]butyrylamino}propyl)carbamic acid tert-butyl ester.
HPLC-MS (Method C): m/z: 515 (M+1); Rt = 3.79 min.
(3-{4-[4-(2,4-Dioxothiazolidin-5-ylidenemethy!)naphthalen-1-yloxy]butyrylamino}- propyl)carbamic acid tert-butyl ester (4.9 g, 9.5 mmol) was added dichloromethane (50 mL) and trifluoroacetic acid (50 mL) and the resulting mixture was stirred at room temperature for 45 minutes. The mixture was concentrated in vacuo and co-evaporated with toluene. To the residue was added ethyl acetate (100 mL) and the mixture was filtered and dried in vacuo to afford the title compound as the trifluoroacetic acid salt.
HPLC-MS (Method C): m/z: 414 (M+1); Rt = 2,27 min.
Compounds of the invention includes:
Example 493
Figure imgf000188_0001
Example 494
Figure imgf000188_0002
Example 495
Figure imgf000188_0003
Example 496
Figure imgf000188_0004
Example 497
Figure imgf000188_0005
Example 498
Figure imgf000188_0006
Example 499
Figure imgf000189_0001
Example 500
Figure imgf000189_0002
Example 501
Figure imgf000189_0003
Example 502
Figure imgf000189_0004
Example 503
Figure imgf000189_0005
Example 504 (Prepared analogously to General Procedure (D)) 2-{5-[4-(2,4-Thiazolidindion-5-ylidenemethyl)naphthalen-1-yloxy]pentyl}malonic acid
Figure imgf000190_0001
A solution of 4-hydroxy-1-naphtaldehyde (1.0 g, 5.81 mmol), 2-(5-bromopentyl)malonic acid diethyl ester (2.07 g, 6.68 mmol) and potassium carbonate (4.01 g, 29 mmol) in DMF (50 mL) was stirred at 100° C for 3 hours. The mixture was cooled and the salt was filtered off. The solvent was then removed under reduced pressure to afford 2.9 g of crude 2-[5-(4- formylnaphtalen-1-yloxy)pentyl]malonic acid diethyl ester which was used for the next reaction without further purification.
HPLC-MS (Method C): m/z: 401 (M+1); Rt = 5.16 min. 1H-NMR (DMSO-d6): δ = 1.18 (t, 6 H), 1 ,39 (m, 2 H), 1.55 (m, 2 H), 1.87 (m, 4 H), 3.48 (t, 1 H), 4.13 (m, 4 H), 4.27 (t, 2 H), 7.17 (d, 1 H), 7.64(t, 1 H), 7.75 (t, 1 H), 8.13 (d, 1 H), 8.29 (d, 1 H), 9.24 (d, 1 H), 10.19 (s, 1 H).
1.4 g (3.5 mmol) of crude 2-[5-(4-formylnaphtalen-1-yloxy)pentyl]malonic acid diethyl ester was treated with aqueous sodium hydroxide (1N, 8.75 mL, 8.75 mmol) and methanol (50 mL). The solution was stirred at 70° C for 5 hours and the mixture was concentrated under reduced pressure. Hydrochloric acid (6 N) was added until pH <2. The resulting slurry was stirred untill it solidified. The crystals were filtered off, washed with water and then dried in vacuo to afford 1.1 g (92%) of 2-[5-(4-formylnaphtalen-1-yloxy)pentyl]malonic acid. The product was used in the next step without further purification.
HPLC-MS (Method C): m/z: 345 (M+1); Rt = 3.52 min. 1H-NMR(DMSO-d6): δ = 1,40 (m, 2 H), 1.55 (m, 2 H), 1.80 (m, 2 H), 1.90 (m, 2 H), 3.24 (t, 1 H), 4.29 (t, 2 H), 7.19 (d, 1 H), 7.64(t, 1 H), 7.75 (t, 1 H), 8.14 (d, 1 H), 8.30 (d, 1 H), 9.23 (d, 1 H), 10.18 (s, 1 H), 12.69 (s, 2 H).
To a solution of 2-[5-(4-formylnaphtalen-1-yloxy) pentyl]malonic acid (0.36 g, 1.05 mmol) in acetic acid (10 mL) was added 2,4-thiazolidindione (0.16 g,1.36 mmol) and piperidine (0.52 mL, 5.25 mmol). The solution was heated to 105 °C for 24 hours. After cooling to room temperature, the solvents were removed in vacuo. Water was added to the residue. The precipi- tate was filtered off and washed with water. Recrystalisation from acetonitrile afforded 200 mg (43%) of the title compound as a solid.
HPLC-MS (Method C): m/z: 422 (M-C02+Na); Rt = 4.08 min. 1H-NMR(DMSO-d6): δ = 1 ,41
(m, 2 H), 1.55 (m, 4 H), 1.88 (m, 2 H), 2.23 (I, 1 H), 4.24 (t, 2 H), 7.61-7.74 (m, 3 H), 8.12 (d, 1 H), 8.28 (d, 1 H), 8.38 (s, 1 H), 12.00 (s, 1 H), 12.59 (s, 2 H).
The following compounds are commercially available and may be prepared according to general procedure (D): Example 505
Figure imgf000191_0001
Example 506
Figure imgf000191_0002
Example 507
Figure imgf000191_0003
Example 508
Figure imgf000192_0001
Example 509
Figure imgf000192_0002
Example 510
Figure imgf000192_0003
Example 511
Figure imgf000192_0004
The following salicylic acid derivatives do all bind to the His B10 Zn2+ site of the insulin hexamer: Example 512
Salicylic acid
Figure imgf000193_0001
Example 513
Thiosalicylic acid (or: 2-Mercaptobenzoic
Figure imgf000193_0002
Example 514 2-Hydroxy-5-nitrobenzoic acid
Figure imgf000193_0003
Example 515 3-Nitrosalicyclic acid
Figure imgf000193_0004
Example 516 5,5'-Methylenedisalicylic acid
Figure imgf000193_0005
Example 517
2-Amino-5-trifluoromethylbenzoesyre
Figure imgf000194_0001
Example 518
2-Amino-4-chlorobenzoic acid
Figure imgf000194_0002
Example 519
2-Amino-5-methoxybenzoesyre
Figure imgf000194_0003
Example 520
Figure imgf000194_0004
Example 521
Figure imgf000194_0005
Example 522
Figure imgf000195_0001
Example 523
Figure imgf000195_0002
Example 524
Figure imgf000195_0003
Example 525
Figure imgf000195_0004
Example 526
5-lodosalicylic acid
Figure imgf000195_0005
Example 527
5-Chloros alicylic acid
Figure imgf000196_0001
Example 528
1-Hydroxy-2-naphthoic acid
Figure imgf000196_0002
Example 529
3,5-Dihydroxy-2-naphthoic acid
Figure imgf000196_0003
Example 530
3-Hydroxy-2-naphthoic acid
Figure imgf000196_0004
Example 531
3,7-Dihydroxy-2-naphthoic acid
Figure imgf000196_0005
Example 532 2-Hydroxybenzo[a]carbazole-3-carboxylic acid
Figure imgf000197_0001
Example 533 7-Bromo-3-hydroxy-2-naphthoic acid
Figure imgf000197_0002
This compound was prepared according to Murphy et al., J. Med. Chem. 1990, 33, 171-8. HPLC-MS (Method A): m/z: 267 (M+1); Rt: = 3.78 min.
Example 534
1 ,6-Dibromo-2-hydroxynaphthalene-3-carboxylic acid
Figure imgf000197_0003
This compound was prepared according to Murphy et al., J. Med. Chem. 1990, 33, 171-8. HPLC-MS (Method A): m/z: 346 (M+1); Rt: = 4,19 min.
Example 535
7-Formyl-3-hydroxynaphthalene-2-carboxylic Acid
Figure imgf000197_0004
A solution of 7-bromo-3-hydroxynaphthalene-2-carboxylic acid (15.0 g, 56.2 mmol) (example 533) in tetrahydrofuran (100 mL) was added to a solution of lithium hydride (893 mg, 112 mmol) in tetrahydrofuran (350 mL). After 30 minutes stirring at room temperature, the resulting solution was heated to 50 °C for 2 minutes and then allowed to cool to ambient temperature over a period of 30 minutes. The mixture was cooled to -78 °C, and butyllithium (1.6 M in hexanes, 53 mL, 85 mmol) was added over a period of 15 minutes. Λ/,Λ/-Dimethylformamide (8.7 mL, 8.2 g, 112 mmol) was added after 90 minutes additional stirring. The cooling was discontinued, and the reaction mixture was stirred at room temperature for 17 hours before it was poured into 1 N hydrochloric acid (aq.) (750 mL). The organic solvents were evaporated in vacuo, and the resulting precipitate was filtered off and rinsed with water (3 x 100 mL) to yield the crude product (16.2 g). Purification on silica gel (dichloromethane / methanol / acetic acid = 90:9:1) furnished the title compound as a solid.
1H-NMR (DMSO-de): 511.95 (1H, bs), 10.02 (1H, s), 8.61 (1H, s), 8.54 (1H, s), 7.80 (2H, bs),
7.24 (1H, s); HPLC-MS (Method (A)): m/z: 217 (M+1); Rt = 2.49 min.
Example 536
3-Hydroxy-7-methoxy-2-naphthoic acid
Figure imgf000198_0001
Example 537
4-Amino-2-hydroxybenzoic acid
Figure imgf000198_0002
Example 538 5-Acetylamino-2-hydroxybenzoic acid
Figure imgf000198_0003
Example 539 2-Hydroxy-5-methoxybenzoic acid
Figure imgf000199_0001
The following compounds were prepared as described below: Example 540
4-Bromo-3-hydroxynaphthalene-2-carboxylic acid
Figure imgf000199_0002
3-Hydroxynaphthalene-2-carboxylic acid (3.0 g, 15.9 mmol) was suspended in acetic acid (40 mL) and with vigorous stirring a solution of bromine (817 μL, 15.9 mmol) in acetic acid (10 mL) was added drop wise during 30 minutes. The suspension was stirred at room temperature for 1 hour, filtered and washed with water. Drying in vacuo afforded 3.74 g (88%) of 4-bromo-3-hydroxynaphthalene-2-carboxylic acid as a solid.
1 H-NMR (DMSO-d6): δ 7.49 (1H, t), 7.75 (1H, t), 8.07 (2H, "t"), 8.64 (1H, s). The substitution pattern was confirmed by a COSY experiment, showing connectivities between the 3 (4 hydrogen) "triplets". HPLC-MS (Method A): m/z: 267 (M+1); Rt = 3.73 min.
Example 541
3-Hydroxy-4-iodonaphthalene-2-carboxylic acid
Figure imgf000199_0003
3-Hydroxynaphthalene-2-carboxylic acid (0.5 g, 2.7 mmol) was suspended in acetic acid (5 mL) and with stirring iodine monochloride (135 μL, 2.7 mml) was added. The suspension was stirred at room temperature for 1 hour, filtered and washed with water. Drying afforded 0.72 g (85%) of 4-iodo-3-hydroxynaphthalene-2-carboxylic acid as a solid. 1H-NMR (DMSO-de): δ 7.47 (1H, t), 7.73 (1H, t), 7.98 (1H, d), 8.05 (1H, d), 8.66 (1H, s). HPLC-MS (Method A): m/z: 315 (M+1); Rt = 3.94 min. Example 542 2-Hydroxy-5-[(4-methoxyphenylamino)methyl]benzoic acid
Figure imgf000200_0001
p-Anisidine (1.3 g, 10.6 mmol) was dissolved in methanol (20 mL) and 5-formylsalicylic acid (1.75 g, 10.6 mmol)was added and the resulting mixture was stirred at room temperature for 16 hours. The solid formed was isolated by filtration, re-dissolved in N-methyl pyrrolidone (20 mL) and methanol (2 mL). To the mixture was added sodium cyanoborohydride (1.2 g) and the mixture was heated to 70 °C for 3 hours. To the cooled mixture was added ethyl acetate (100 mL) and the mixture was extracted with water (100 mL) and saturated aqueous ammonium chloride (100 mL). The combined aqueous phases were concentrated in vacuo and a 2 g aliquot was purified by SepPac chromatography eluting with mixtures of aetonitrile and water containing 0.1% trifluoroacetic acid to afford the title compound.
HPLC-MS (Method A): m/z: 274 (M+1); Rt = 1.77 min.
1H-NMR (methanol-d4): δ 3.82 (3H, s), 4.45 (2H, s), 6.96 (1H, d), 7.03 (2H, d), 7.23 (2H, d),
7.45 (1H, dd), 7.92 (1 H, d).
Example 543 2-Hydroxy-5-(4-methoxyphenylsulfamoyl)benzoic acid
Figure imgf000200_0002
A solution of 5-chlrosulfonylsalicylic acid (0.96 g, 4.1 mmol) in dichloromethane (20 mL) and triethylamine (1.69 mL, 12.2 mmol) was added p-anisidine (0.49 g, 4.1 mmol) and the resulting mixture was stirred at room temperature for 16 hours. The mixture was added dichloromethane (50 mL) and was washed with water (2 x 100 mL). Drying (MgS04) of the organic phase and concentration in vacuo afforded 0.57 g crude product. Purification by column chromatography on silica gel eluting first with ethyl acetate:heptane (1 :1) then with methanol afforded 0.1 g of the title compound.
HPLC-MS (Method A): m/z: 346 (M+23); Rt = 2.89 min.
1H-NMR (DMSO-d6): δ 3.67 (3H, s), 6.62 (1 H, d), 6.77 (2H, d), 6.96 (2H, d), 7.40 (1H, dd),
8.05 (1H, d), 9.6 (1 H. bs).
General procedure (E) for preparation of compounds of general formula l-j
Figure imgf000201_0001
wherein Lea is a leaving group such as Cl, Br, I or OS02CF3, R is hydrogen or d-Cβ-alkyl, optionally the two R-groups may together form a 5-8 membered ring, a cyclic boronic acid ester, and J is as defined above.
An analogous chemical transformation has previously been described in the literature (Bumagin et al., Tetrahedron, 1997, 53, 14437-14450). The reaction is generally known as the Suzuki coupling reaction and is generally performed by reacting an aryl halide or triflate with an arylboronic acid or a heteroarylboronic acid in the presence of a palladium catalyst and a base such as sodium acetate, sodium carbonate or sodium hydroxide. The solvent can be water, acetone, DMF, NMP, HMPA, methanol, ethanol toluene or a mixture of two or more of these solvents. The reaction is performed at room temperature or at elevated temperature.
The general procedure (E) is further illustrated in the following example: Example 544 (General Procedure (E)) 7-(4-Acetylphenyl)-3-hydroxynaphthalene-2-carboxylic Acid
Figure imgf000201_0002
To 7-bromo-3-hydroxynaphthalene-2-carboxylic acid (100 mg, 0.37 mmol) (example 533) was added a solution of 4-acetylphenylboronic acid (92 mg, 0.56 mmol) in acetone (2.2 mL) followed by a solution of sodium carbonate (198 mg, 1.87 mmol) in water (3.3 mL). A suspension of palladium(ll) acetate (4 mg, 0.02 mmol) in acetone (0.5 mL) was filtered and added to the above solution. The mixture was purged with N2 and stirred vigorously for 24 hours at room temperature. The reaction mixture was poured into 1 N hydrochloric acid (aq.) (60 mL) and the precipitate was filtered off and rinsed with water (3 x 40 mL). The crude product was dissolved in acetone (25 mL) and dried with magnesium sulfate (1 h). Filtration followed by concentration furnished the title compound as a solid (92 mg). 1H-NMR (DMSO-d6): 512.60 (1H, bs), 8.64 (1H, s), 8.42 (1H, s), 8.08 (2H, d), 7.97 (2H, d), 7.92 (2H, m), 7.33 (1H, s), 2.63 (3H, s); HPLC-MS (Method (A): m/z: 307 (M+1); Rt = 3.84 min.
The compounds in the following examples were prepared in a similar fashion. Optionally, the compounds can be further purified by recrystallization from e.g. ethanol or by chromatography.
Example 545 (General Procedure (E)) 3-Hydroxy-7-(3-methoxyphenyl)naphthalene-2-carboxylic acid
Figure imgf000202_0001
HPLC-MS (Method (A)): m/z: 295 (M+1); Rt = 4.60 min.
Example 546 (General Procedure (E)) 3-Hydroxy-7-phenylnaphthalene-2-carboxylic acid
Figure imgf000202_0002
HPLC-MS (Method (A)): m/z: 265 (M+1); Rt = 4.6 min.
Example 547 (General Procedure (E)) 3-Hydroxy-7-p-tolylnaphthalene-2-carboxylic acid
Figure imgf000203_0001
Example 548 (General Procedure (E)) 7-(4-Formylphenyl)-3-hydroxynaphthalene-2-carboxylic acid
Figure imgf000203_0002
HPLC-MS (Method (A)): m/z: 293 (M+1); Rt = 4.4 min.
Example 549 (General Procedure (E)) 6-Hydroxy-[1 ,2]binaphthalenyl-7-carboxylic acid
Figure imgf000203_0003
HPLC-MS (Method (A)): m/z: 315 (M+1); Rt = 5.17 min.
Example 550 (General Procedure (E)) 7-(4-Carboxy-phenyl)-3-hydroxynaphthalene-2-carboxylic acid
Figure imgf000203_0004
HPLC-MS (Method (A)): m/z: 309 (M+1); Rt = 3.60 min.
Example 551 (General Procedure (E)) 7-Benzofuran-2-yl-3-hydroxynaphthalene-2-carboxylic acid
Figure imgf000204_0001
HPLC-MS (Method (A)): m/z: 305 (M+1); Rt = 4.97 min.
Example 552 (General Procedure (E)) 3-Hydroxy-7-(4-methoxyphenyl)-naphthalene-2-carboxylic acid
Figure imgf000204_0002
HPLC-MS (Method (A)): m/z: 295 (M+1); Rt = 4.68 min.
Example 553 (General Procedure (E)) 7-(3-Ethoxyphenyl)-3-hydroxynaphthalene-2-carboxylic acid
Figure imgf000204_0003
HPLC-MS (Method (A)): m/z: 309 (M+1); Rt = 4.89 min.
Example 554 (General Procedure (E))
7-Benzo[1 ,3]dioxol-5-yl-3-hydroxynaphthalene-2-carboxylic acid
Figure imgf000204_0004
HPLC-MS (Method (A)): m/z: 309 (M+1); Rt = 5.61 min.
Example 555 (General Procedure (E)) 7-Biphenyl-3-yl-3-hydroxynaphthaIene-2-carboxylic acid
Figure imgf000204_0005
HPLC-MS (Method (A)): m/z: 341 (M+1); Rt = 5.45 min.
General procedure (F) for preparation of compounds of general formula Is.
Figure imgf000205_0001
wherein R is hydrogen or Cι-C6-alkyl and T is as defined above
This general procedure (F) is further illustrated in the following example:
Example 556 (General procedure (F)) 3-Hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylic Acid
Figure imgf000205_0002
7-Formyl-3-hydroxynaphthalene-2-carboxylic acid (40 mg, 0.19 mmol) (example 535) was suspended in methanol (300 μL). Acetic acid (16 μL, 17 mg, 0.28 mmol) and 4-(2- propyl)aniline (40 μL, 40 mg, 0.30 mmol) were added consecutively, and the resulting mixture was stirred vigorously at room temperature for 2 hours. Sodium cyanoborohydride (1.0 M in tetrahydrofuran, 300 μL, 0.3 mmoJ) was added, and the stirring was continued for another 17 hours. The reaction mixture was poured into 6 N hydrochloric acid'(aq.) (6 mL), and the precipitate was filtered off and rinsed with water (3 x 2 mL) to yield the title compound (40 mg) as its hydrochloride salt. No further purification was necessary.
1H-NMR (DMSO-d6): 510.95 (1H, bs), 8.45 (1H, s), 7.96 (1H, s), 7.78 (1H, d), 7.62 (1H, d), 7.32 (1H, s), 7.13 (2H, bd), 6.98 (2H, bd), 4.48 (2H, s), 2.79 (1H, sept), 1.14 (6H, d); HPLC- MS (Method (A)): m/z: 336 (M+1); Rt = 3.92 min. The compounds in the following examples were made using this general procedure (F).
Example 557 (General procedure (F)) 7-{[(4-Bromophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic cid
Figure imgf000206_0001
HPLC-MS (Method C): m/z: 372 (M+1); Rt = 4.31 min.
Example 558 (General procedure (F)) 7-{[(3,5-Dichlorophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic cid
Figure imgf000206_0002
HPLC-MS (Method C): m/z: 362 (M+1); Rt = 4.75 min.
Example 559 (General procedure (F)) 7-{[(Benzothiazol-6-yl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid
Figure imgf000206_0003
HPLC-MS (Method C): m/z: 351 (M+1); Rt = 3.43 min.
Example 560 (General procedure (F)) 3-Hydroxy-7-{[(quinolin-6-yl)amino]methyl}naphthalene-2-carboxylic Acid
Figure imgf000206_0004
HPLC-MS (Method C): m/z: 345 (M+1); Rt = 2.26 min.
Example 561 (General procedure (F)) 3-Hydroxy-7-{[(4-methoxyphenyl)amino]methyl}naphthalene-2-carboxylic Acid
Figure imgf000207_0001
Example 562 (General procedure (F)) 7-{[(2,3-Dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic
Acid
Figure imgf000207_0002
HPLC-MS (Method C): m/z: 350 (M+1); Rt = 2.22 min.
Example 563 (General procedure (F))
7-{[(4-Chlorobenzyl)amino]methyI}-3-hydroxynaphthalene-2-carboxylic Acid
Figure imgf000207_0003
HPLC-MS (Method C): m/z: 342 (M+1); Rt = 2.45 min.
Example 564 (General procedure (F))
3-Hydroxy-7-{[(naphthalen-1-ylmethyI)amino]methyl}naphthalene-2-carboxylic Acid
Figure imgf000207_0004
HPLC-MS (Method C): m/z: 357 (M+1); Rt = 2.63 min.
Example 565 (General procedure (F))
7-{[(Biphenyl-2-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic cid
Figure imgf000207_0005
HPLC-MS (Method C): m/z: 384 (M+1); Rt = 2.90 min.
Example 566 (General procedure (F)) 3-Hydroxy-7-{[(4-pheno) ybenzyl)amino]methyI}naphthalene-2-carboxyIic cid
Figure imgf000208_0001
HPLC-MS (Method C): m/∑: 400 (M+1); Rt = 3.15 min.
Example 567 (General procedure (F))
3-Hydroxy-7-{[(4-methoxybenzyl)amino]methyl}naphthalene-2-carboxylic Acid
Figure imgf000208_0002
HPLC-MS (Method C): m/z: 338 (M+1); Rt = 2.32 min.
General procedure (G) for preparation of compounds of general formula l6
+ ( ,-C6-alkanoyl)20
Figure imgf000208_0003
Figure imgf000208_0004
wherein J is as defined above and the moiety
Figure imgf000208_0005
is an anhydride.
The general procedure (G) is illustrated by the following example: Example 568 (General procedure (G))
/V-Acetyl-3-hydroxy-7-[(4-(2-propyl)phenylamino)methylJnaphthalene-2-carboxylicAcid
Figure imgf000208_0006
3-Hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylic acid (25 mg, 0.07 mmol) (example 556) was suspended in tetrahydrofuran (200 μL). A solution of sodium hy- drogencarbonate (23 mg, 0.27 mmol) in water (200 μL) was added followed by acetic anhydride (14 μL, 15 mg, 0.15 mmol). The reaction mixture was stirred vigorously for 65 hours at room temperature before 6 N hydrochloric acid (4 mL) was added. The precipitate was filtered off and rinsed with water (3 x 1 L) to yield the title compound (21 mg). No further purification was necessary.
1H-NMR (DMSO-ds): 510.96 (1H, bs), 8.48 (1H, s), 7.73 (1H, s), 7.72 (1H, d), 7.41 (1H, dd), 7.28 (1H, s), 7.23 (2H, d), 7.18 (2H, d), 4.96 (2H, s), 2.85 (1H, sept), 1.86 (3H, s), 1.15 (6H, d); HPLC-MS (Method (A)): m/z: 378 (M+1); Rt = 3.90 min.
The compounds in the following examples were prepared in a similar fashion.
Example 569 (General procedure (G)) /V-Acetyl-7-{[(4-bromophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid
Figure imgf000209_0001
HPLC-MS (Method C): m/z: 414 (M+1); Rt = 3.76 min.
Example 570 (General procedure (G)) V-Acetyl-7-{[(2,3-dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2- carboxylic Acid
Figure imgf000209_0002
HPLC-MS (Method C): m/z: 392 (M+1); Rt = 3.26 min.
Example 571 (General procedure (G))
/V-Acetyl-7-{[(4-chlorobenzyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic cid
HPLC-MS (Method C): m/z: 384 (M+1); Rt = 3.67 min.
Compounds of the invention may also include tetrazoles: Example 572 5-(3-(Naphthalen-2-yloxymefhyl)-phenyl)-1H-tetrazole
Figure imgf000210_0001
To a mixture of 2-naphthol (10 g, 0.07 mol) and potassium carbonate (10 g, 0.073 mol) in acetone (150 mL), alpha-bromo-m-tolunitril (13.6 g, 0.07 mol) was added in portions. The reaction mixture was stirred at reflux temperature for 2.5 hours. The cooled reaction mixture was filtered and evaporated in vacuo affording an oily residue (19 g) which was dissolved in diethyl ether (150 mL) and stirred with a mixture of active carbon and MgS04 for 16 hours. The mixture was filtered and evaporated in vacuo affording crude 18.0 g (100 %) of 3- (naphthalen-2-yloxymethyl)-benzonitrile as a solid.
12 g of the above benzonitrile was recrystallised from ethanol (150 mL) affording 8.3 g (69 %) of 3-(naphthalen-2-yloxymethyl)-benzonitrile as a solid.
M.p. 60 - 61 °C.
Calculated for C18Hι3N0:
C, 83.37 %; H, 5.05 %; N, 5.40 %; Found
C, 83.51 %; H, 5.03 %; N, 5.38 %.
To a mixture of sodium azide (1.46 g, 22.5 mmol) and ammonium chloride (1.28 g, 24.0 mmol) in dry dimethylformamide (20 mL) under an atmosphere of nitrogen, 3-(naphthalen-2- yloxymethy -benzonitrile (3.9 g, 15 mmol) was added and the reaction mixture was stirred at 125 °C for 4 hours. The cooled reaction mixture was poured on to ice water (300 mL) and acidified to pH = 1 with 1 N hydrochloric acid. The precipitate was filtered off and washed with water, dried at 100 °C for 4 hours affording 4.2 g (93 %) of the title compound.
M.p. 200 - 202 °C.
Calculated for C18H14N40:
C, 71.51 %; H, 4.67 %; N, 18.54 %; Found
C, 72.11 %; H, 4.65 %; N, 17.43 %. 1H NMR (400 MHz, DMSO-d6) δH 5.36 (s, 2H), 7.29 (dd, 1H), 7.36 (dt, 1H), 7.47 (m, 2H), 7.66 (t, 1 H), 7.74 (d, 1H), 7.84 (m, 3H), 8.02 (d, 1 H), 8.22 (s, 1 H).
Example 573 N-(3-(Tetrazol-5-yl)phenyl)-2-naphtoic acid amide
Figure imgf000211_0001
2-Naphtoic acid (10 g, 58 mmol) was dissolved in dichloromethane (100 mL) and N,N- dimethylformamide (0.2 mL) was added followed by thionyl chloride (5.1 ml, 70 mmol). The mixture was heated at reflux temperature for 2 hours. After cooling to room temperature, the mixture was added dropwise to a mixture of 3-aminobenzonitril (6.90 g, 58 mmol) and triethyl amine (10 mL) in dichloromethane (75 mL). The resulting mixture was stirred at room temperature for 30 minutes. Water (50 mL) was added and the volatiles was exaporated in vacuo. The resulting mixture was filtered and the filter cake was washed with water followed by heptane (2 x 25 mL). Drying in vacuo at 50 °C for 16 hours afforded 15.0 g (95 %) of N-(3- cyanophenyl)-2-naphtoic acid amide.
M.p. 138-140 °C
The above naphthoic acid amide (10 g. 37 mmol) was dissolved in N,N-dimethyIformamide (200 mL) and sodium azide (2.63 g, 40 mmol) and ammonium chloride (2.16 g, 40 mmol) were added and the mixture heated at 125 °C for 6 hours. Sodium azide (1.2 g) and ammonium chloride (0.98 g) were added and the mixture heated at 125 °C for 16 hours. After cooling, the mixture was poured into water (1.5 I) and stirred at room temperature for 30 minutes. The solid formed was filtered off, washed with water and dried in vacuo at 50 °C for 3 days affording 9.69 g (84 %) of the title compound as a solid which could he further purified by treatment with ethanol at reflux temperature.
H NMR (200 MHz, DMSO-d6): δH 7.58-7.70 (m, 3H), 7.77 (d, 1H), 8.04-8.13 (m, 5H), 8.65 (d, 1H), 10.7 (s, 1H). Calculated for Cι8H13N50, 0.75 H20:
C, 65.74 %; H, 4.44 %; N, 21.30 %. Found:
C, 65.58 %; H, 4.50 %; N, 21.05 %.
Example 574 5-[3-(Biphenyl-4-yloxymethyl)phenyl]-1H-tetra∑ole
Figure imgf000212_0001
To a solution of 4-phenylphenol (10.0 g, 59 mmol) in dry N,N-dimethyl-formamide (45 mL) kept under an atmosphere of nitrogen, sodium hydride (2.82 g, 71 mmol, 60 % dispersion in oil) was added in portions and the reaction mixture was stirred until gas evolution ceased. A solution of m-cyanobenzyl bromide (13 g, 65 mmol) in dry N,N-dimethylformamide (45 mL) was added dropwise and the reaction mixture was stirred at room temperature for 18 hours.
The reaction mixture was poured on to ice water (150 mL). The precipitate was filtered of and washed with 50 % ethanol
(3 x 50 mL), ethanol (2 x 50 mL), diethyl ether (80 mL), and dried in vacuo at
50 °C for 18 hours affording crude 17.39 g of 3-(biphenyl-4-yloxymethyl)-benzonitrile as a solid.
1H NMR (200 MHz, CDCI3) δH 5.14 (s, 2H), 7.05 (m, 2H), 7.30 - 7.78 (m, 11H).
To a mixture of sodium azide (2.96 g, 45.6 mmol) and ammonium chloride (2.44 g, 45.6 mmol) in dry N,N-dimethylformamide (100 mL) under an atmosphere of nitrogen, 3-(biphenyl- 4-yloxymethyl)-benzonJtrile (10.0 g, 35.0 mmol) was added and the reaction mixture was stirred at 125 °C for 18 hours. The cooled reaction mixture was poured on to a mixture of 1 N hydrochloric acid (60 mL) and ice water (500 mL). The precipitate was filtered off and washed with water (3 x 100 mL), 50 % ethanol (3 x 100 mL), ethanol (50 L), diethyl ether (50 mL), ethanol (80 mL), and dried in vacuo at 50 °C for 18 hours affording 8.02 g (70 %) of the title compound.
1H NMR (200 MHz, DMSO-d6) δH 5.31 (s, 2H), 7.19 (m, 2H), 7.34 (m, 1H), 7.47 (m, 2H), 7.69 (m, 6H), 8.05 (dt, 1H), 8.24 (s, 1H). Example 575 5-(3-Phenoxymethyl)-phenyl)-tetrazole
Figure imgf000213_0001
3-Bromomethylbenzonitrile (5.00 g, 25.5 mmol) was dissolved in N,N-dimethylformamide (50 mL), phenol (2.40 g, 25.5 mmol) and potassium carbonate (10.6 g, 77 mmol) were added. The mixture was stirred at room temperature for 16 hours. The mixture was poured into water (400 mL) and extracted with ethyl acetate (2 x 200 mL). The combined organic extracts were washed with water (2 x 100 mL), dried (MgS04) and evaporated in vacuo to afford 5.19 g (97 %) 3-(phenoxymethyl)benzonitrile as an oil.
TLC: Rf = 0.38 (Ethyl acetate/heptane = 1:4)
The above benzonitrile (5.19 g, 24.8 mmol) was dissolved in N,N-dimethyIformamide (100 L) and sodium azide (1.93 g, 30 mmol) and ammonium chloride (1.59 g, 30 mmol) were added and the mixture was heated at 140 °C for 16 hours. After cooling, the mixture was poured into water (800 mL). The aqeous mixture was washed with ethyl acetate (200 mL). The pH of the aqueous phase was adjusted to 1 with 5 N hydrochloric acid and stirred at room temperature for 30 minutes. Filtration, washing with water and drying in vacuo at 50 °C afforded 2.06 g (33 %) of the title compound as a solid.
1H NMR (200 MHz, CDCI3 + DMSO-d6) δH 5.05 (s, 2H), 6.88 (m, 3H), 7.21 (m, 2H), 7.51 (m, 2H), 7.96 (dt, 1H), 8.14 (s, 1H).
Example 576 5-[3-(Biphenyl-4-ylmethoxy)phenyl]-1 H-tetrazole
Figure imgf000213_0002
To a solution of 3-cyanophenol (5.0 g, 40.72 mmol) in dry N,N-dimethylformamide (100 mL) kept under an atmosphere of nitrogen, sodium hydride (2 g, 48.86 mmol, 60 % dispersion in oil) was added in portions and the reaction mixture was stirred until gas evolution ceased, p- Phenylbenzyl chloride (9.26 g, 44.79 mmol) and potassium iodide (0.2 g, 1.21 mmol) were added and the reaction mixture was stirred at room temperature for 60 hours. The reaction mixture was poured on to a mixture of saturated sodium carbonate (100 mL) and ice water (300 mL). The precipitate was filtered of and washed with water (3 x 100 mL), n-hexane (2 x 80 rnL) and dried in acuo at 50 °C for 18 hours affording 11.34 g (98 %) of 3-(biphenyl-4- ylmethoxy)-benzonitrile as a solid.
To a mixture of sodium azide (2.37 g, 36.45 mmol) and ammonium chloride (1.95 g, 36.45 mmol) in dry N,N-dimethylformamide (100 mL) under an atmosphere of nitrogen, 3-(biphenyl- 4-ylmethoχy)-benzonitriIe (8.0 g, 28.04 mmol) was added and the reaction mixture was stirred at
125 °C for 18 hours. To the cooled reaction mixture water (100 mL) was added and the reaction mixture stirred for 0.75 hour. The precipitate was filtered off and washed with water, 96 % ethanol (2 x 50 mL), and dried in vacuo at 50°C for 18 hours affording 5.13 g (56 %) of the title compound.
1H NMR (200 MHz, DMSO-d6) δH 5.29 (s, 2H), 7.31 (dd, 1H), 7.37 - 7.77 (m, 12H).
Example 577 5-[4-(BiphenyI-4-ylmethoxy)-3-methoxyphenyl]-1H-tetrazol
Figure imgf000214_0001
This compound was made similarly as described in example 576.
Example 578
Figure imgf000214_0002
Example 579 5-(2-Naphtylmethyl)- 1 H-tetrazole
Figure imgf000215_0001
This compound was prepared similarly as described in example 572, step 2.
Example 580 5-(1-Naphtylmethyl)-1H-tetrazole
Figure imgf000215_0002
This compound was prepared similarly as described in example 572, step 2.
Example 581 5-[4-(Biphenyl-4-yloxymethyl)phenyl]-1H-tetrazole
Figure imgf000215_0003
A solution of alpha-bromo-p-tolunitrile (5.00 g, 25.5 mmol), 4-phenylphenol (4.56 g, 26.8 mmol), and potassium carbonate (10.6 g, 76.5 mmol) in N-dimethylformamide (75 mL) was stirred vigorously for 16 hours at room temperature. Water (75 mL) was added and the mixture was stirred at room temperature for 1 hour. The precipitate was filtered off and washed with thoroughly with water. Drying in vacuo over night at 50 °C afforded 7.09 g (97 %) of 4- (biphenyl-4-yloxymethyl)benzonitrile as a solid.
The above benzonitrile (3.00 g, 10.5 mmol) was dissolved in N,N-dimethylformamide (50 mL), and sodium azide (1.03 g, 15.8 mmol) and ammonium chloride (0.84 g, 15.8 mmol) were added and the mixture was stirred 16 hours at 125 °C. The mixture was cooled to room temperature and water (50 mL) was added. The suspension was stirred overnight, filtered, washed with water and dried in vacuo at 50 °C for 3 days to give crude 3.07 g (89 %) of the title compound. From the mother liquor crystals were colected and washed with water, dried by suction to give 0.18 g (5 %) of the title compound as a solid. 1H NMR (200 MHz, DMSO-d6): δH 5.21 (s, 2H), 7.12 (d, 2H), 7.30 (t, 1H), 7.42 (t, 2H), 7.56- 7.63 (m, 6H), 8.03 (d, 2H).
Calculated for C2oH16 40, 2H20:
C, 65.92 %; H, 5.53 %; N, 15.37 %. Found: C, 65.65 %; H, 5.01 %; N, 14.92 %.
Example 582
Figure imgf000216_0001
This compound was prepared similarly as described in example 576.
Example 583
Figure imgf000216_0002
Example 584
Figure imgf000216_0003
Example 585
Figure imgf000217_0001
Example 586 5-(3-(Biphenyl-4-yloxymethyl)-benzyl)-1H-tetrazol€
Figure imgf000217_0002
Example 587
5-(1 -Naphthyl)- 1 H-tetrazole
Figure imgf000217_0003
This compound was prepared similarly as described in example 572, step 2.
Example 588 5-[3-Methoxy-4-(4-methylsulfonylbenzyloxy)phenyl]-1r7,-tetrazole
Figure imgf000217_0004
This compound was made similarly as described in example 576.
Example 589 5-(2-Naphthyl)-1 H-tetrazole
Figure imgf000218_0001
This compound was prepared similarly as described in example 572, step 2.
Example 590 2-Amino-N~(1H-tetrazol-5-yl)-ben∑amide
Figure imgf000218_0002
Example 591 5-(4-Hydroxy-3-methoxyphenyl)-1H-tetrazole
Figure imgf000218_0003
This compound was prepared similarly as described in example 572, step 2.
Example 592
4-(2H-Tetrazol-5-ylmethoxy)benzoic acid
Figure imgf000218_0004
To a mixture of methyl 4-hydroxybenzoate (30.0 g, 0.20 mol), sodium iodide (30.0 g, 0.20 mol) and potassium carbonate (27.6 g, 0.20 mol) in acetone (2000 mL) was added chloroacetonitrile (14.9 g , 0.20 mol). The mixture was stirred at RT for 3 days. Water was added and the mixture was acidified with 1 N hydrochloric acid and the mixture was extracted with diethyl ether. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was dissolved in acetone and chloroacetonitrile (6.04 g,0.08 mol), so- dium iodide (12.0 g, 0.08 mol) and potassium carbonate (11.1 g, 0.08 mol) were added and the mixture was stirred for 16 hours at RT and at 60 °C. More chloroacetonitrile was added until the conversion was 97%. Water was added and the mixture was acidified with 1 N hydrochloric acid and the mixture was extracted with diethyl ether. The combined organic layers were dried over Na2S04 and concentrated in vacuo to afford methyl 4- cyanomethyloxybenzoate in quantitative yield. This compound was used without further purification in the following step.
A mixture of methyl 4-cyanomefhyloxyben∑oate (53.5 g,0.20 mol), sodium azide (16.9 g, 0.26 mol) and ammonium chloride (13.9 g, 0.26 mol) in DMF 1000 (mL) was refluxed overnight under N2. After cooling, the mixture was concentrated in vacuo. The residue was suspended in cold water and extracted with ethyl acetate. The combined organic phases were washed with brine, dried over Na2S0 and concentrated in vacuo, to afford methyl 4-(2H-tetrazol-5- ylmethoxy)benzoate. This compound was used as such in the following step.
Methyl 4-(2H-Tetrazol-5-ylmethoxy)-benzoate was refluxed in 3N sodium hydroxide. The reaction was followed by TLC (DCM:MeOH = 9:1). The reaction mixture was cooled, acidified and the product filtered off. The impure product was washed with DCM, dissolved in MeOH, filtered and purified by column chromatography on silica gel (DCM:MeOH = 9:1).The resulting product was recrystallised from DCM:MeOH=95:5. This was repeated until the product was pure. This afforded 13.82 g (30 %) of the title compound.
1H-NMR (DMSO-dβ): 4.70 (2H, s), 7.48 (2H, d), 7.73 (2H, d), 13 (1 H, bs).
Example 593
4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoic acid
Figure imgf000219_0001
To a solution of sodium hydroxide (10.4 g, 0.26 mol) in degassed water (600 mL) was added 4-mercaptobenzoic acid (20.0 g, 0.13 mol). This solution was stirred for 30 minutes. To a solution of potassium carbonate (9.0 g, 65 mmol) in degassed water (400 mL) was added chloroacetonitrile (9.8 g, (0.13 mol) portion-wise. These two solutions were mixed and stirred for 48 hours at RT under N2. The mixture was filtered and washed with heptane. The aque- ous phase was acidified with 3N hydrochloric acid and the product was filtered off, washed with water and dried, affording 4-cyanomethylsulfanylbenzoic acid (27.2 g, 88%). This compound was used without further purification in the following step.
A mixture of 4-cyanomethylsulfanylbenzoic acid (27.2 g, 0.14 mol), sodium azide (11.8 g,
0,18 mol) and ammon urn chloride (9.7 g, 0.18 mol) in DMF (1000 mL) was refluxed over- night under N2. The m xture was concentrated in vacuo. The residue was suspended in cold water and extracted wi th diethyl ether. The combined organic phases were washed with brine, dried over Na2SO_ι and concentrated in vacuo. Water was added and the precipitate was filtered off. The aqueous layer was concentrated in vacuo, water was added and the precipitate filtered off. The combined impure products were purified by column chromatography using DCM:MeOH = 9:1 as eluent, affording the title compound (5.2 g, 16%).
1H-NMR (DMSO-dβ): 5.58 (2H, s), 7.15 (2H, d), 7.93 (2H, d), 12.7 (1H, bs).
Example 594 3-(2H-Tetrazol-5-yl)-9H-carbazole
Figure imgf000220_0001
3-Bromo-9H-carbazole was prepared as described by Smith ef al. in Tetrahedron 1992, 48, 7479-7488.
A solution of 3-bromo-9H-carbazole (23.08 g, 0.094 mol) and cuprous cyanide (9.33 g, 0.103 mol) in Λ/-methyl-pyrrolidone (300 ml) was heated at 200 °C for 5 h. The cooled reaction mixture was poured on to water (600 ml) and the precipitate was filtered off and washed with ethyl acetate (3 x 50 ml). The filtrate was extracted with ethyl acetate (3 x 250 ml) and the combined ethyl acetate extracts were washed with water (150 ml), brine (150 ml), dried (MgSO4) and concentrated in vacuo. The residue was crystallised from heptanes and recrys- tallised from acetonitrile (70 ml) affording 7.16 g (40 %) of 3-cyano-9H-carbazole as a solid. M.p. 180 - 181 °C.
3-Cyano-9H-carba∑ole (5.77 g, 30 mmol) was dissolved in W,/V-dimethylformamide (150 ml), and sodium azide (9.85 g, 152 mmol), ammonium chloride (8.04 g, 150 mmol) and lithium chloride (1.93 g, 46 mmol) were added and the mixture was stirred for 20 h at 125 °C. To the reaction mixture was added an additional portion of sodium azide (9.85 g, 152 mmol) and ammonium chloride (8.04 g, 150 mmol) and the reaction mixture was stirred for an additional 24 h at 125 °C. The cooled reaction mixture was poured on to water (500 ml). The suspension was stirred for 0.5 h, and the precipitate was filtered off and washed with water (3 x 200 ml) and dried in vacuo at 50 °C. The dried crude product was suspended in diethyl ether (500 ml) and stirred for 2 h, filtered off and washed with diethyl ether (2 x 200 ml) and dried in vacuo at 50 °C affording 5.79 g (82 %) of the title compound as a solid.
1 H-NMR (DMSO-d6): 511.78 (1H, bs), 8.93 (1H, d), 8.23 (1H, d), 8.14 (1H, dd), 7.72 (1H, d), 7.60 (1H, d), 7.49 (1H, t), 7.28 (1H, t); HPLC-MS (Method C): m/z: 236 (M+1); Rt = 2.77 min.
The following commercially available tetrazoles do all bind to the His B10 Zn2+ site of the insulin hexamer:
Example 595 5-(3-Tolyl)-1 H-tetrazole
Figure imgf000221_0001
Example 596
5-(2-Bromophenyl)tetrazole
Figure imgf000221_0002
Example 597 5-(4-Ethoxalylamino-3-nitrophenyl)tetrazoIe
Figure imgf000222_0001
Example 598
Figure imgf000222_0002
Example 599
Figure imgf000222_0003
Example 600
Figure imgf000222_0004
Example 601
Figure imgf000223_0001
Example 602 Tetrazole
Figure imgf000223_0002
Example 603 5-Methyltetrazole
H H3C^
N-N
Example 604 5-Benzyl-2H-tetrazole
Example 605 4-(2H-Tetrazol-5-yl)benzoic acid
Figure imgf000223_0004
Example 606 5-Phenyl-2H-tetrazole
Figure imgf000223_0005
Example 607 5-(4-Chlorophenylsulfanylmethyl)-2H-tetrazole
Figure imgf000224_0001
Example 608 5-(3-Benzyloxyphenyl)-2H-tetrazole
Figure imgf000224_0002
Example 609 2-Phenyl-6-(1H-tetrazoI-5-yl)-chromen-4-one
Figure imgf000224_0003
Example 610
Figure imgf000224_0004
Example 61
Figure imgf000225_0001
Example 612
Figure imgf000225_0002
Example 613
Figure imgf000225_0003
Example 614
Figure imgf000225_0004
Example 615 5-(4-Bromo-phenyl)-1 H-tetrazole
Figure imgf000226_0001
Example 616
Figure imgf000226_0002
Example 617
Figure imgf000226_0003
Example 618
Figure imgf000226_0004
Example 619
Figure imgf000226_0005
Example 620
Figure imgf000227_0001
Example 621
Figure imgf000227_0002
Example 622
Figure imgf000227_0003
Example 623
Figure imgf000227_0004
Example 624
Figure imgf000228_0001
Example 625
Figure imgf000228_0002
Example 626
Figure imgf000228_0003
Example 627
Figure imgf000228_0004
Example 628
Figure imgf000228_0005
Example 629
Figure imgf000229_0001
Example 630
Figure imgf000229_0002
Example 631
Figure imgf000229_0003
Example 632
Figure imgf000229_0004
Example 633
Figure imgf000230_0001
Example 634
Figure imgf000230_0002
Example 635
Figure imgf000230_0003
Example 636
Figure imgf000230_0004
Example 637
Figure imgf000231_0001
Example 638
Figure imgf000231_0002
Example 639
Figure imgf000231_0003
Example 640
Figure imgf000231_0004
Example 641
Figure imgf000232_0001
Example 642
Figure imgf000232_0002
Example 643
Figure imgf000232_0003
Example 644
Figure imgf000232_0004
Example 645
Figure imgf000233_0001
Example 646 5-(2,6-Dichlorobenzyl)-2H-tetrazc
Figure imgf000233_0002
General procedure (H) for preparation of compounds of general formula l7:
NaCBH3
Figure imgf000233_0003
wherein K, M, and T are as defined above.
The reaction is generally known as a reductive alkylation reaction and is generally performed by stirήng an aldehyde with an amine at low pH (by addition of an acid, such as acetic acid or formic acid) in a solvent such as THF, DMF, NMP, methanol, ethanol, DMSO, dichloromethane, 1 ,2-dichloroethane, trimethyl orthoformate, triethyl orthoformate, or a mixture of two or more of these. As reducing agent sodium cyano borohydride or sodium triacetoxy borohydride may be used. The reaction is performed between 20°C and 120°C, preferably at room temperature.
When the reductive alkylation is complete, the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art.
The general procedure (H) is further illustrated in the following example 647: Example 647 (General procedure (H)) Biphenyl-4-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000234_0001
A solution of 5-(3-aminophenyl)-2H-tetrazole (example 873, 48 mg, 0.3 mmol) in DMF (250 μL) was mixed with a solution of 4-biphenylylcarbaldehyde (54 mg, 0.3 mmol) in DMF (250 μL) and acetic acid glacial (250 μL) was added to the mixture followed by a solution of sodium cyano borohydride (15 mg, 0.24 mmol) in methanol (250 μL). The resulting mixture was shaken at room temperature for 2 hours. Water (2 mL) was added to the mixture and the resulting mixture was shaken at room temperature for 16 hours. The mixture was centrifugated (6000 rpm, 10 minutes) and the supernatant was removed by a pipette. The residue was washed with water (3 mL), centrifugated (6000 rpm, 10 minutes) and the supernatant was removed by a pipette. The residue was dried in vacuo at 40 °C for 16 hours to afford the title compound as a solid.
HPLC-MS (Method C): m/z: 328 (M+1), 350 (M+23); Rt = 4.09 min.
Example 648 (General procedure (H)) Benzyl-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000234_0002
HPLC-MS (Method D): m/z: 252 (M+1); Rt = 3,74 min.
Example 649 (General procedure (H)) (4-Methoxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000234_0003
HPLC-MS (Method D): m/z: 282,2 (M+1); Rt = 3,57min. Example 650 (General procedure (H)) 4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenol
Figure imgf000235_0001
HPLC-MS (Method D): m/z: 268,4 (M+1); Rt = 2,64 min.
Example 651 (General procedure (H)) (4-Nitrobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000235_0002
HPLC-MS (Method D); m/z: 297,4 (M+1); Rt = 3,94 min.
Example 652 (General procedure (H)) (4-Chlorobenzyl)~[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000235_0003
HPLC-MS (Method D): m/z: 287,2 (M+1); Rt = 4,30 min.
Example 653 (General procedure (H)) (2-Chlorobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000235_0004
HPLC-MS (Method D): m/z: 286 (M+1); Rt = 4,40 min.
Example 654 (General procedure (H)) (4-Bromobenzyl)-[3-(2H-tetra∑ol-5-yl)phenyl]amine
Figure imgf000236_0001
HPLC-MS (Method D): m/∑:332 (M+1); Rt = 4,50 min.
Example 655 (General procedure (H)) (3-Benzyloxybenzyl)-[3-(2H-tetrazol-5-yI)phenyl]amine
Figure imgf000236_0002
HPLC-MS (Method D): m/z: 358 (M+1); Rt = 4,94 min.
Example 656 (General procedure (H)) Naphthalen-1-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000236_0003
HPLC-MS (Method D): m/z: 302 (M+1); Rt = 4,70 min.
Example 657 (General procedure (H)) Naphthalen-2-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000236_0004
HPLC-MS (Method D): m/z: 302 (M+1); Rt = 4,60 min.
Example 658 (General procedure (H)) 4-{[3-(2H-Tetrazol-5-yl)phenyIamino]methyl}benzoic acid
Figure imgf000236_0005
HPLC-MS (Method D): m/z: 296 (M+1); Rt = 3,24 min. Example 659 (General procedure (H)) [3-(2H-Tetrazoi-5-yl)-phenyl]-[3-(3-trifluoromethyl-phenoxy)benzyl]amine
Figure imgf000237_0001
HPLC-MS (Method D): m/z: 412 (M+1); Rt = 5,54 min.
Example 660 (General procedure (H)) (3-Phenoxybenzyl)-[3-(2H-tetrazoI>5-yl)phenyl]amine
Figure imgf000237_0002
HPLC-MS (Method D): m/z: 344 (M+1); Rt = 5,04 min.
Example 661 (General procedure (H)) (4-Phenoxy-benzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000237_0003
HPLC-MS (Method D): m/z: 344 (M+1); Rt = 5,00 min.
Example 662 (General procedure (H)) (4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenoxy)acetic acid
Figure imgf000237_0004
HPLC-MS (Method D): m/z: 326 (M+1); Rt = 3,10 min.
Example 663 (General procedure (H)) (4-Benzyloxybenzyl)-|3-(2H-tetrazol-5-yI)phenyl]amine
Figure imgf000238_0001
HPLC-MS (Method D): m/z: 358 (M+1); Rt = 4,97 min.
Example 664 (General procedure (H)) 3-(4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyI}phenyl)acrylic acid
Figure imgf000238_0002
HPLC-MS (Method D): m/z: 322 (M+1); Rt = 3,60 min.
Example 665 (General procedure (H)) Dimethyl-(4-{[3-(2H-tetrazol-5-yl)phenylamino]methyl}naphthalen-1-yl)amine
Figure imgf000238_0003
HPLC-MS (Method D): m/z: 345 (M+1); Rt = 3,07 min.
Example 666 (General procedure (H)) (4'-Methoxybiphenyl-4-ylmethyl)-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000238_0004
HPLC-MS (Method D): m/z: 358 (M+1); Rt = 4,97 min.
Example 667 (General procedure (H)) (2'-Chlorobiphenyl-4-ylmethyI)-[3-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000239_0001
HPLC-MS (Method D): m/z: 362 (M+1); Rf = 5,27 min.
Example 668 (General procedure (H)) Ben∑yl-[4-(2H-tetra∑ol-5-yl)phenyl]amine
Figure imgf000239_0002
For preparation of starting material, see example 874. HPLC-MS (Method D): m/z: 252 (M+1); Rt = 3,97 min.
Example 669 (General procedure (H)) (4-Methoxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000239_0003
HPLC-MS (Method D): m/z: 282 (M+1); Rt = 3,94 min.
Example 670 (General procedure (H)) 4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyI}phenol
Figure imgf000239_0004
HPLC-MS (Method D): m/z: 268 (M+1); Rt = 3,14 min.
Example 671 (General procedure (H)) (4-Nitrobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000240_0001
HPLC-MS (Method D): m/z: (M+1); Rt = 3,94 min.
Example 672 (General procedure (H)) (4-Chloroben∑yl)-[4-(2H-tetrazol-5-yl)phenyl]aminc
Figure imgf000240_0002
HPLC-MS (Method D): m/z: (M+1); Rt = 4,47 min.
Example 673 (General procedure (H)) (2-Chlorobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000240_0003
HPLC-MS (Method D): m/z: 286 (M+1); Rt = 4,37 min.
Example 674 (General procedure (H)) (4-Bromobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000240_0004
HPLC-MS (Method D): m/z: 331 (M+1); Rt = 4,57 min.
Example 675 (General procedure (H)) (3-BenzyloxybenzyI)-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000240_0005
HPLC-MS (Method D): m/z: 358 (M+1); Rt = 5,07min.
Example 676 (General procedure (H)) Naphthalen-1-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000241_0001
HPLC-MS (Method D): m/z: 302 (M+1); Rt = 4,70 min.
Example 677 (General procedure (H)) Naphthalen-2-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000241_0002
HPLC-MS (Method D): m/z: 302 (M+1); Rt = 4,70 min.
Example 678 (General procedure (H)) B/phenyl-4-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000241_0003
HPLC-MS (Method D): m/z: 328 (M+1); Rt = 5,07 min.
Example 679 (General procedure (H)) 4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid
Figure imgf000241_0004
HPLC-MS (Method D): m/z: 296 (M+1); Rt = 3,34 min. Example 680 (General procedure (H)) [4-(2H-Tetrazol-5-yl)phenyl]-[3-(3-trifluoromethylphenoxy)benzyl]amine
Figure imgf000242_0001
HPLC-MS (Method D): m/z: 412 (M+1); Rt = 5,54 min.
Example 681 (General procedure (H)) (3-Phenoxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000242_0002
HPLC-MS (Method D): m/z: 344 (M+1); Rt = 5,07 min.
Example 682 (General procedure (H)) (4-Phenoxybenzyl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine
Figure imgf000242_0003
HPLC-MS (Method D): m/z: 344 (M+1); Rt = 5,03 min.
Example 683 (General procedure (H)) 3-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid
Figure imgf000242_0004
HPLC-MS (Method D): m/z: 286 (M+1); Rt = 3,47 min.
Example 684 (General procedure (H)) (4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenoxy)acetic acid
Figure imgf000243_0001
HPLC-MS (Method D): m/z: 326 (M+1); Rt = 3,40 min.
Examp Ie 685 (Ge sneral procedure (H))
(4-Benzyloxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000243_0002
HPLC-MS (Method D): m/z: 358 (M+1); Rt = 5,14 min.
Example 686 (General procedure (H)) 3-(4-{|4-(2H-Tetrazol-5-yl)phenylamino3methyl}phenyl)acrylic acid
Figure imgf000243_0003
HPLC-MS (Method D): m/z: 322 (M+1); Rt = 3,66 min.
Example 687 (General procedure (H)) Dimethyl-(4-{[4-(2H-tetrazol-5-yl)phenylamino]methyl}naphthalen-1-yl)amine
Figure imgf000243_0004
HPLC-MS (Method D): m/z: 345 (M+1); Rt = 3,10 min.
Example 688 (General procedure (H)) (4'-Methoxybiphenyl-4-ylmethyl)-[4-(2H-tetrazol-5-yl)phenyl]amine
Figure imgf000243_0005
HPLC-MS (Method D): m/z: 358 (M+1); Rt = 5,04 min. Example 689 (General procedure (H)) (2'-Chlorobiphenyl-4-ylmethyl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine
Figure imgf000244_0001
HPLC-MS (Method D): m/z: 362 (M+1); Rt = 5,30 min.
General procedure (I) for preparation of compounds of general formula :
HOAt
Figure imgf000244_0002
wherein K, M and T are as defined above.
This procedure is very similar to general procedure (A), the only difference being the carboxylic acid is containing a tetrazole moiety. When the acylation is complete, the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art.
The general procedure (I) is further illustrated in the following example 690:
Example 690 (General procedure (I)) 4-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic acid
Figure imgf000244_0003
To a solution of 4-(2H-tetrazol-5-yl)benzoic acid (example 605, 4 mmol) and HOAt (4.2 mmol) in DMF (6 mL) was added 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochlo- ride (4.2 mmol) and the resulting mixture was stirred at room temperature for 1 hour. An alquot of this HOAt-ester solution (0.45 mL) was mixed with 0.25 mL of a solution of 4- aminobenzoic acid (1.2 mmol in 1 mL DMF). (Anilines as hydrochlorides can also be utilised, a slight excess of triethylamine was added to the hydrochloride suspension in DMF prior to mixing with the HOAt-ester.) The resulting mixture was shaken for 3 days at room temperature. 1N hydrochloric acid (2 mL) was added and the mixture was shaken for 16 hours at room temperature. The solid was isolated by centrifugation (alternatively by filtration or extraction) and was washed with water (3 mL). Drying in vacuo at 40 °C for 2 days afforded the title compound.
HPLC-MS (Method D): m/z: 310 (M+1); Rt = 2.83 min.
Example 691 (General procedure (I)) 3-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic acid
Figure imgf000245_0001
HPLC-MS (Method D): m/z: 310 (M+1); Rt = 2.89 min.
Example 692 (General procedure (I)) 3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}acrylic acid
Figure imgf000245_0002
HPLC-MS (Method D): m/z: 336 (M+1); Rt = 3.10 min.
Example 693 (General procedure (l))
3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}propionic acid
Figure imgf000245_0003
HPLC-MS (Method D): m/z: 338 (M+1); Rt = 2.97 min.
Example 694 (General procedure (I)) 3-Methoxy-4-[4-(2H-tetrazol-5-yl)benzoyJamino]benzoic acid
Figure imgf000246_0001
HPLC-MS (Method D): m/z: 340 (M+1); Rt = 3.03 min.
Example 695 (General procedure (I)) W-(4-Ben2yloxyphenyl)-4-(2H-tetrazol-5-yl)ben∑amide
Figure imgf000246_0002
HPLC-MS (Method D): m/z: 372 (M+1); Rt = 4.47 min.
Example 696 (General procedure (I)) Λ/-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-yl)benzamide
Figure imgf000246_0003
HPLC-MS (Method D): m/z: 358 (M+1); Rt = 4.50 min.
Example 697 (Genera) procedure (])) Λ/-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-yl)benzamide
Figure imgf000246_0004
HPLC-MS (Method D): m/z: 354 (M+1); Rt = 4.60 min.
Example 698 (General procedure (I)) W-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-yl)benzamids
Figure imgf000247_0001
HPLC-MS (Method D): m/z: 383 (M+1); Rt = 4.60 min.
Example 699 (General procedure (I)) W-Phenyl-4-(2H-tetra∑ol-5-yl)benzamidβ
Figure imgf000247_0002
HPLC-MS (Method D): m/z: 266 (M+1); Rt = 3.23 min.
Example 700 (General procedure (I)) 4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic acid
Figure imgf000247_0003
The starting material was prepared as described in example 592. HPLC-MS (Method D): m/z: 340 (M+1); Rt = 2.83 min.
Example 701 (General procedure (l)) 3-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic acid
Figure imgf000247_0004
HPLC-MS (Method D): m/z: 340 (M+1); Rt = 2.90 min.
Example 702 (General procedure (I)) 3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyI}acrylic acid
Figure imgf000248_0001
HPLC-MS (Method D): m/z: 366 (M+1); Rt = 3.07 min.
Example 703 (General procedure (I)) 3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}propionic acid
Figure imgf000248_0002
HPLC-MS (Method D): m/z: 368 (M+1); Rt = 2.97 min.
Example 704 (General procedure (I)) 3-Methoxy-4-[4-(2H-tetrazol-5-ylmethoxy)benzoylamino]benzoic acid
Figure imgf000248_0003
HPLC-MS (Method D): m/z: 370 (M+1); Rt = 3.07 min.
Example 705 (General procedure (I)) Λ/-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)benzamide
Figure imgf000248_0004
HPLC-MS (Method D): m/z: 402 (M+1); Rt = 4.43 min.
Example 706 (General procedure (I)) M-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)ben∑amide
Figure imgf000248_0005
HPLC-MS (Method D): m/z: 388 (M+1); Rt = 4.50 min.
Example 707 (General procedure (I)) Λ/-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-ylmβthoxy)ben∑amide
Figure imgf000249_0001
HPLC-MS (Method D): m/z: 384 (M+1); Rt = 4.57 min.
Example 708 (General procedure (I)) Λ-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide
Figure imgf000249_0002
HPLC-MS (Method D): m/z: 413 (M+1); Rt = 4.57 min.
Example 709 (General procedure (I)) Λ-Phenyl-4-(2H-tetrazol-5-ylmethoxy)benzamide
Figure imgf000249_0003
HPLC-MS (Method D): m/z: 296 (M+1); Rt = 3.23 min.
Example 710 (General procedure (I))
4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid
Figure imgf000249_0004
The starting material was prepared as described in example 593. HPLC-MS (Method D): m/z: 356 (M+1); Rt = 2.93 min. Example 711 (General procedure (I)) 3-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid
Figure imgf000250_0001
HPLC-MS (Method D): m/z: 356 (M+1); Rt = 3.00 min.
Example 712 (General procedure (I))
3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}acrylic acid
Figure imgf000250_0002
HPLC-MS (Method D): m/z: 382 (M+1); Rt = 3.26 min.
Example 713 (General procedure (I)) 3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}propionic acid
Figure imgf000250_0003
HPLC-MS (Method D): m/z: 384 (M+1); Rt = 3.10 min.
Example 714 (General procedure (I))
3-Methoxy-4-[4-(2H-tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid
Figure imgf000250_0004
HPLC-MS (Method D): m/z: 386 (M+1); Rt = 3.20 min.
Example 715 (General procedure (I)) A-(4-Benzyloxyphenyl)-4-(2H-tetra2ol-5-ylmethylsulfanyl)ben∑amide
Figure imgf000251_0001
HPLC-MS (Method D): m/∑: 418 (M+1); Rt = 4.57 min.
Example 716 (General procedure (I)) N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-ylmethyIsulfanyl)benzamide
Figure imgf000251_0002
HPLC-MS (Method D): m/z: 404 (M+1); Rt = 4.60 min.
Example 717 (General procedure (I)) Λ/-(9r7'-Fluoren-2-yl)-4-(2H-tetrazo/-5-ylmethylsulfanyl)benzamide
Figure imgf000251_0003
HPLC-MS (Method D): m/z: 400 (M+1); Rt = 4.67 min.
Example 718 (General procedure (I)) Λ/-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide
Figure imgf000251_0004
HPLC-MS (Method D): m/z: 429 (M+1); Rt = 4.67 min.
Example 719 (General procedure ({)) A-Phenyl-4-(2H-tetra2ol-5-ylmethylsulfanyl)benzamide
Figure imgf000251_0005
HPLC-MS (Method D): m/z: 312 (M+1); Rt = 3.40 min.
©©neral procedure (J) for solution phase preparation of amides of general formula l9:
Figure imgf000252_0001
wherein T is as defined above.
This general procedure (J) is further illustrated in the following example.
Example 720 (General procedure (J)). 9-(3-Chlorobenzyi)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000252_0002
3-(2H-TetrazoI-5-yl)-9H-carbazole (example 594, 17 g, 72.26 mmol) was dissolved in N,N- dimethylformamide (150 mL). Triphenylmethyl chloride (21.153 g, 75.88 mmol) and triethylamine (20.14 mL, 14.62 g, 144.50 mmol) were added consecutively. The reaction mixture was stirred for 18 hours at room temperature, poured into water (1.5 L) and stirred for an additional 1 hour. The crude product was filtered off and dissolved in dichloromethane (500 mL). The organic phase was washed with water (2 x 250 mL) and dried with magnesium sulfate (1 h). Filtration followed by concentration yielded a solid which was triturated in heptanes (200 mL). Filtration furnished 3-[2-(triphenylmethyl)-2H-tetrazoI-5-yl]-9H-carbazole (31.5 g) which was used without further purification.
1H-NMR (CDCI3): 8.87 (1H, d), 8.28 (1H, bs), 8.22 (1H, dd), 8.13 (1H, d), 7.49 (1H, d), 7.47- 7.19 (18H, m); HPLC-MS (Method C): m/∑: 243 (triphenylmethyl); Rt = 5.72 min. 3-[2-(Triphenylmethyl)-2H-tetrazol-5-yI]-9H-carbazole (200 mg, 0.42 mmol) was dissolved in methyl sulfoxide (1.5 L). Sodium hydride (34 mg, 60 %, 0.85 mmol) was added, and the resulting suspension was stirred for 30 min at room temperature. 3-Chlorobenzyl chloride (85 μL, 108 mg, 0.67 mmol) was added, and the stirring was continued at 40 °C for 18 hours. The reaction mixture was cooled to ambient temperature and poured into 0.1 N hydrochloric acid (aq.) (15 mL). The precipitated solid was filtered off and washed with water (3 x 10 mL) to furnish 9-(3-chlorobenzyl)-3-[2-(triphenylmethyl)-2H-tetrazol-5-yl]-9H-carbazole, which was dissolved in a mixture of tetrahydrofuran and 6 N hydrochloric acid (aq.) (9:1) (10 mL) and stirred at room temperature for 18 hours. The reaction mixture was poured into water (100 mL). The solid was filtered off and rinsed with water (3 x 10 mL) and dichloromethane (3 x 10 mL) to yield the title compound (127 mg). No further purification was necessary. 1H-NMR (DMSO-d6): δ8.89 (1H, d), 8.29 (1H, d), 8.12 (1H, dd), 7.90 (1H, d), 7.72 (1H, d), 7.53 (1 H, t), 7.36-7.27 (4H, m), 7.08 (1 H, bt), 5.78 (2H, s); HPLC-MS (Method B): m/z: 360 (M+1); Rt = 5.07 min.
The compounds in the following examples were prepared in a similar fashion. Optionally, the compounds can be further purified by recrystallization from e.g. aqueous sodium hydroxide (1 N) or by chromatography.
Example 721 (General Procedure (J)). 9-(4-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000253_0001
HPLC-MS (Method C): m/z: 360 (M+1); Rt = 4.31 min.
Example 722 (General Procedure (J)). 9-(4-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000253_0002
HPLC-MS (Method C): m/z: 340 (M+1); Rt = 4.26 min.
Example 723 (General Procedure (J)). 3-(2H-Tetrazol-5-yl)-9-(4-trifluoromethylben∑ -9H-carba∑ole
Figure imgf000254_0001
HPLC-MS (Method C): m/z: 394 (M+1); Rt = 4.40 min.
Example 724 (General Procedure (J)). 9-(4-Benzyloxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000254_0002
HPLC-MS (Method C): m/z: 432 (M+1); Rt = 4.70 min.
Example 725 (General Procedure (J)). 9-(3-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazoIe
Figure imgf000254_0003
HPLC-MS (Method C): m/z: 340 (M+1); Rt = 4.25 min.
Example 726 (General Procedure (J)). 9-Benzyl-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000255_0001
1H-NMR (DMSO-d6): £8.91 (1H, dd), 8.30 (1H, d), 8.13 (1H, dd), 7.90 (1H, d), 7.73 (1H, d), 7.53 (1H, t), 7.36-7.20 (6H, m), 5.77 (2H, s).
Example 727 (General Procedure (J)). 9-(4-Phenylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carba∑ole
Figure imgf000255_0002
1H-NMR (DMSO-de): £8.94 (1 H, s), 8.33 (1 H, d), 8.17 (1 H, dd), 7.95 (1 H, d), 7.77 (1 H, d), 7.61-7.27 (11H, m), 5.82 (2H, s).
Example 728 (General Procedure (J)). 9-(3-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000255_0003
HPLC-MS (Method C): m/z: 356 (M+1); Rt = 3.99 min.
Example 729 (General Procedure (J)). 9-(Naphthalen-2-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazoIe
Figure imgf000256_0001
HPLC-MS (Method C): m/z: 376 (M+1); Rt = 4.48 min.
Example 730 (General Procedure (J)). 9-(3-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000256_0002
HPLC-MS (Method C): m/z: 404 (M+1); Rt = 4.33 min.
Example 731 (General Procedure (J)). 9-(Biphenyl-2-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000256_0003
HPLC-MS (Method C): m/z: 402 (M+1); Rt = 4.80 min.
Example 732 (General Procedure (J)). 3-(2H-Tetrazol-5-yl)-9-[4-(1,2,3-thiadiazol-4-yl)benzyl]-9H-carbazole
Figure imgf000257_0001
Example 733 (General Procedure (J)). 9-(2'-Cyanobiphenyl-4-ylmethyl)-3-(2H-tetrazol-5- -9H-carba∑ole
Figure imgf000257_0002
1H-NMR (DMSO-de): £8.91 (1H, d), 8.31 (1H, d), 8.13 (1H, dd), 7.95 (1H, d), 7.92 (1H, d), 7.78 (1H, d), 7.75 (1H, dt), 7.60-7.47 (5H, m), 7.38-7.28 (3H, m), 5.86 (2H, s); HPLC-MS (Method C): m/z: 427 (M+1); Rt = 4.38 min.
Example 734 (General Procedure (J)). 9-(4-lσdobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000257_0003
HPLC-MS (Method C): m/z: 452 (M+1); Rt = 4.37 min.
Example 735 (General Procedure (J)). 9-(3,5-Bis(trifluoromethyl)benzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000258_0001
HPLC-MS (Method C): m/z: 462 (M+1); Rt = 4.70 min.
Example 736 (General Procedure (J)). 9-(4-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000258_0002
1H-NMR (DMSO-de): £8.89 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.88 (1H, d), 7.70 (1H, d), 7.52 (1H, t), 7.49 (2H, d), 7.31 (1H, t), 7.14 (2H, d), 5.74 (2H, s); HPLC-MS (Method C): m/z: 404 (M+1); Rt = 4.40 min.
Example 737 (General Procedure (J)). 9-(Anthracen-9-yfmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000258_0003
HPLC-MS (Method C): m/z: 426 (M+1); Rt = 4.78 min.
Example 738 (General Procedure (J)). 9-(4-Garboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000259_0001
3.6 fold excess sodium hydride was used.
1H-NMR (DMSO-de): £12.89 (1H, bs), 8.89 (1H, d), 8.30 (1H, d), 8.10 (1H, dd), 7.87 (1H, d), 7.86 (2H, d), 7.68 (1H, d), 7.51 (1H, t), 7.32 (1H, t), 7.27 (2H, d), 5.84 (2H, s); HPLC-MS (Method C): m/z: 370 (M+1); Rt = 3.37 min.
Example 739 (General Procedure (J)). 9-(2-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000259_0002
HPLC-MS (Method B): m/z: 360 (M+1); Rt = 5.30 min.
Example 740 (General Procedure (J)). 9-(4-Fluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000259_0003
1H-NMR (DMSO-d6): £8.88 (1H, d), 8.28 (1H, d), 8.10 (1H, dd), 7.89 (1H, d), 7.72 (1H, d), 7.52 (1H, t), 7.31 (1H, t), 7.31-7.08 (4H, m), 5.74 (2H, s); HPLC-MS (Method C): m/z: 344 (M+1); Rt = 4.10 min.
Example 741 (General Procedure (J)). 9-(3-Fluoroben∑yl)-3-(2H-tetra∑ol-5-yl)-9H-carbazole
Figure imgf000260_0001
1 H-NMR (DMSO-de): £8.89 (1 H, d), 8.29 (1H, d), 8. (1H, dd), 7.90 (1H, d), 7.72 (1 H, d), 7.53 (1H, t), 7.37-7.27 (2H, m), 7.12-7.02 (2H, m), € 7 (1 H, d), 5.78 (2H, s); HPLC-MS (Method C): m/z: 344 (M+1); Rt = 4.10 min.
Example 742 (General Procedure (J)). 9-(2-lodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000260_0002
HPLC-MS (Method C): m/z: 452 (M+1); Rt = 4.58 min.
Example 743 (General Procedure (J)). 9-(3-Carboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000260_0003
3.6 fold excess sodium hydride was used.
1H-NMR (DMSO-de): £ 12.97 (1 H, bs), 8.90 (1 H, bs), 8.30 (1 H, d), 8.12 (1H, bd), 7.89 (1H, d), 7.82 (1 H, m), 7.77 (1H, bs), 7.71 (1 H, d), 7.53 (1H, t), 7.46-7.41 (2H, m), 7.32 (1 H, t), 5.84 (2H, s); HPLC-MS (Method C): m/z: 370 (M+1); Rt = 3.35 min. Example 744 (General Procedure (J)). 9-[4-(2-Propyl)benzyl]-3-(2H-tetrazol-5-yl)-9H-carbazoIe
Figure imgf000261_0001
1H-NMR (DMSO-de): £8.87 (1H, d), 8.27 (1H, d), 8.10 (1H, dd), 7.87 (1H, d), 7.71 (1H, d), 7.51 (1H, t), 7.31 (1H, t), 7.15 (2H, d), 7.12 (2H, d), 5.69 (2H, s), 2.80 (1H, sept), 1.12 (6H, d); HPLC-MS (Method C): m/z: 368 (M+1); Rt = 4.73 min.
Example 745 (General Procedure (J)). 9-(3,5-Dimethoxybenzy|)-3-(2H-tetrazo!-5-yl)-9H-carbazole
Figure imgf000261_0002
HPLC-MS (Method C); m/z: 386 (M+1); Rt = 4.03 min.
Example 746 (General Procedure (J)). 3-(2H-Tetrazol-5-yl)-9-(2,4,5-trifluorobenzyl)-9H-carbazole
Figure imgf000261_0003
HPLC-MS (Method B): m/z: 380 (M+1); Rt = 5.00 min.
Example 747 (General Procedure (J)). /V-Methyl-W-phenyI-2-[3-(2H-tetra2ol-5-yl)carbazol-9-yl]acetamide
Figure imgf000262_0001
HPLC-MS (Method B): m/z: 383 (M+1); Rt = 4.30 min.
Example 748 (General Procedure (J)). 9-(4-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000262_0002
1H-NMR (DMSO-de): £8.86 (1 H, d), 8.26 (1H, d), 8.10 (1 H, dd), 7.90 (1H, d), 7.73 (1 H, d), 7.51 (1 H, t), 7.30 (1 H, t), 7.18 (2H, d), 6.84 (2H, d), 5.66 (2H, s), 3.67 (3H, s); HPLC-MS (Method B): m/z: 356 (M+1 ); Rt = 4.73 min.
Example 749 (General Procedure (J)). 9-(2-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000262_0003
1H-NMR (DMSO-de): £8.87 (1 H, d), 8.27 (1 H, d), 8.09 (1 H, dd), 7.77 (1 H, d), 7.60 (1 H, d), 7.49 (1 H, t), 7.29 (1 H, t), 7.23 (1 H, bt), 7.07 (1 H, bd), 6.74 (1 H, bt), 6.61 (1 H, bd), 5.65 (2H, s), 3.88 (3H, s); HPLC-MS (Method B): m/z: 356 (M+1); Rt = 4.97 min.
Example 750 (General Procedure (J)). 9-(4-Cyanoben∑yl)-3-(2H-tetra∑ol-5-yl)-9H-carba∑ole
Figure imgf000263_0001
HPLC-MS (Method C): m/z: 351 (M+1); Rt = 3.. mm.
Example 751 (General Procedure (J)). 9-(3-Cyanobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000263_0002
HPLC-MS (Method C): m/z: 351 (M+1); Rt = 3.73 min.
Example 752 (General Procedure (J)). 9-(5-Chloro-2-methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000263_0003
1H-NMR (DMSO-de): £8.87 (1H, d), 8.35 (1H, d), 8.10 (1H, dd), 7.73 (1H, d), 7.59 (1H, d), 7.49 (1H, t), 7.29 (1H, t), 7.27 (1H, dd), 7.11 (1H, d), 6.51 (1H, d), 5.63 (2H, s), 3.88 (3H, s); HPLC-MS (Method C): m/z: 390 (M+1); Rt = 4.37 min.
Example 753 (General Procedure (J)). W-Phenyl-2-[3-(2H-tetrazol-5-yl)carba∑ol-9-yl]acetamide
Figure imgf000264_0001
1H-NMR (DMSO-de): £ 10.54 (1H, s), 8.87 (1H, bs), 8.27 (1H, d), 8.12 (1H, bd), 7.83 (1 H, d), 7.66 (1H, d), 7.61 (2H, d), 7.53 (1H,t), 7.32 (1H, t), 7.32 (2H, t), 7.07 (1H, t), 5.36 (2H, s); HPLC-MS (Method C): m/∑: 369 (M+1 ); Rt = 3.44 min.
Example 754 (General Procedure (J)). /V-Butyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide
Figure imgf000264_0002
1H-NMR (DMSO-de): £8.85 (1 H, d), 8.31 (1 H, t), 8.25 (1 H, d), 8.10 (1 H, dd), 7.75 (1H, d), 7.58 (1H, d), 7.52 (1H, t), 7.30 (1H, t), 5.09 (2H, s), 3.11 (2H, q), 1.42 (2H, quint), 1.30 (2H, sext), 0.87 (3H, t); HPLC-MS (Method C): m/z: 349 (M+1); Rt = 3.20 min.
Example 755 (General Procedure (J)). 9-(2,4-Dichlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000264_0003
1H-NMR (DMSO-de): £8.92 (1H, d), 8.32 (1H, d), 8.09 (1H, dd), 7.76 (1 H, d), 7.74 (1H, d), 7.58 (1H, d), 7.51 (1H, t), 7.33 (1H, t), 7.23 (1H, dd), 6.42 (1H, d), 5.80 (2H, s); HPLC-MS (Method B): m/z: 394 (M+1); Rt = 5.87 min. Example 756 (General Procedure (J)). 9-(2-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000265_0001
1H-NMR (DMSO-de): £8.92 (1 H, d), 8.32 (1 H, d), 8.08 (1 H, dd), 7.72 (1 H, d), 7.55 (1 H, d), 7.48 (1 H, t), 7.32 (1 H, t), 7.26 (1 H, d), 7.12 (1 H, t), 6.92 (1 H, t), 6.17 (1H, d), 5.73 (2H, s), 2.46 (3H, s); HPLC-MS (Method B): m/z: 340 (M+1); Rt = 5.30 min.
Example 757 (General Procedure (J)). 9-(3-Nitrobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000265_0002
HPLC-MS (Method C): m/z: 371 (M+1); Rt = 3.78 min.
Example 758 (General Procedure (J)). 9-(3,4-Dichlorobenzyl)-3-(2H-tetrazo)-5-yl)-9H-carbazole
Figure imgf000265_0003
HPLC-MS (Method B): m/z: 394 (M+1); Rt = 5.62 min.
Example 759 (General Procedure (J)). 9-(2,4-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazoIe
Figure imgf000266_0001
H-NMR (DMSO-de): £8.89 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.88 (1H, d), 7.69 (1H, d), 7.52 (1 H, t), 7.36-7.24 (2H, m), 7.06-6.91 (2H, m), 5.78 (2H, s); HPLC-MS (Method B): m/z: 362 (M+1 ); Rt = 5.17 min.
Example 760 (General Procedure (J)). 9-(3,5-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carba∑ole
Figure imgf000266_0002
1H-NMR (DMSO-de): £8.90 (1 H, bs), 8.31 (1 H, d), 8.13 (1H, bd), 7.90 (1 H, d), 7.73 (1H, d), 7.54 (1 H, t), 7.34 (1 H, t), 7.14 (1 H, t), 6.87 (2H, bd), 5.80 (2H, s); HPLC-MS (Method B): m/z: 362 (M+1); Rt = 5.17 min.
Example 761 (General Procedure (J)). 9-(3,4-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000266_0003
1H-NMR (DMSO-de): £8.89 (1H, bs), 8.29 (1H, d), 8.12 (1H, bd), 7.92 (1H, d), 7.74 (1H, d), 7.54 (1H, t), 7.42-7.25 (3H, m), 6.97 (1H, bm), 5.75 (2H, s); HPLC-MS (Method B): m/z: 362 (M+1); Rt = 5.17 min.
Example 762 (General Procedure (J)). 9-(3-lodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000267_0001
HPLC-MS (Method B): m/z: 452 (M+1); Rt = 5.50 min.
Example 763 (General Procedure (J)). 3-(2H-Tetrazol-5-yl)-9-[3-(trifluoromethyl)benzyl]-9H-carbazole
Figure imgf000267_0002
Η-NMR (DMSO-dβ): £8.89 (1H, d), 8.30 (1H, d), 8.11 (1H, dd), 7.90 (1H, d), 7.72 (1H, d), 7.67 (1H, bs), 7.62 (1H, bd), 7.53 (1H, t), 7.50 (1H, bt), 7.33 (1H, bd), 7.32 (1H, t), 5.87 (2H, s); HPLC-MS (Method B): m/z: 394 (M+1); Rt = 5.40 min.
Example 764 (General Procedure (J)). ιV-(4-Carboxyphenyl)-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide
Figure imgf000267_0003
3.6 fold excess sodium hydride was used.
HPLC-MS (Method B): m/z: 413 (M+1); Rt = 3.92 min.
Example 765 (General Procedure (J)). N-(2-Propyl)-2-[3-(2H-tetrazol-5-yJ)carbazol-9-yl]acetamide
Figure imgf000268_0001
HPLC-MS (Method B): m/z: 335 (M+1); Rt = 3.70 min.
Example 766 (General Procedure (J)). /V-Benzyl-/V-phenyI-2-[3-(2H-tetra2ol-5-yl)carbazol-9-yl]acetamide
Figure imgf000268_0002
HPLC-MS (Method B): m/z: 459 (M+1 ); Rt = 5.37 min.
Example 767 (General Procedure (J)). Λ/-[4-(2-Methyl-2-propyl)phenyl]-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide
Figure imgf000268_0003
HPLC-MS (Method B): m/z: 425 (M+1); Rt = 5.35 min.
Example 768 (General Procedure (J)). /V-Phenethyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide
Figure imgf000269_0001
HPLC-MS (Method C): m/z: 397 (M+1); Rt = 3.43 min.
Example 769 (General Procedure (J)). 3-(2H-Tetrazol-5-yl)-9-[2-(trifluoromethyl)bβn∑yl]-9H-carba2θle
Figure imgf000269_0002
HPLC-MS (Method C): m/z: 394 (M+1); Rt = 4.44 min.
Example 770 (General Procedure (J)). 9-[2-Fluoro-6-(trifluoromethyl)ben2yl]-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000269_0003
HPLC-MS (Method C): m/z: 412 (M+1); Rt = 4.21 min.
Example 771 (General Procedure (J)). 9-[2,4-Bis(trifluoromethyl)benzyl)]-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000270_0001
HPLC-MS (Method C): m/z: 462 (M+1); Rf = 4.82 min.
Example 772 (General Procedure (J)). 3-(2H-Tetrazol-5-yl)-9-(2,4,6-trimethylbenzyl)-9H-carba∑ole
Figure imgf000270_0002
HPLC-MS (Method C): m/z: 368 (M+1); Rt = 4.59 min.
Example 773 (General Procedure (J)). 9-(2,3,5,6-TetramethylbenzyI)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000270_0003
HPLC-MS (Method C): m/z: 382 (M+1); Rt = 4.47 min.
Example 774 (General Procedure (J)).
9-[(Naph thalen- 1 -yl)methyl]-3-(2H-tetrazol-5-yl)-9H-carha∑ole
Figure imgf000271_0001
HPLC-MS (Method C): m/z: 376 (M+1); Rt = 4.43 min.
Example 775 (General Procedure (J)). 9-[Bis(4-fluorophenyl)methyl]-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000271_0002
HPLC-MS (Method C): m/z: 438 (M+1); Rt = 4.60 min.
Example 776 (General Procedure (J)). 9-(2-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000271_0003
HPLC-MS (Method C): m/z: 404 (M+1); Rt = 4.50 min.
Example 777 (General Procedure (J)). 9-(2-Fluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000272_0001
HPLC-MS (Method C): m/z: 344 (M+1); Rt = 4.09 min.
Example 778 (General Procedure (J)). 9-(4-Carboxy-2-methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000272_0002
In this preparation, a 3.6-fold excess of sodium hydride was used. HPLC-MS (Method C): m/z: 384 (M+1); Rt = 3.56 min.
Example 779 (General Procedure (J)). 9-(2-Phenylethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000272_0003
HPLC-MS (Method C): m/z: 340 (M+1); Rt = 4.08 min.
Example 780 (General Procedure (J)). 9-[2-Fluoro-5-(trifluoromethyl)ben2yl]-3-(2H-tetrazol-5-yl)-9H-carbazolβ
Figure imgf000273_0001
HPLC-MS (Method C): m/z: 412 (M+1); Rt = 4.34 min.
Example 781 (General Procedure 9-(4-Carboxy-2-fluorobenzyl)-3-(2H-tetra∑ol-5-yl)-9H-carba∑ole
Figure imgf000273_0002
3-Fluoro-4-methylbenzoic acid (3.0 g, 19.5 mmol) and benzoyl peroxide (0.18 g, 0.74 mmol) were suspended in benzene. The mixture was purged with N2 and heated to reflux. N- Bromosuccinimide (3.47 g, 19.5 mmol) was added portionwise, and reflux was maintained for 18 hours. The reaction mixture was concentrated, and the residue was washed with water (20 mL) at 70 °C for 1 hour. The crude product was isolated by filtration and washed with additional water (2 x 10 mL). The dry product was recrystallized from heptanes. Filtration furnished 4-bromomethyl-3-fluorobenzoic acid (1.92 g) which was used in the following step according to General Procedure (J).
In this preparation, a 3.6-fold excess of sodium hydride was used. HPLC-MS (Method C): m/z: 388 (M+1); Rt = 3.49 min.
Example 782 (General Procedure (J)). 5-{4-[[(3-(2H-Tetrazol-5-yl)carbazol-9-yl)methyl]naphthalen-1-yl]oxy}pentanoic Acid
Figure imgf000273_0003
5-[(4-Formylnaphthalen-1-yl)oxy]pentanoic acid intermediate obtained in example 470(3.0 g, 11.0 mmol) was dissolved in a mixture of methanol and tetrahydrofuran (9:1) (100 mL), and sodium borohydride (1.67 g, 44.1 mmol) was added portionwise at ambient temperature. After 30 minutes, the reaction mixture was concentrated to 50 mL and added to hydrochloric acid (0.1 N, 500 mL). Additional hydrochloric acid (1 N, 40 mL) was added, and 5-[(4- hydroxymethyl-naphthalen-1-yl)oxy]pentanoic acid (2.90 g) was collected by filtration. To the crude product was added concentrated hydrochloric acid (100 mL), and the suspension was stirred vigorously for 48 hours at room temperature. The crude product was filtered off and washed with water, until the pH was essentially neutral. The material was washed with heptanes to furnish 5-[(4-chloromethylnaphthalen-1-yl)oxy]pentanoic acid (3.0 g) which was usec in the following step according to General Procedure (J).
In this preparation, a 3.6-fold excess of sodium hydride was used. HPLC-MS (Method C): /z: 492 (M+1); Rt = 4.27 min.
Example 783 (General procedure (J)) 9-(2,3-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000274_0001
HPLC-MS (Method C): m/z= 362 (M+1); Rt = 4.13 min.
Example 784 (General procedure (J)) 9-(2,5-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000274_0002
HPLC-MS (Method C): m/z = 362 (M+1); Rt = 4.08 min. Example 785 (General procedure (J)) 9-Pentafluorophenylmethyl-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000275_0001
HPLC-MS (Method C): m/z = 416 (M+1); Rt = 4.32 min.
Example 786 (General procedure (J)) 9-(2,6-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole
Figure imgf000275_0002
HPLC-MS (Method C): m/z = 362 (M+1); Rt = 3.77 min.
Further compounds of the invention that may be prepared according to general procedure (J), and includes:
Example 787 Example 788 Example 789
Figure imgf000276_0001
Example 790 Example 791 Example 792
Figure imgf000276_0002
Example 793 Example 794 Example 795
Figure imgf000276_0003
Example 796 Example 797 Example 798
Figure imgf000276_0004
Example 799
Figure imgf000276_0005
The following compounds of the invention may be prepared eg. from 9-(4-bromobenzyl)-3- (2H-tetrazol-5-yl)-9H-carbazole (example 736) or from 9-(3-bromobenzyl)-3-(2H-tetrazol-5- yl)-9H-carbazole (example 730) and aryl boronic acids via the Suzuki coupling reaction eg as described in Littke, Dai & Fu J. Am. Chem. Soc, 2000, 122, 4020-8 (or references cited therein), or using the methodology described in general procedure (E), optionally changing the palladium catalyst to bis(tri-ferf-butylphosphine)palladium (0).
Figure imgf000277_0001
General procedure (K) for preparation of compounds of general formula l10:
Figure imgf000277_0002
wherein T is as defined above.
The general procedure (K) is further illustrated by the following example: Example 806 (General procedure (K)). 1 -Benzyl-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000277_0003
5-Cyanoindole (1.0 g, 7.0 mmol) was dissolved in A , V-dimethylformamide (14 mL) and cooled in an ice-water bath. Sodium hydride (0.31 g, 60 %, 7.8 mmol) was added, and the resulting suspension was stirred for 30 min. Benzyl chloride (0.85 L, 0.94 g, 7.4 mmol) was added, and the cooling was discontinued. The stirring was continued for 65 hours at room temperature. Water (150 mL) was added, and the mixture was extracted with ethyl acetate (3 x 25 mL). The combined organic phases were washed with brine (30 mL) and dried with sodium sulfate (1 hour). Filtration and concentration yielded the crude material. Purification by flash chromatography on silica gel eluting with ethyl acetate/heptanes = 1 :3 afforded 1.60 g 1 -benzyl-1 H-indole-5-carbonitrile.
HPLC-MS (Method C): m/z: 233 (M+1); Rt = 4.17 min.
1-BenzyI-1H-indole-5-carbonitrile was transformed into 1-benzyl-5-(2H-tetrazol-5-yl)-1H- indole by the method described in general procedure (J) and in example 594. Purification was done by flash chromatography on silica gel eluting with dichloromethane/methanol = 9:1.
HPLC-MS (Method C): m/z: 276 (M+1); Rt = 3.35 min.
The compounds in the following examples were prepared by the same procedure.
Example 807 (General procedure (K)).
1 -(4-Bromobenzyl)-5-(2H-tetrazoI-5-yl)-1 H-indole
Figure imgf000278_0001
HPLC-MS (Method C): m/z: 354 (M+1); Rt = 3.80 min.
Example 808 (General procedure (K)).
1 -(4-Phenylbenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000278_0002
Η-NMR (200 MHz, DMSO-d6): δ = 5.52 (2H, s), 6.70 (1H, d), 7.3-7.45 (6H, m), 7.6 (4H, m), 7.7-7.8 (2H, m), 7.85(1 H, dd), 8.35 (1H, d). Calculated for C22H17N5, H20: 73.32% C; 5.03% H; 19.43% N. Found: 73.81% C; 4.90% H; 19.31% N.
Example 809 4'-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carboxylic acid
Figure imgf000279_0001
5-(2H-Tetrazol-5-yl)-1 H-indole (Syncom BV, Groningen, NL) (1.66g, 8.9 mmol) was treated with trityl chloride (2.5 g, 8.9 mmol) and triethyl amine (2.5 mL, 17.9 mmol) in DMF(25 mL) by stirring at RT overnight. The resulting mixture was treated with water. The gel was isolated, dissolved in methanol, treated with activated carbon; filtered and evaporated to dryness in vacuo. This afforded 3.6 g (94%) of crude 5-(2-trityl-2H-tetrazol-5-yl)-1 H-indole.
HPLC-MS (Method C): m/z = 450 (M+23); Rt. = 5.32 min.
4-Methylphenylbenzoic acid (5 g, 23.5 mmol) was mixed with CCI4 (100 mL) and under an atmosphere of nitrogen, the slurry was added Λ/-Bromosuccinimide (4.19 g, 23.55 mmol) and dibenzoyl peroxide (0.228 g, 0.94 mmol). The mixture was subsequently heated to reflux for 0.5 hour. After cooling, DCM and water (each 30 mL) were added. The resulting precipitate was isolated, washed with water and a small amount of methanol. The solid was dried in vacuo to afford 5.27 g (77%) of 4'-bromomethylbiphenyl-4-carboxylic acid.
HPLC-MS (Method C): m/z = 291 (M+1); Rt. = 3.96 min.
5-(2-Trity|-2H-tetrazol-5-yl)-1 H-indole (3.6 g, 8.4 mmol) was dissolved in DMF (100 mL). Under nitrogen, NaH (60 % suspension in mineral oil, 34 mmol) was added slowly. 4'- Bromomethylbiphenyl-4-carboxylic acid (2.7 g, 9.2 mmol) was added over 5 minutes and the resulting slurry was heated at 40 °C for 16 hours. The mixture was poured into water (100mL) and the precipitate was isolated by filtration and treated with THF/6N HCI (9/1) (70 mL) at room temperature for 16 hours. The mixture was subsequently evaporated to dryness in vacuo, the residue was treated with water and the solid was isolated by filtration and washed thoroughly 3 times with DCM. The solid was dissolved in hot THF (400 mL) treated with activated carbon and filtered. The filtrate was evaporated in vacuo to dryness. This afforded 1.6 g (50%o) of the title compound.
HPLC-MS (Method C): m/∑ = 396 (M+1); Rt. = 3.51 min.
Example 810 (General procedure (K)). 5-(2H-Tetrazo!-5-yJ)-1 H-indole
Figure imgf000280_0001
5-(2H-Tetrazol-5-yl)-1 H-indole was prepared from 5-cyanoindole according to the method described in example 594.
HPLC-MS (Method C): m/z: 186 (M+1); Rt = 1.68 min.
Example 811 (General procedure (K)). 1 -BenzyI-4-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000280_0002
1-Benzyl-1H-indole-4-carbonitrile was prepared from 4-cyanoindole according to the method described in example 806.
HPLC-MS (Method C): m/z: 233 (M+1); Rt = 4.24 min.
1-Benzyl-4-(2H-tetra∑ol-5-yl)-1 H-indole was prepared from 1-ben∑yl-1H-indole-4-carbonitrile according to the method described in example 594. HPLC-MS (Method C): m/z: 276 (M+1); Rt = 3.44 min.
Figure imgf000281_0001
wherein T is as defined above and
Pol- is a polystyrene resin loaded with a 2-chlorotrityl linker, graphically shown below:
Figure imgf000281_0002
This general procedure (L) is further illustrated by the following example: Example 812 (General procedure (L)). 5-(2H-Tetrazol-5-yl)-1 -[3-(trif(uoromethyl)benzyl]-1 H-indole
Figure imgf000281_0003
2-ChIorotritylchloride resin (100 mg, 0.114 mmol active chloride) was swelled in dichloromethane (2 mL) for 30 min. The solvent was drained, and a solution of 5-(2H-tetrazol-5-yl)- 1 H-indole (example 810) (63 mg, 0.34 mmol) in a mixture of Λ/,Λ/-dimethylformamide, dichloromethane and Λ/,Λ/-di(2-propyl)ethylamine (DIPEA) (5:5:2) (1.1 mL) was added. The reaction mixture was shaken at room temperature for 20 hours. The solvent was removed by filtration, and the resin was washed consecutively with W,W-dimethylformamide (2 x 4 mL), dichloromethane (6 x 4 mL) and methyl sulfoxide (2 x 4 mL). Methyl sulfoxide (1 mL) was added, followed by the addition of a solution of lithium bis(trimethylsilyl)arnide in tetrahydrofuran (1.0 M, 0.57 mL, 0.57 mmol). The mixture was shaken for 30 min at room temperature, before 3-(trifluoromethyl)benzyl bromide (273 mg, 1.14 mmol) was added as a solution in methyl sulfoxide (0.2 mL). The reaction mixture was shaken for 20 hours at room temperature. The drained resin was washed consecutively with methyl sulfoxide (2 x 4 mL), dichloromethane (2 x 4 mL), methanol (2 x 4 mL), dichloromethane (2 x 4 mL) and tetrahydrofuran (4 L). The resin was treated with a solution of hydrogen chloride in tetrahydrofuran, ethyl ether and ethanol = 8:1:1 (0.1 M, 3 mL) for 6 hours at room temperature. The resin was drained and the filtrate was concentrated in vacuo. The crude product was re-suspended in dichloromethane (1.5 mL) and concentrated three times to afford the title compound (35 mg). No further purification was necessary.
HPLC-MS (Method B): m/z: 344 (M+1); Rt = 4.35 min.
1 H-NMR (DMSO-de): £8.29 (1H, s), 7.80 (1H, dd), 7.72 (2H, m), 7.64 (2H, bs), 7.56 (1 H, t),
7.48 (1H, d), 6.70 (1 H, d), 5.62 (2H, s).
The compounds in the following examples were prepared in a similar fashion. Optionally, the compounds can be further purified by recrystallization or by chromatography.
Example 813 (General procedure (L)).
1 -(4-Chlorobenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000282_0001
HPLC-MS (Method B): m/z: 310 (M+1); Rt = 4.11 min.
Example 814 (General procedure (L)).
1 -(2-Chlorobenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000283_0001
HPLC-MS (Method B): m/z: 310 (M+1); Rt = 4.05 min.
Example 815 (General procedure (L)).
1 -(4-Methoxybenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000283_0002
HPLC-MS (Method B): m/z: 306 (M+1); Rt = 3.68 min.
Example 816 (General procedure (L)).
1 -(4-Methylbenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000283_0003
HPLC-MS (Method B): m/z: 290 (M+1); Rt = 3.98 min.
Example 817 (General procedure (L)). 5-(2H-Tetrazol-5-yl)-1 -[4-(trifluoromethyl)benzyl]-1 H-indole
Figure imgf000283_0004
HPLC-MS (Method B): m/∑: 344 (M+1); Rt = 4.18 min. Example 818 (General procedure (L)).
1 -(3-ChlorobenzyI)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000284_0001
HPLC-MS (Method B): m/z: 310 (M+1); Rt = 4.01 min.
Example 819 (General procedure (L)).
1 -(3-Methylbenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000284_0002
HPLC-MS (Method B): m/z: 290 (M+1); Rt = 3.98 min.
Example 820 (General procedure (L)).
1 -(2,4-Dichlorobenzyl)-5-(2H-tetrazoI-5-yI)-1 H-indole
Figure imgf000284_0003
HPLC-MS (Method B): m/z: 344 (M+1); Rt = = 4.41 min
Example 821 (General procedure (L)).
1 -(3-MethoxybenzyI)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000285_0001
HPLC-MS (Method B): m/z: 306 (M+1); Rt = 3.64 min.
Example 822 (General procedure (L)). 1-(4-Fluorobenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000285_0002
HPLC-MS (Method B): m/z: 294 (M+1); Rt = 3.71 min.
Example 823 (General procedure (L)).
1 -(3-Fluorobenzyl)-5-(2H-tetrazol-5-yl)-1 H-indoie
Figure imgf000285_0003
HPLC-MS (Method B): m/z: 294 (M+1); Rt = 3.68 min.
Example 824 (General procedure (L)).
1 -(2-lodobenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000285_0004
HPLC-MS (Method B): m/z: 402 (M+1); Rt = 4.11 min. Example 825 (General procedure (L)).
1 -[(Naphthalen-2-yl)methyl]-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000286_0001
HPLC-MS (Method B): m/z: 326 (M+1); Rt = 4.18 min.
Example 826 (General procedure (L)).
1 -(3-Bromobenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000286_0002
HPLC-MS (Method B): m/z: 354 (M+1); Rt = 4.08 min.
Example 827 (General procedure (L)).
1 -(4-Carboxybenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000286_0003
In this preparation, a larger excess of lithium bis(trimethylsilyl)amide in tetrahydrofuran (1.0 M, 1.7 mL, 1.7 mmol) was used. HPLC-MS (Method B): m/z: 320 (M+1); Rt = 2.84 min.
Example 828 (General procedure (L)).
1 -(3-Carboxybenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000287_0001
In this preparation, a larger excess of lithium bis(trimethylsilyl)arnide in tetrahydrofuran (1.0
M, 1.7 mL, 1.7 mmol) was used.
HPLC-MS (Method B): m/z: 320 (M+1); Rt = 2.91 min.
Example 829 (General procedure (L)).
1-(2,4-Difluorobenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000287_0002
HPLC-MS (Method B): m/z: 312 (M+1); Rt = 3.78 min.
Example 830 (General procedure (L)).
1-(3,5-Difluorobenzyl)-5-(2H-tetrazol-5-yi)-1H-indole
Figure imgf000287_0003
HPLC-MS (Method B): m/z: 312 (M+1); Rt = 3.78 min.
Example 831 (General procedure (L)).
1 -(3,4-Difluoroben∑yl)-5-(2H-tetra2ol-5-yl)-1 H-indole
Figure imgf000288_0001
HPLC-MS (Method B): m/z: 312 (M+1); Rt = 3.81 min.
Example 832 (General procedure (L)).
1 -[4-(2-Propyl)benzyl]-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000288_0002
HPLC-MS (Method B): m/z: 318 (M+1); Rt = 4.61 min.
Example 833 (General procedure (L)).
1 -(3,5-Dimethoxybenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000288_0003
HPLC-MS (Method B): m/z: 336 (M+1); Rt = 3.68 min.
Example 834 (General procedure (L)). 1-(2'-Cyanobiphenyl-4-ylmethyl)-5-(2H-tetrazol-5-yl)-1H-indo)e
Figure imgf000289_0001
HPLC-MS (Method B): m/z: 377 (M+1); Rt = 4.11 min.
Example 835 (General procedure (L)).
1 -(2-Methylbenzyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000289_0002
HPLC-MS (Method B): m/z: 290 (M+1); Rt = 3.98 min.
Further compounds of the invention that may be prepared according to general procedure (K) and/or (L) includes:
Figure imgf000290_0001
Example 857 Example 858 Example 859
Figure imgf000291_0001
The following compounds of the invention may be prepared eg. from 1-(4-bromoben∑yl)-5- (2H-tetrazol-5-yl)-1 H-indole (example 807) or from the analogue 1-(3-bromoben∑yl)-5-(2H- tetrazol-5-yl)-1H-indo!e and aryl boronic acids via the Suzuki coupling reaction eg as described in Littke, Dai & Fu J. Am. Chem. Soc, 2000, 122, 4020-8 (or references cited therein), or using the methodology described in general procedure (E), optionally changing the palladium catalyst to bis(tri-fe/ -butylphosphine)palladium (0).
Example 860
Figure imgf000291_0002
Example 861 Example 862 Example 863
Figure imgf000291_0003
General procedure (M) for preparation of compounds of general formula lι2:
Figure imgf000291_0004
"12 wherein T is as defined above.
The general procedure (M) is further illustrated by the following example: Example 864 (General procedure (M)). 1 -Benzoyl-5-(2H-tetrazol-5-yI)-1 H-indole
Figure imgf000292_0001
To a solution of 5-cyanoindole (1.0 g, 7.0 mmol) in dichloromethane (8 mL) was added 4- (dimethyla ino)pyridine (0.171 g, 1.4 mmol), triethylamine (1.96 mL, 1.42 g, 14 mmol) and benzoyl chloride (0.89 mL, 1.08 g, 7.7 mmol). The resulting mixture was stirred for 18 hours at room temperature. The mixture was diluted with dichloromethane (80 mL) and washed consecutively with a saturated solution of sodium hydrogencarbonate (40 L) and brine (40 mL). The organic phase was dried with magnesium sulfate (1 hour). Filtration and concentration furnished the crude material which was purified by flash chromatography on silica gel, eluting with ethyl acetate/heptanes = 2:3. 1 -Benzoyl- H-indole-5-carbonitrile was obtained as a solid.
HPLC-MS (Method C): m/z: 247 (M+1); Rt = 4.07 min.
1-Benzoyl-1H-indole-5-carbonitrile was transformed into 1-benzoyl-5-(2H-tetrazol-5-yl)-1H- indole by the method described in example 594.
HPLC (Method C): Rt = 1.68 min.
The compound in the following example was prepared by the same procedure.
Example 865 (General procedure (M)). 1 -Benzoyl-4-(2H-tetra∑ol-5-yl)-1 H-indole
Figure imgf000293_0001
1-Ben∑oyl-1H-indole-4-carbonitrile was prepared from 4-cyanoindole according to the method described in example 864.
HPLC-MS (Method C): m/z: 247 (M+1); Rt = 4.24 min.
1-Benzoyl-4-(2H-tetrazol-5-yl)-1 H-indole was prepared from 1-benzoyl-1H-indole-4- carbonitrile according to the method described in example 594.
HPLC (Method C): Rt = 1.56 min.
Example 866 (General procedure (M)) (2-Fluoro-3-trifluoromethylphenyl)-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000293_0002
HPLC-MS (Method B): m/z = 376 (M+1); Rt = 4.32 min.
Example 867 (General procedure (M)) (4-Methoxyphenyl)-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000293_0003
HPLC-MS (Method B): m/z = 320 (M+1); Rt = 3.70 min. Example 868 (General procedure (M)) (3-Nitrophenyl)-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000294_0001
HPLC-MS (Method B): m/z = 335 (M+1); Rt = 3.72 min.
Example 869 (General procedure (M)) (4-Nitrophenyl)-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000294_0002
HPLC-MS (Method B): m/z = 335 (M+1); Rt = 3.71 min.
Example 870 (General procedure (M)) Naphthalen-2-yl-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000294_0003
HPLC-MS (Method C): m/z = 340 (M+1); Rt = 4.25 min.
Example 871 (General procedure (M)) (2,3-Difluorophenyl)-[5-(2H-tefrazol-5-yl)-indol-1-yl]-methanon€
Figure imgf000295_0001
HPLC-MS (Method B: m/z = 326 (M+1); Rt = 3.85 min.
The following known and commercially available compounds do all bind to the His B10 Zn 2+ site of the insulin hexamer:
Example 872
1 -(4-Fluorophenyl)-5-(2H-tetrazol-5-yl)-1 H-indole
Figure imgf000295_0002
Example 873 1-Amino-3-(2H-tetrazol-5-yl)benzene
Figure imgf000295_0003
Example 874 1-Amino-4-(2H-tetrazol-5-yl)benzene
Figure imgf000295_0004
A mixture of 4-aminobenzonitrile (10 g, 84.6 mmol), sodium azide (16.5 g, 254 mmol) and ammonium chloride (13.6 g, 254 mmol) in DMF was heated at 125 °C for 16 hours. The cooled mixture was filtered and the filtrate was concentrated in vacuo. The residue was added water (200 mL) and diethyl ether (200 mL) which resulted in crystallisation. The mixture was filtered and the solid was dried in vacuo at 40 °C for 16 hours to afford 5-(4- aminophenyl)-2H-tetra∑ole.
1H NMR DMSO-de): δ = 5.7 (3H, bs), 6.69 (2H, d), 7.69 (2H, d). HPLC-MS (Method C): m/∑: 162 (M+1); Rt = 0,55 min.
Example 8751 -Nitro-4-(2H-tetrazol-5-yl)benzene
Figure imgf000296_0001
Example 8761 -Bromo-4-(2H-tetrazol-5-yl)benzene
Figure imgf000296_0002
General procedure (N) for solution phase preparation of amides of general formula 3-
Frag — U-OH + HN R ^ Frag-U-N R
R' R.
I .3 wherein Frag is any fragment carrying a carboxylic acid group, R is hydrogen, optionally substituted aryl or C^-alkyl and R' is hydrogen or C^-alkyl.
Frag-G02H may be prepared eg by general procedure (D) or by other similar procedures described herein, or may be commercially available.
The procedure is fuήher illustrated in the following example 877: Example 877 (General procedure (N)) Λ/-(4-Chlorobenzyl)-2-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)-1H-indol-1-yl]acetamide
Figure imgf000297_0001
[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]acelic acid (example 478, 90.7 mg, 0.3 mmol) was dissolved in NMP (1 mL) and added to a mixture of 1-ethyl-3-(3-dimethylamino- propyl)carbodiimide, hydrochloride (86.4 mg, 0.45 mmol) and 1-hydroxybenzotriazol (68.8 mg, 0.45 mmol) in NMP (1 mL). The resulting mixture was shaken at RT for 2 h. 4- Chlorobenzylamine (51 mg, 0.36 mmol) and DIPEA (46.4 mg, 0.36 mmol) in NMP (1 mL) were added to the mixture and the resulting mixture shaken at RT for 2 days. Subsequently ethyl acetate (10 mL) was added and the resulting mixture washed with 2x10 mL water followed by saturated ammonium chloride (5 mL). The organic phase was evaporated to dryness giving 75 mg (57%) of the title compound.
HPLC-MS (Method C): m/z: 426 (M+1); Rt. = 3.79 min.
Example 878 (General procedure (N))
Λ/-(4-Chlorobenzyl)-4-t2-chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide
Figure imgf000297_0002
HPLC-MS (Method A): m/z: 465 (M+1); Rt = 4.35 min.
Example 879 (General procedure (N)) Λ-(4-Chlorobenzyl)-4-t4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide
Figure imgf000297_0003
HPLC-MS (Method A): m/z: 431 (M+1); Rt = 3.68 min. Example 880 (General procedure (N)) 2-[2-Bromo-4-(2,4-dioχothiazolidin-5-ylidenemethyl)phenoxy]-Λ/-(4-chlorobenzyl)acetamide
Figure imgf000298_0001
HPLC-MS (Method A): /z: 483 (M+1); Rt = 4.06 min.
Example 881 (General procedure (N)) /V-(4-Chlorobenzyl)-2-[3-(2,4-dioxothia∑olidin-5-ylidenemethyl)phenoxy]acetamid.
Figure imgf000298_0002
HPLC-MS (Method A): m/z: 403 (M+1); Rt = 4.03 min.
Example 882 (General procedure (N))
Λ/-(4-Chlorobenzyl)-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyI)phenyl]acrylamide
Figure imgf000298_0003
HPLC-MS (Method A): m/z: 399 (M+1); Rt = 3.82.
Example 883 (General procedure (N)) Λ/-(4-Chlorobenzyl)-4-[3-(2,4-dioxothiazolidin-5-ylidenemethyI)phenoxy]butyramide
Figure imgf000298_0004
HPLC-MS (Method A): m/z: 431 (M+1); Rt = 3.84 min.
Example 884 (General procedure (N)) 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]-W-(4-chlorobenzyl)butyramide
Figure imgf000298_0005
HPLC-MS (Method A): m/z: 511 (M+1); Rt = 4.05 min. Example 885 (General procedure (N))
4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-phenoxy]-/V-(4-chloroben2yl)- butyramide
Figure imgf000299_0001
Example 886 (General procedure (N)) W-(4-Chlorobenzyl)-2-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetamide
Figure imgf000299_0002
HPLC-MS (Method C): /z: 431 (M+1); Rt. = 4.03 min.
Example 887 (General procedure (N)) Λ/-(4-Chlorobenzyl)-3-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)-1H-indol-1-yl]propionamide
Figure imgf000299_0003
HPLC-MS (Method C): m/z: 440 (M+1); Rt. = 3.57 min.
Example 888 (General procedure (N)) A/-(4-ChIorobenzyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyramide
Figure imgf000299_0004
HPLC-MS (Method C): m/z: 481 (M+1); Rt = 4.08 min. Example 889 (General procedure (N)) 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-V-hexylbutyramide
Figure imgf000300_0001
HPLC-MS (Method C): m/z: 441 (M+1); Rt = 4.31 min.
Example 890 (General Procedure (N))
4-({[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carbonyl]amino}methyl)ben∑oic acid methyl ester
Figure imgf000300_0002
HPLC-MS (Method C): m/z: 436 (M+1); Rt.= 3.55 min.
Example 891 (General procedure (N)) Λ/-(4-Chlorobenzyl)-4~[3-(2H-tetra2oI-5-yl)carbazol-9-ylmethyl]benzamide
Figure imgf000300_0003
HPLC-MS (Method C): m/z:493 (M+1); Rt = 4.19 min.
Example 892 (General procedure (N)) V-(4-Chlorobenzyl)-3-[3-(2H-tetra∑ol-5-yl)carbazol-9-ylmethyl]benzamide
Figure imgf000301_0001
HPLC-MS (Method C): m/z: 493 (M+1); Rt = 4.20 min.
Example 893 (General Procedure (N)) W-(4-Chlorobenzyl)-3-methyl-4-[3-(2H-tetrazol-5-yl)-carbazol-9-ylmethyl]benzamide
Figure imgf000301_0002
HPLC-MS (Method C): m/z: 507 (M+1); Rt = 4.37min.
Example 894 (General procedure (N))
5-{2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-acetylamino}-isophthalic acid dimethyl ester
Figure imgf000301_0003
HPLC-MS (Method C): m/z = 521 (M+1); Rt. = 4.57 min.
Example 895 (General procedure (N)) 5-{2-[4-(2,4-Dioxothia2olidin-5-ylidenemethyl)-naphthalen-1-yloxy]-acetylamino}-isophthalic acid
Figure imgf000302_0001
HPLC-MS (Method C): m/z = 515 (M+23); Rt. = 3.09 min.
Example 896 (General procedure (N))
5-(3-{2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-ethyl}-ureido)- isophthalic acid monomethyl ester
Figure imgf000302_0002
HPLC-MS (Method C): m/z = 536 (M+1); Rt = 3,58 min.
Example 897 (General Procedure (N)). 2-{4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}succinic acid dimethyl ester
Figure imgf000302_0003
4-[3-(1H-Tetrazol-5-yl)carbazol-9-ylmethyl]ben∑oic acid (2.00 g, 5.41 mmol), 1- hydroxyben∑otria∑ole (1.46 g, 10.8 mmol) and N,N-di(2-propyl)ethylamine (4.72 mL, 3.50 g, 27.1 mmol) were dissolved in dry N,N-dimethylformamide (60 mL). The mixture was cooled in an ice-water bath, and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.45 g, 7.56 mmol) and (S)-aminosuccinic acid dimethyl ester hydrochloride (1.28 g, 6.48 mmol) were added. The cooling was discontinued, and the reaction mixture was stirred at room temperature for 18 hours before it was poured into hydrochloric acid (0.1 N, 600 mL).
The solid was collected by filtration and washed with water (2 X 25 mL) to furnish the title compound.
HPLC-MS (Method C): m/z: 513 (M+1); Rt = 3.65 min.
1H-NMR (DMSO-d6): δ 8.90 (1H, d), 8.86 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.87 (1H, d),
7.75 (2H, d), 7.69 (1H, d), 7.51 (1H, t), 7.32 (1H, t), 7.28 (2H, d), 5.82 (2H, s), 4.79 (1H, m),
3.61 (3H, s), 3.58 (3H, s), 2.92 (1H, dd), 2.78 (1H, dd).
Example 898 (General Procedure ' 2-{4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}succinic acid
Figure imgf000303_0001
2-{4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}succinic acid dimethyl ester (1.20 g, 2.34 mmol) was dissolved in tetrahydrofuran (30 mL). Aqueous sodium hydroxide (1 N, 14 mL) was added, and the resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was poured into hydrochloric acid (0.1 N, 500 mL). The solid was collected by filtration and washed with water (2 X 25 mL) and diethyl ether (2 X 25 mL) to furnish the title compound.
HPLC-MS (Method C): m/z: 485 (M+1 ); Rt = 2.94 min.
1H-NMR (DMSO-d6): δ 12.44 (2H, s (br)), 8.90 (1H, d), 8.68 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.87 (1H, d), 7.75 (2H, d), 7.68 (1H, d), 7.52 (1H, t), 7.32 (1H, t), 7.27 (2H, d), 5.82 (2H, s), 4.70 (1H, m), 2.81 (1H, dd), 2.65 (1H, dd).
The compounds in the following examples were prepared in a similar fashion.
Example 899 (General procedure (N)) 2-{4-[3-(2H-Tetrazol-5-yl)-carbazol-9-yImethyl]-ben∑oylamino}-succinic acid dimethyl ester
Figure imgf000304_0001
HPLC-MS (Method C): m/z = 513 (M+1); Rt = 3.65min.
Example 900 (General procedure (N)) 2-{4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}pentanedioic acid dimethyl ester
Figure imgf000304_0002
HPLC-MS (Method C): m/z = 527 (M+1); Rt = 3.57min.
Example 901 (General procedure (N))
(MethoxycarbonyImethyl-{4-[3-(2H-tetrazol-5-yl)-carbazol-9-ylmethyl]-benzoyl}-amino)-acetic acid methyl ester
Figure imgf000304_0003
HPLC-MS (Method C): m/z = 513 (M+1); Rt = 3,55min.
Example 902 (General procedure (N)) 2-{4-[3-(2H-Tetra∑ol-5-yl)carba∑ol-9-ylmethyl]benzoyIamino}pentanedioic acid
Figure imgf000305_0001
HPLC-MS (Method C): m/∑ = 499 (M+1); Rt = 2.87min.
Example 903 (General procedure (N))
(Ethoxycarbonylmethyl-{4-[3-(2H-tetra∑ol-5-yl)-carbazol-9-ylmethyl]-ben∑oyl}-amino)-acetic acid ethyl ester
Figure imgf000305_0002
HPLC-MS (Method C): m/z = 541 (M+1); Rt = 3.91 min.
Example 904 (General procedure (N))
3-(3-{4-[4-(2,4-Dioxo-thiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyrylamino}- propylamino)-hexanedioic acid dimethyl ester
Figure imgf000305_0003
HPLC-MS (Method C: m/z = 585 (M+1); Rt = 2,81 min.
Example 905 (General procedure (N))
3-(3-{4-[4-(2,4-Dioxo-thiazolidin-5-yIidenemethyl)-naphthalen-1-yloxy]-butyrylamino}- propyJamino)-hexanedioie acid
Figure imgf000306_0001
HPLC-MS (Method C): m/z = 554 (M-3); Rt = 3,19 rnin.
Example 906 (General procedure (N)) (Carboxymethyl-{4-[3-(2H-tetra∑ol-5-yl)-carba∑ol-9-ylmethyl]-benzoyl}-amino)-acetic acid
Figure imgf000306_0002
HPLC-MS (Method C); /z = 485 (M+1); Rt = 3.04 min.
Example 907 (General procedure (N))
4-(3-{4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyrylamino)- propylamino)-cyclohexane-1 ,3-dicarboxyIic acid dimethyl ester
Figure imgf000306_0003
HPLC-MS (Method C): m/z = 612 (M+1); Rt = 3,24 min.
Example 908 (General procedure (N)) 2-{3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}pentanedioic acid dimethyl ester
Figure imgf000306_0004
HPLC-MS (Method C): m/z = 527 (M+1); Rt = 3.65min. Example 909 (General procedure (N)) 2-{3-[3-(2H-Tetrazol-5-yI)carbazol-9-ylmethyl]benzoylamino}ρentanedioic acid dimethyl ester
Figure imgf000307_0001
HPLC-MS (Method C): m/z = 527 (M+1); Rt = 3.65min.
Example 910 (General procedure 2-{3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}pentanedioic acid dimethyl ester
Figure imgf000307_0002
HPLC-MS (Method C): m/z = 527 (M+1); Rt = 3.65min.
Example 911 (General procedure (N)) 2-{3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}pentanedioic acid
Figure imgf000307_0003
HPLC-MS (Method C): m/z = 499 (M+1); Rt = 3.00 min.
Example 912 (General procedure (N))
(Methoxycarbonylmethyl-{3-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoyI}amino)acetic acid methyl ester
Figure imgf000308_0001
1H-NMR (DMSO-de): δ 8.88 (1H, d), 8.29 (1H, d), 8.10 (1H, dd), 7.85 (1H, d), 7.67 (1H, d), 7.52 (1H, t), 7.39 (1H, t), 7.30 (2H, m), 7.17 (2H, ), 5.79 (2H, s), 4.17 (2H, s), 4.02 (2H, s), 3.62 (3H, s), 3.49 (3H, s).
Example 913 (General procedure (N)) 2-{3-[3-(2H-Tetrazol-5-yJ)carbazo)-9-ylmethyl]benzoylamino}succinic acid dimethyl ester
Figure imgf000308_0002
HPLC-MS (Method C): m/z = 513 (M+1); Rt = 3.70 min.
Example 914 (General procedure (N)) 2-{3-[3-(2H-Tetrazol-5-yl)-carbazol-9-ylmethylj-benzoylamino}-succinic acid
Figure imgf000308_0003
HPLC-MS (Method C): m/z = 485 (M+1); Rt = 2.96 min.
Example 915 (General procedure (N)) (Carboxymethyl-{3-[3-(2H-tetrazol-5-yl)carba∑oI-9-ylmethyl]benzoyl}amino)acetic acid
Figure imgf000309_0001
HPLC-MS (Method C): m/z = 485 (M+1); Rt = 2.87 min.
Example 916 (General procedure (N))
4-(4-(3-Carboxy-propylcarbamoyl)4-{4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]- benzoylamino}-butyrylamino)-butyric acid
Figure imgf000309_0002
The title compound was prepared by coupling of (S)-2-{4-[3-(2H-tetrazol-5-yl)carbazol-9- ylmethy.]benzoylamino}pentanedioic acid bis-(2,5-dioxopyrrolidin-1-yl) ester (prepared from (S)-2-{4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}pentanedioic acid by essentially the same procedure as described for the synthesis of 4-[3-(2H-tetrazol-5-yl)carbazol-9- y.me.hy.]benzoic acid 2,5-diσxopyrrolidin-1-yl ester) with 4-aminobutyric acid according to the procedure described for the preparation of 4-{4-[3-(2H-tetrazol-5-yl)carbazol-9-yImethyl]- benzoylamino}butyric acid .
HPLC-MS (Method C): m/z: 669 (M+1); Rt = 2.84 min.
Example 917 (General procedure (N)) [2-(2-{4-[3-(2H-Tetrazol-5-yl)-carbazol-9-ylmethyl]benzoylamino}ethoxy)ethoxy]acetic acid
Figure imgf000310_0001
HPLC-MS (Method C): m/z: 515 (M+1); Rf = 3.10 min.
Example 918 (General procedure (N)) 2-{4-[3-(2H-Tetrazol-5-yl)-carbazol-9-ylmethyl]-benzoylamino}-pentanedioic acid di-tert-butyl ester
Figure imgf000310_0002
HPLC-MS (Method C): m/z = 611 (M+1); Rt = 4.64 min.
Example 919 (General Procedure (N)). 4-{4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoylamino}butyric Acid
Figure imgf000310_0003
HPLC-MS (Method C): m/z: 455 (M+1); Rt = 3.13 min.
Example 920 (General Procedure (N)). [2-(2-{4-[3-(2H-Tetra2ol-5-yI)carbazol-9-ylmethyl]benzoylamino}ethoxy)ethoxy]acetic acid
Figure imgf000311_0001
The title compound was prepared by coupling of 4-[5-(2 -tetrazol-5-yl)carbazol-9- ylmethyl]ben∑oic acid 2,5-dioxopyrrolidin-1-yl ester with [2-(2-aminoethoxy)ethoxy]acetic acid (prepared from [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid by treatment with PS-Trisamine resin in DMF). HPLC-MS (Method C): m/∑: 515 (M+1); Rt = 3.10 min.
The commercially available compounds in the following examples do all bind to the HisBlO Zn2+site:
Example 921
1 -(4-Bromo-3-methylphenyl)-1 ,4-dihydrotetrazole-5-thione
Figure imgf000311_0002
Example 922 1-(4-lodophenyl)-1 ,4-dihydrotetra2ole-5-thione
Figure imgf000311_0003
Example 923
1 -(2,4,5-Trichlorophenyl)-1 H-tetrazole-5-thiol
Figure imgf000311_0004
Example 924
1 -(2,6-Dimethylphenyl)-1 ,4-dihydrotetrazole-5-thione
Figure imgf000312_0001
Example 925
1 -(2,4,6-Trirnethylpheny.)- 1 ,4-dihydrotetrazoIe-5-thion€
Figure imgf000312_0002
Example 926
1 -(4-Dimethylaminophenyl)-1 H-tetrazole-5-thiol
Figure imgf000312_0003
Example 927
1-(3,4-Dichlorophenyl)-1 ,4-dihydro-1 H-tetrazole-5-thione
Figure imgf000312_0004
Example 928
1-(4-PropylphenyI)-1 ,4-dihydro-1 H-tetra∑ole-5-thione
Figure imgf000312_0005
Example 929
1-(3-Chlorophenyl)-1 ,4-dihydro-1 H-tetrazole-5-thione
Figure imgf000313_0001
Example 930
1 -(2-Fluorophenyl)-1 ,4-dihydro-1 H-tetrazo)e-5-thione
Figure imgf000313_0002
Example 931
1-(2,4-Dichlorophenyl)-1 ,4-dihydro-1 H-tetrazole-5-thione
Figure imgf000313_0003
Example 932
1 -(4-Trifluoromethoxyphenyl)-1 ,4-dihydro-1 H-tetrazole-5-thione
Figure imgf000313_0004
Example 933 N-[4-(5-Mercaptotetrazol-1-yl)-phenyl]-acetamide
Figure imgf000313_0005
Example 934
1 -(4-Chlorophenyl)-1 ,4-dihydrotetrazole-5-thione
Figure imgf000314_0001
Example 935
1 -(4-Methoxyphenyl)-1 ,4-dihydrotetrazole-5-thione
Figure imgf000314_0002
Example 936
1 -(3-Fluoro-4-pyrrolidin- 1 -ylphenyl)- ,4-dihydrotetrazole-5-thione
Figure imgf000314_0003
Example 937 Λ/-[3-(5-Mercaptotetrazol-1-yl)phenyl]acetamide
Figure imgf000314_0004
Example 938 1-(4-Hydroxyphenyl)-5-mercaptotetrazole
Figure imgf000315_0001
Example 939
Figure imgf000315_0002
Preparation of 1-aryl-1,4-dihydrotetrazole-5-th)ones (or the tautomeric 1-aryltetrazole-5- thiols) is described in the literature (eg. by Kauer & Sheppard, J. Org. Chem., 32, 3580-92 (1967)) and is generally performed eg. by reaction of aryl-isothiocyanates with sodium azide followed by acidification
1-Aryl-1 ,4-dihydrotetrazole-5-thiones with a carboxylic acid tethered to the aryl group may be prepared as shown in the following scheme:
Figure imgf000315_0003
Step 1 is a phenol alkylation and is very similar to steps 1 and 2 of general procedure (D) and may also be prepared similarly as described in example 481. Step 2 is a reduction of the nitro group. SnCI2, H2 over Pd/C and many other procedures known to those skilled in the art may be utilised.
Step 3 is formation of an arylisothiocyanate from the corresponding aniline. As reagents CS2, CSCI2, or other reagents known to those skilled in the art, may be utilised.
Step 4 is a conversion to mercaptotetrazole as described above.
Compounds of the invention include:
Example 940 Example 941
Figure imgf000316_0001
Example 942 Example 943
Figure imgf000316_0002
Example 944 Example 945
Figure imgf000316_0003
Example 946
Figure imgf000316_0004
Example 947
4-(4-Hydroxyphenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000317_0001
Phenylsulphonyl acetonitrile (2.0 g, 11.04 mmol) was mixed with 4-hydroxybenzaldehyde (1.35 g, 11.04 mmol) in DMF (10 mL) and toluene (20 mL). The mixture was refluxed for 3 hours and subsequently evaporated to dryness in vacuo. The residue was treated with diethyl ether and toluene. The solid formed was filtered to afford 2.08 g (66%) of 2- ben∑enesulfonyl-3-(4-hydroxyphenyl)acrylonitrile. HPLC-MS (Method C): m/z: 286 (M+1); Rt. = 3.56 min.
A mixture of 2-benzenesulfonyl-3-(4-hydroxyphenyl)acrylonitrile (2.08 g, 7.3 mmol) and sodium azide (0.47g,7.3 mmol) in DMF (50 mL) was heated at reflux temperature 2 hours. After cooling, the mixture was poured on ice. The mixture was evaporated in vacuo to almost dryness and toluene was added. After filtration, the organic phase was evaporated in vacuo. The residue was purified by silicagel chromatography eluting with a mixture of ethyl acetate and heptane (1 :2). This afforded 1.2 g (76%) of the title compound.
1H NMR (DMSO-d6): 10.2 (broad, 1H); 7.74 (d,2H); 6.99 (d,2H); 3.6-3.2 (broad, 1 H). HPLC-MS (Method C) m/z: = 187 (M+1); Rt. = 1.93 min
General procedure (O) for preparation of compounds of general formula lι :
Figure imgf000317_0002
'u
wherein
AA is as defined above,
Steps 1 and 2 are described in the literature (eg Beck & GQnther, Chem. Ber., 106, 2758-66 (1973)) Step 1 is a Knoevenagel condensation of the aldehyde AA-CHO with phenylsulfonyl- acetonitrile and step 2 is a reaction of the vinylsulfonyl compound obtained in step 1 with sodium azide. This reaction is usually performed in DMF at 90 - 110 °C.
This general procedure is further illustrated in the following example 948:
Example 948 (General Procedure (O)) [4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)phenoxy]acetic acid
Figure imgf000318_0001
Phenylsulphonylacetonitrile (0.1 g, 0.55 mmol) was mixed with 4-formylphenoxyactic acid (0.099 g, 0.55 mmol) in DMF (3 mL) and heated to 110 °C for 3 h and subsequently cooled to RT. Sodium azide (0.036 g, 0.55 mmol) was added and the resulting mixture was heated to 110 °C for 3 h and cooled to RT. The mixture was poured into water (20 mL) and centrifuged. The supernatant was discarded, ethanol (5 L) was added and the mixture was centrifuged again. After discarding the supernatant, the residue was dried in vacuo to afford 50 mg (37%) of [4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)phenoxy]acetic acid.
HPLC-MS (Method C): m/z: 245 (M+1) Rt. 2.19 min.
Example 949 (General Procedure (O)) 5-(Naphthalen-1 -yl)-3H-[1 ,2,3]triazole-4-carbonitrile
Figure imgf000318_0002
HPLC-MS (Method C): m/z: 221 (M+1); Rt. 3.43 min. Example 950 (General Procedure (O)) 5-(Naphthalen-2-yI)-3H-[1,2,3]triazole-4-carbonitrile
Figure imgf000319_0001
HPLC-MS (Method C): m/z: 221 (M+1); Rt = 3.66 min.
Example 951 (General procedure (O))
4-[3-(5-Cyano-[1 ,2,3]triazol-4-yl)-1 ,4-dimethylcarbazol-9-ylmethyl]-benzoic acid
Figure imgf000319_0002
HPLC-MS (Method C): m/z = 422 (M+1); Rt = 3.85 min.
Preparation of intermediary aldehyde:
1,4 DimethyIcarbazol-3-carbaldehyde (0.68 g, 3.08 mmol) was dissolved in dry DMF (15 mL), NaH (diethyl ether washed) (0.162 g, 6.7 mol) was slowly added under nitrogen and the mixture was stirred for 1 hour at room temperature. 4-Bromomethylbenzoic acid (0.73 g, 3.4 mmol) was slowly added and the resulting slurry was heated to 40 °C for 16 hours. Water (5 mL) and hydrochloric acid (6N, 3 mL) were added. After stirring for 20 min at room temperature, the precipitate was filtered off and washed twice with acetone to afford after drying 0.38 g (34%) of 4-(3-formyI-1 ,4-dimethylcarbazol-9-ylmethyl)benzoic acid.
HPLC-MS (Method C) : m/z = 358 (M+1), RT. = 4.15 min.
Example 952 (General Procedure (O)) 5-(Anthracen-9-yl)-3H-[1 ,2,3]triazole-4-carbonitrile
Figure imgf000320_0001
HPLC-MS (Method C): m/z: 271 (M+1); Rt = 3.87 min.
Example 953 (General Procedure (O)) 5-(4-Methoxynaphthalen-1-yl)-3H-[1,2,3]triazole-4-carbonitrile
Figure imgf000320_0002
HPLC-MS (Method C): m/z: 251 (M+1); Rt = 3.57 min.
Example 954 (General Procedure (O)) 5-(1,4-Dimethyl-9H-carbazol-3-yl)-3H-[1,2,3]triazole-4-carbonitrile
Figure imgf000320_0003
HPLC-MS (Method C): m/z: 288 (M+1); Rt = 3.67 min.
Example 955 (General procedure (0)) δ^'-Methoxybiphenyl^-y -SH-tl^.Sltriazole^-carbonitrile
Figure imgf000321_0001
HPLC-MS (Method C): m/z = 277 (M+1); Rt = 3.60 min.
Example 956 (General procedure (O)) 5-(4-StyryIphenyl)-3H-[1 ,2,3]triazole-4-carbonitrile
Figure imgf000321_0002
HPLC-MS (Method C): m/z = 273 (M+1); Rt = 4.12 min.
Example 957 (General procedure (O))
5-(2,6-Dichloro-4-dibenzylaminophenyl)-3H-[1 ,2,3]triazole-4-carbonitrile
Figure imgf000321_0003
Example 958 (General procedure (O))
5-(1 -Bromonaphthalen-2-yl)-3H-[1 ,2,3]triazole-4-carbonitrile
Figure imgf000322_0001
HPLC-MS (Method C: m/z = 300 (M+1); Rt. = 3.79 min.
Example 959
4-(4-Bromophenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000322_0002
This compound is commercially available (MENAI).
Example 960
N-[4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)-phenyl]-acetamide
Figure imgf000322_0003
This compound is commercially available (MENAI).
Example 961 (General procedure (O)) 5-(4'-Chlorobiphenyl-4-yl)-3H-[1,2,3]triazole-4-carbonitrile
Figure imgf000323_0001
HPLC-MS (Method C): m/z = 281 (M+1); Rt = 4.22 min.
The compounds in the following examples are commercially available and may be prepared using a similar methodology:
Example 962
4-(4-Trifluoromethoxyphenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000323_0002
Example 963
4-Benzo[1 ,3]dioxol-5-yl-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000323_0003
Example 964
4-(3-TrifluoromethyIphenyl)-1 H-[1 ^.Sltriazole-S-carbonitrile
Figure imgf000324_0001
Example 965
4-Pyridin-3-yl-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000324_0002
Example 966
4-(2,6-Dichlorophenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000324_0003
Example 967
4-Thiophen-2-yl-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000324_0004
Example 968 3,5-Dimethylisoxa2oIe-4-carboxylic acid 4-(5-cyano-1H-[1 ,2,3]triazol-4-yl)phenyl ester
Figure imgf000325_0001
Example 969 3,3-Dimethyl-l acid 4-(5-cyano-1H-[1,2,3Jtriazol-4-y.)phenyl ester
Figure imgf000325_0002
Example 970
4-Methyl-[1 ,2,3]thiadia2θle-5-carboxylic acid 4-(5-cyano-1H-[1 ,2,3]tria2ol-4-yl)phenyl ester
Figure imgf000325_0003
Example 971
4-Chlorobenzoic acid 4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl ester
Figure imgf000325_0004
Example 972
4-(3-Phenoxyphenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000326_0001
Example 973
4-(5-Bromo-2-methoxyphenyl)-1 H-[1 ,2,3]triazole-5-carbonitril,
Figure imgf000326_0002
Example 974
4-(2-Chloro-6-fluorophenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile
Figure imgf000326_0003
The following cyanotriazoles are also compounds of the invention:
4-(2-Chloro-6-fluorophenyl)-1 H-[1 ,2,3]triazoIe-5-carbonitrile. Terephthalic acid mono[ 4-(5-cyano-1 H-[1 ,2,3]triazol-4-yl)phenyl] ester. N- f4-(5-cyano-1H-[1,2,3]triazol-4-yl)-phenyl]terephthalamic acid 4-(4-Octyloxyphenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile 4-(4-Styrylphenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile. 4-(4'-Trifluoromethylbiphenyl-4-yl)-1H-[1,2,3]trtazσle-5-carbonitrile. 4-(4'-ChIorobiphenyl-4-yI)-1 H-[1 ,2,3]triazole-5-carbonitrile. 4-(4'-Methoxybiphenyl-4-yl)-1 H-[1 ,2,3]triazoIe-5-carbonitrile. 4-(1 -Naphthyl)-1 H-[1 ,2,3]triazole-5-carbonitrile. 4-(9-Anthranyl)-1 H-[1 ,2,3]triazole-5-carbonitrile. 4-(4-Methoxy-1-naphthyl)-1 H-[1 ,2,3]triazole-5-carbonitrile. 4-(4-Aminophenyl)-1 H-[1 ,2,3]triazole-5-carbonitrile. 4-(2-Naphthyl)-1 H-[1 ,2,3]triazoIe-5-carbonitrile.
©eneral procedure (P) for preparation of compounds of general formula l15:
Figure imgf000327_0001
Step 3
Figure imgf000327_0002
wherein n is 1 or 3-20,
AA is as defined above,
R" is a standard carboxylic acid protecting group, such as d-Ce-alkyl or benzyl and Lea is a leaving group, such as chloro, bromo, iodo, methanesulfonyloxy, toluenesulfonyloxy or the like.
This procedure is very similar to general procedure (D), steps 1 and 2 are identical.
Steps 3 and 4 are described in the literature (eg Beck & GOnther, Chem. Ben, 106, 2758-66 (1973))
Step 3 is a Knoevenagel condensation of the aldehyde obtained in step 2 with phenylsulfon- ylacetonitrile and step 4 is a reaction of the vinylsulfonyl compound obtained in step 3 with sodium azide. This reaction is usually performed in DMF at 90 - 110 °C. This General procedure (P) is further illustrated in the following two examples
Example 975 (General procedure (P))
5-[6-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)-naphthalen-2-yloxy3-pentanoic acid ethyl ester
Figure imgf000328_0001
6-Hydroxynaphtha)ene-2-carbaldehyde (Syncom BV. NL, 15.5 g, 90 mmol) and K2C03 (62.2 g, 450 mmol) were mixed in DMF (300mL) and stirred at room temperature for 1hour. Ethyl 5-bromovalerate (21.65 g, 103.5 mmol) was added and the mixture was stirred at room temperature for 16 hours. Activated carbon was added and the mixture was filtered. The filtrate was evaporated to dryness in vacuo to afford 28.4 g of crude 5-(6-formylnaphthalen-2- yloxy)pentanoic acid ethyl ester, which was used without further purification.
HPLC-MS (Method C ): m/z = 301 (M+1); Rt. = 4.39 min.
5-(6-Formylnaphthalen-2-yfoxy)pentanoic acid ethyl ester (28.4 g, 94.5 mmol), phenylsulfon- ylacetonitrile (20.6 g, 113.5 mmol), and piperidine (0.94 mL) were dissolved in DMF (200 mL) and the mixture was heated at 50 °C for 16 hours. The resulting mixture was evaporated to dryness in vacuo and the residue was dried for 16 hours at 40 °C in vacuo. The solid was recrystallised from 2-propanol (800 mL) and dried again as described above. This afforded 35 g (80%) of 5-[6-(2-benzenesulfonyl-2-cyanovinyl)naphthalen-2-yloxy]pentanoic acid ethyl ester.
HPLC-MS (Method C): m/z = 486 (M+23); Rt. = 5.09 min.
5-[6-(2-Benzenesulfonyl-2-cyanovinyl)naphlhalen-2-yloxy]pentanoic acid ethyl ester (35 g, 74.6 mmol) and sodium azide (4.9 g, 75.6 mmol) were dissolved in DMF (100 mL) and stirred for 16 hours at 50 °C. The mixture was evaporated to dryness in vacuo, redissolved in THF / ethanol and a small amount of precipitate was filtered off. The resulting filtrate was poured into water (2.5 L). Filtration afforded after drying 24.5 g (88%) of 5-[6-(5-cyano-1H- [1,2,3]triazol-4-yl)naphthalen-2-yloxy]pentanoic acid ethyl ester (24.5 g, 88%).
HPLC-MS (Method C): m/z = 365 (M+1); Rt. = 4.36 min.
Example 976 (General procedure (B))
5-[6-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)-naphthalen-2-yloxy]-pentanoic acid
Figure imgf000329_0001
5-[6-(5-Cyano-1H-[1,2,3]triazol-4-yl)naphthalen-2-yloxy]pentanoicacid ethyl ester (24.5 g, 67.4 mmol) was dissolved in THF (150 mL) and mixed with sodium hydroxide (8.1 g, 202 mmol) dissolved in water (50 mL). The mixture was stirred for 2 days and the volatiles were evaporated in vacuo. The resulting aqueous solution was poured into a mixture of water (1 L) and hydrochloric acid (1N, 250 mL). The solid was isolated by filtration, dissolved in sodium hydroxide (1N, 200 mL), and the solution was washed with DCM and then ethyl acetate, the aquous layer was acidified with hydrochloric acid (12N). The precipitate was isolated by filtration, dissolved in THF / diethyl ether, the solution was treated with MgS0 and activated carbon, filtrated and evaporated in vacuo to almost dryness followed by precipitation by addition of pentane (1L). This afforded after drying in vacuo 17.2 g ( 76%) of the title compound.
HPLC-MS (Method C): m/z = 337 (M+1); Rt. = 3.49 min.
Example 977 (General procedure (P))
6-I6-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)naphthalen-2-yloxy]hexanoic acid
Figure imgf000330_0001
HPLC-MS (Method C): m/z = 351 (M+1); Rf = 3.68 min.
Example 978 (General procedure (P))
11-[6-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)-naphthalen-2-yIoxy]-undecanoic acid
Figure imgf000330_0002
HPLC-MS (Method C): m/z = 443 (M+23); Rt = 4.92 min.
Example 979 (General procedure (P))
2-{3-[6-(5-Cyano- H-(;i ,2,3]tr.azo)-4-yl)-naphthaJen-2-yJoxy]-propyl}-malonic acid diethyl ester
Figure imgf000330_0003
HPLC-MS (Method C): m/z = 465 (M+1); Rt. = 4.95 min.
Example 980 (General procedure (P))
2-{5-[6-(5-Cyano-1H-[1 ,2,3]triazol-4-yl)-naphthalen-2-yloxy]-pentyl}-maIonic acid diethyl ester
Figure imgf000331_0001
HPLC-MS (Method C): m/z = 465 (M+1); Rt. = 4.95 min.
Example 981 (General procedure (P))
2-{3-[6-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)-naphthalen-2-yloxy)-propyl)-malonic acid
Figure imgf000331_0002
HPLC-MS (Method C): m/z = 381 (M+1); Rt. = 3.12 min.
Example 982 (General procedure (P))
2-{5-[6-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)-naphthalen-2-yloxy]-pentyl}-malonic acid
Figure imgf000331_0003
HPLC-MS (Method C): m/z 0409 (M+1); Rt. a 3.51 min.
Example 983 (General procedure (P)) 4-[4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)-phen ic acid
Figure imgf000332_0001
HPLC-MS (Method C): m/z = 273 (M+1); Rt = 2.44 min.
The following compounds may be prepared according to this general procedure (P):
4-(4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)phenoxy)butyric acid:
Figure imgf000332_0002
2-(4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)phenoxy)acetic acid:
Figure imgf000332_0003
4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyric acid ethyl ester 5-(4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)phenoxy)pentanoic acid 8-(4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)phenoxy)octanoic acid 10-(4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yl)phenoxy)decanoic acid 12-(4-(5-Cyano-1 H-[1 ,2,3]triazol-4-yI)pheno)cy)dodecanoic acid General procedure (R) for preparation of compounds of general formula lι2:
Figure imgf000333_0001
Figure imgf000333_0002
wherein T is as defined above and R2 and R3 are hydrogen, aryl or lower alkyl, both optionally substituted.
The general procedure (R) is further illustrated by the following example:
Example 984 (General procedure (R)) Phenyl-[3-(2H-tetrazol-5-yl)-carbazol-9-yl]-methanone
Figure imgf000333_0003
2-Chlorotritylchloride resin (100 mg, 0.114 mmol active chloride) was swelled in dichloromethane (4 mL) for 30 minutes. The solvent was drained, and a solution of 3-(2H-tetrazol-5- yl)-9H-carba∑ole (80 mg, 0.34 mmol) in a mixture of N,N-dimethylformamide / dichloromethane / N,N-di(2-propyl)ethylamine (5:5:1) (3 mL) was added. The reaction mixture was shaken at room temperature for 20 hours. The solvent was removed by filtration, and the resin was washed thoroughly with N,N-dimethylformamide (2 x 4 mL) and dichloromethane (6 x 4 mL). A solution of 4-(dimethylamino)pyridine (14 mg, 0.11 mmol) and N,N-di(2- propyl)ethylamine (0.23 mL, 171 mg, 1.32 mmol) in N,N-dimethylformamide (2 mL) was added followed by benzoyl chloride (0.13 mL, 157 mg, 1.12 mmol). The mixture was shaken for 48 hours at room temperature. The drained resin was washed consecutively with dichloromethane (2 mL), methanol (2 x 4 mL) and tetrahydrofuran (4 rnL). The resin was treated for 2 hours at room temperature with a solution of dry hydrogen chloride in tetrahydrofuran / ethyl ether / ethanol = 8:1:1 (0.1 M, 3 mL). The reaction mixture was drained and concentrated. The crude product was stripped with dichloromethane (1.5 mL) three times to yield the title compound.
HPLC-MS (Method C): m/z: 340 (M+1); Rt = 3.68 min.
1H-NMR (DMSO-dβ): δ 8.91 (1H, s), 8.34 (1H, d), 8.05 (1H, d), 7.78 (3H, ), 7.63 (3H, ni),
7.46 (2H, m), 7.33 (1 H, dd).
The compounds in the following examples were prepared in a similar fashion.
Example 985 (General procedure (R)) PhenyI-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000334_0001
HPLC-MS (Method C): m/z: 290 (M+1); Rt = 3.04 min.
1H-NMR (DMSO-de): δ 8.46 (1H, d), 8.42 (1H, d), 8.08 (1H, dd), 7.82 (2H, d), 7.74 (1H, t),
7.64 (2H, t), 7.55 (1H, d), 6.93 (1H, d).
Example 986 (General procedure (R)) (2,3-DifJuorophenyl)-[5-(2H-ieirazoJ-5-yl)-indol-1-yl]-methanone
Figure imgf000335_0001
3LC- ■MS (Method B): m/z = 326 (M+1); Rt = 3.85 min.
Example 987 (General procedure (R)) (2-Fluoro~3-trifluoromethylphenyl)-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000335_0002
HPLC-MS (Method B): m/z = 376 (M+1); Rt = 4.32 min.
Example 988 (General procedure (R)) (3-Nitrophenyl)-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000335_0003
HPLC-MS (Method B): m/z = 335 (M+1); Rt = 3.72 min.
Example 989 (General procedure (R)) (4-Nitrophenyl)-[5-(2H-tetrazol-5-yl)-indol-1-yl]-methanone
Figure imgf000335_0004
HPLC-MS (Method B): m/z = 335 (M+1); Rt = 3.71 min. Example 990 (General procedure (R)) Naphthalen-2-yl-[5-(2H-tetrazoI-5-yl)-indol-1- 1-methanone
Figure imgf000336_0001
HPLC-MS (Method C): m/z = 340 (M+1); Rt = 4.25 min.
Example 991 (General procedure (R))
Figure imgf000336_0002
HPLC-MS (Method C): m/z: 354 (M+1); Rt = 3.91 min.
Example 992 (General procedure (R))
Figure imgf000336_0003
HPLC-MS (Method C): m/z: 418 (M+1); Rt = 4.39 min.
Example 993 (General procedure (R))
Figure imgf000336_0004
HPLC-MS (Method C): m/z: 370 (M+1); Rt = 4.01 min. Example 994 (General procedure (R))
Figure imgf000337_0001
HPLC-MS (Method C): m/z: 374 (M+1); Rt = 4.28 min.
Example 995 (General procedure (R))
Figure imgf000337_0002
HPLC-MS (Method C): m/z: 416 (M+1); Rt = 4.55 min.
Example 996 (General procedure (R))
Figure imgf000337_0003
HPLC-MS (Method C): m/z: 354 (M+1); Rt = 4.22 min. Example 997 (General procedure (R))
Figure imgf000338_0001
HPLC-MS (Method C): m/z: 358 (M+1); Rt = 3.91 min.
Example 998 (General procedure (R))
Figure imgf000338_0002
HPLC-MS (Method C): m/z: 390 (M+1); Rt = 4.38 min.
Example 999 (General procedure (R))
Figure imgf000338_0003
HPLC-MS (Method C): m/z: 418 (M+1); Rt = 4.36 min.
Example 1000 (General procedure (R))
Figure imgf000338_0004
HPLC-MS (Method C): m/z: 304 (M+1); Rt = 3.32 min. Example 1001 (General procedure (R))
Figure imgf000339_0001
HPLC-MS (Method C): m/z: 368 (M+1); Rt = 3.84 min.
Example 1002 (General procedure (R))
Figure imgf000339_0002
HPLC-MS (Method C): m/z: 320 (M+1); Rt = 3.44 min.
Example 1003 (General procedure (R))
Figure imgf000339_0003
HPLC-MS (Method C): m/z: 324 (M+1); Rt = 3.73 min. Example 1004 (General procedure (R))
Figure imgf000340_0001
HPLC-MS (Method C): m/z: 304 (M+1); Rf = 3.64 min.
Example 1005 (General procedure (R))
Figure imgf000340_0002
HPLC-MS (Method A): m/z: 308 (M+1); Rt = 3.61 min.
Example 1006 (General procedure (R))
Figure imgf000340_0003
HPLC-MS (Method C): m/z: 368 (M+1); Rt = 3.77 min.
Example 1007 (General procedure (R))
Figure imgf000340_0004
HPLC-MS (Method A): (sciex) m/z: 326 (M+1); Rt = 3.73 min. HPLC-MS (Method C): lz: 326 (M+1); Rt = 3.37 min.
Example 1008 (General procedure (R))
Figure imgf000341_0001
HPLC-MS (Method C): m/z: 374 (M+1); Rt = 4.03 min.
Example 1009
Preparation of NPH-insulin in the presence of ligands for the His810 Zn2+-site of the R-state insulin hexamer.
Preparations are prepared by mixing equal volumes of the following two solutions: a) 1.2 mM human insulin, 0.46 mM Zn2+, 28 mM phosphate, 1.6 % glycerol, 0.15 % m-cresol, 0.065 % phenol, and 0.46 mM ligand for the His610 2n2+-site (see below), optionally the ligand was added as a 9.2 mM DMSO solution, pH 7.5; and b) 0.636 mg/mL protamine sulphate 1.6 % glycerol, 0.15 % m-cresol, 0.065 % phenol, pH 6. The NPH-crystals grow overnight from the resulting suspension, pH 7.3.
Figure imgf000341_0002
Figure imgf000342_0001
Example 1010
Formulation of ligand-incorporated NPH-insulin preparation by addition of ligand for the His810 Zn2+-siie of the R-state insulin hexamer to pre-crystalli∑ed NPH-insulin.
The following four solutions are prepared: A. 2.4 mM Human Insulin 0.92 mM Zn2+
12.8 mM Hydrochloric acid 1.29 mg/ml Protamine sulphate 16 mg/ml Glycerol 1.5 mg/ml m-Crβsol 0.65 mg/ml Phenol
B. 28 mM Disodium hydrogen phosphate 1.2 mM Sodium hydroxide
16 mg/ml Glycerol 1.5 mg/ml m-Cresol 0.65 mg/ml Phenol
C. 0.92 mM 4-[3-( 1 H-Tetrazo)-5-yJ)-carbazol-9-ylmethyO-benzoic acid
(added as a 9.2 mM solution in DMSO) 14 mM Disodium hydrogen phosphate 16 mg/ml Glycerol 1.5 mg/ml m-Cresol 0.65 mg/ml Phenol pH adjusted to 7.3 with Hydrochloric acid.
D. 0.21 mg/ml Protamine sulphate
14 mM Disodium hydrogen phosphate
16 mg/ml Glycerol
1.5 mg/ml m-Cresol
0.65 mg/ml Phenol pH adjusted to 7.3 with Hydrochloric acid.
The ligand-incorporated NPH-insulin preparation is prepared by mixing equal volumes of the four solutions in the following manner:
Solutions A and B are mixed and the resulting suspension is adjusted to pH 7.3 and left overnight at 20-23°C for crystallisation. Solution C is then added with gentle agitation and after 30 minutes standing solution D is admixed. Example 1011
The glucose utilization effect following a subcutaneous injection of the NPH-insulin preparations of the present invention were characterized using a pig clamp model as described in Kurtzhals & Ribel, Diabetes 44, 1381-1385, 1995.
Figure 1 compares a regular NPH preparation to two NPH preparations formulated with different (stoichiometric/excess) concentrations compared to Zn2+ of 4-[3-(1 H-Tetrazol-5- yl)-carbazol-9-ylmethyl]-ben∑oic acid as described in the table below.
Figure imgf000345_0002
Figure imgf000345_0001
6 12 18 24 iur
Figure 1: Glucose utili∑ation after subcutaneous injection of a) 144nmol NPH (7 pigs), b) 144 nmol of NPH preparation with stoichiometric concentration of 4-[3-(1H-Tetrazol-5-yϊ)- carbazol-9-ylmethyl]-benzoic acid compared to Zn + (8 pigs) and c) 144nmol of NPH preparation with excess concentration of 4-[3-(1 H-Tetrazol-5-yl)-carbazol-9-ylmethyl]-benzoic acid compared to Zn2+ (8 pigs). The results are expressed as means ± SE.
ANALYTICAL METHODS
TZD-assay for quantitatiαn of ligands binding to the R-state HϊsB10Zn2+:
The binding affinity of ligands to the metal site of insulin R6 hexamers are measured in a fluo- rescense based displacement assay. The fluorescence of 5-(4- dimethylaminobenzylidene)thiazolidine-2,4-dione (TZD) which is a ligand for the metal site of insulin R6 is quenched upon displacement from the metal site to the solution. Titration of a ligand to a stock solution of insulin R6 hexamers with this compound mounted in the metal site allows the binding affinity of these ligands to be determined measuring the fluorescence at 455nm upon excitation at 410nm.
Preparation
Stock solution: 0.02 mM human insulin, 0.007 mM Zn-acetate, 40 mM phenol, 0.01 mM TZD in 50mM tris buffer adjusted to pH=8.0 with NaOH/CI04 ".
The ligand is dissolved in DMSO to a concentration of 5 mM and added in aliquots to the stock solution to final concentrations of 0-250 μM.
Measurements
Fluorescence measurements were carried out on a Perkin Elmer Spectrofluorometer LSδOB.The main absorption band was excited at 410 nm and emission was detected at 455 nm. The resolution was 10 nm and 2.5 nm for excitation and emission, respectively.
Data analysis
This equation is fitted to the datapoints
ΔF(455nm)) = ΔFmax * [ligand]free/( KD(app) * ( 1 +|TZD]/KTZD )+ [ligand]free))
KD(app) is the apparent dissociation constant and Fmax is the fluorescence at maximal ligand concentration. The value of JZD is measured separately to 230 nM Two different fitting-procedures can be used. One in which both parameters, KD(app) and Fmax, are adjusted to best fit the data and a second in which the value of Fmax is fixed (Fmax=1) and only KD(app) is adjusted. The given data are from the second fitting procedure. The Solver module of Microsoft Excel can be used to generate the fits from the datapoints.
H3M-assay:
The binding affinity of ligands to the metal site of insulin Re hexamers are measured in a UV/vis based displacement assay. The UV/vis spectrum of 3-hydroxy-4-nitro benzoic acid (4H3N) which is a known ligand for the metal site of insulin R6 shows a shift in absorption maximum upon displacement from the metal site to the solution (Huang et al., 1997, Biochemistry 36, 9878-9888). Titration of a ligand to a solution of insulin R6 hexamers with 4H3N mounted in the metal site allows the binding affinity of these ligands to be determined following the reduction of absorption at 444 nm.
A stock solution with the following composition 0.2 mM human insulin, 0.067 mM Zn-acetate, 40 mM phenol, 0.101 mM 4H3N is prepared in a 10mL quantum as described below. Buffer is always 50mM tris buffer adjusted to pH=8.0 with NaOH/CI04 ".
1000 μL of 2.0mM human insulin in buffer 66.7 μL of 10mM Zn-acetate in buffer 800 μL of 500mM phenol in H20 201 μL of 4H3N in H20 7.93 ml buffer
The ligand is dissolved in DMSO to a concentration of 20 mM.
The ligand solution is titrated to a cuvette containing 2 mL stock solution and after each addition the UV/vis spectrum is measured. The titration points are listed in Table 3 below.
Table 3
Figure imgf000348_0001
The UV/vis spectra resulting from a titration of the compound 3-hydroxy-2-naphthoic acid is shown in Figure 5. Inserted in the upper right corner is the absorbance at 444nm vs. the concentration of ligand.
The following equation is fitted to these datapoints to determine the two parameters KD(obs), the observed dissociation constant, and absmax the absorbance at maximal ligand concentration.
abs gandjfree) = (absmax * [ligand]free)/ (KD(obs) + [ligand]free)
The observed dissociation constant is recalculated to obtain the apparent dissociation constant
KD(app) = KD(obs) / ( 1 +[4H3N]/K4H3N )
The value of K4H3N=50 /M is taken from Huang et al., 1997, Biochemistry 36, 9878-9888.

Claims

1. Pharmaceutical preparation comprising o Insulin o Protamine o Zinc ions o ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer, wherein said ligand is selected from the group consisting of carboxylates, dithiocar- boxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imida∑oles, tria- zoles, 4-cyano-1 ,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thymines, thiazolidinediones, tetrazoles, 5-mercaptotetra∑oles, rhodanines, N-hydroxya∑oles, hydantoines, thiohydantoines, naphthoic acids and salicylic acids, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
2. A pharmaceutical preparation according to claim 1 wherein the insulin preparation comprises 60 to 3000 nmol/ml of insulin.
3. A pharmaceutical preparation according to claim 2 wherein the insulin preparation comprises 240 to 1200 nmol/ml of insulin.
4. A pharmaceutical preparation according to claim 3 wherein the insulin preparation comprises about 600 nmol/ml of insulin.
5. A pharmaceutical preparation according to any one of the claims 1 to 4 wherein the insulin is selected from the group consisting of human insulin, an analogue of human insulin, a derivative of human insulin, and combinations of any of these
6. A pharmaceutical preparation according to claim 5 wherein the insulin is an analogue of human insulin selected from the group consisting of i. An analogue wherein position B28 is Asp, Glu, Lys, Leu, Val, or Ala and position
B29 is Lys or Pro; ii. An analogue wherein position B3 is Lys and position B29 is Glu; and iii. des(B28-B30), des(B27) or des(B30) human insulin.
7. A pharmaceutical preparation according to claim 6, wherein the insulin is an analogue of human insulin wherein position B28 is Asp or Lys, and position B29 is Lys or Pro.
8. A pharmaceutical preparation according to claim 6 wherein the insulin is des(B30) human insulin.
9. A pharmaceutical preparation according to claim 5 wherein the insulin is a derivative of human insulin having one or more lipophilic substituents.
10. A pharmaceutical preparation according to claim 9 wherein the insulin derivative is selected from the group consisting of B29-Nc-myristoyl-des(B30) human insulin, B29-Nε- palmitoyl-des(BSO) human insulin, B29-Nε-myristoyl human insulin, B29-Nε-palmitoyl human insulin, B28-Nε-myristoyl LysB28 ProB29 human insulin, B28-Nε-palmitoyl LysB28ProB29 human insulin, BSO-N^myristoyl-Th^Lys630 human insulin, B30-Nε-palmitoyl-ThrB29LysB30 human insulin, B29-Nε-(N-palmitoyl-γ-glutamyl)-des(B30) human insulin, B29-Nε-(N-lithocholyl-γ- glutamyl)-des(BSO) human insulin, B29-Nε-(co-carboxyheptadecanoyl)-des(B30) human insulin and B29-Nε-(ω-carboxyheptadecanoyl) human insulin.
11. A pharmaceutical preparation according to claim 10 wherein the insulin derivative is B29- Nε-myristoyl-des(B30) human insulin.
12. A pharmaceutical preparation according to claim 10 wherein the insulin derivative is B29- Nε-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin
13. A pharmaceutical preparation according to any one of the claims 1 to 12 wherein the protamine is protamine sulphate.
14. A pharmaceutical preparation according to claim 13 wherein the concentration of protamine sulphate is from 0.05-3 mg/mL.
15. A pharmaceutical preparation according to claim 14 wherein the concentration of protamine sulphate is from 0.1-0.6 mg/mL.
16. A pharmaceutical preparation according to any one of the claims 1 to 15 wherein the amount of zinc ions is 2-6 moles per mole putative insulin hexamer.
17. A pharmaceutical preparation according to claim 16 wherein the amount of zinc ions is 2 to 3 moles per mole putative insulin hexamer.
18. A pharmaceutical preparation according to any one of the claims 1 to 17 wherein the ratio of ligand that binds reversibly to a HisB1° Zn2+ site of an R-state insulin hexamer to zinc ions is 1:3 to 3:1.
19. A pharmaceutical preparation according to claim 18 wherein the ratio of ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer to zinc ions is 1:2 to 2:1.
20. A pharmaceutical preparation according to claim 19 wherein the ratio of ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer to zinc ions is 1 :.2 to 1.2:1.
21. A pharmaceutical preparation according to any one of the claims 1 to 20 wherein the ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer is a chemical structure selected from the group consisting of carboxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles, 4-cyano-1,2,3-triazoles, benzimida∑oles, benzotria∑oles, purines, thymines, thiazolidinediones, tetra∑oles, 5- mercaptotetra∑oles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, naphthoic acids and salicylic acids.
22. A pharmaceutical preparation according to claim 21 wherein the ligand that binds reversibly to a His810 Zn2+ site of an R-state insulin hexamer is a chemical structure selected from the group consisting of benzotriazoles, 3-hydroxy 2-napthoic acids, salicylic acids, tetrazoles or thiazolidinediones
23. A pharmaceutical preparation according to claim 22 wherein the zinc-binding ligand is
Figure imgf000351_0001
wherein
X is =0, =S or =NH
Y is -S-, -O- or -NH-
R\ R1A and R4 are independently selected from hydrogen or d-Ce-alkyl, R2 and R2* are hydrogen or Cι-C6-alkyl or aryl, R1 and R2 may optionally be combined to form a double bond, R1A and R2* may optionally be combined to form a double bond, R3, R3A and R5 are independently selected from hydrogen, halogen, aryl optionally substituted with one or more substituents independently selected from R16, CrCe-alkyl, or -C(0)NR11R12,
A, A1 and B are independently selected from d-Ce-alkyl, aryl, aryl-C C6-alkyl, -NR11-aryl, aryl-C2-C6-alkenyl or heteroaryl, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R6 and the aryl or heteroaryl is optionally substituted with up to four substituents R7, R8, R9, and R10,
A and R3 may be connected through one or two valence bonds, B and R5 may be connected through one or two valence bonds, R6 is independently selected from halogen, -CN, -CF3, -OCF3, aryl, -COOH and -NH2, R7, R8, R9 and R10 are independently selected from
jen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3 -OCF2CHF2, -S(0)2CF3, -OS(0)2CF3, -SCF3, -N02, -OR11, -NR11R12, -SR11 -NR11S(0)2R12, -S(0)2NR11R12, -S(0)NR11R12, -S(0)R11, -S(0)2R11, -OS(0)2 R11 -C(0)NR11R12, -OC(O)NR11R12, -NR11C(0)R12, -CH2C(0)NR11R12
-Od-C6-alkyl-C(0)NR11R12, -CH2OR11, -CH2OC(0)R11, -CH2NR11R12, -OC(0)R11 -Od-C15-alkyl-C(0)OR11, -OCι-C6-alkyl-OR11, -SC C6-alkyl-C(0)OR11 -C2-C6-alkenyl-C(=0)OR11, -NR11-C(=0)-C1-C6-alkyl-C(=0)OR11
-NR11-C(=0)-C C6-alkenyl-C(=0)OR11 , -C(0)OR11, C(0)R11, or -C2-C6-alkenyl- C(=0)R11, =0, or -C2-C6-alkenyI-C(=0)-NR11R12,
• CrC6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, each of which may optionally be substituted with one or more substituents independently selected from R13,
• aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-d-C6-alkoxy, aryl-CrCβ-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryJ-d- Ce-alkyl, heteroaryl-C2-C6-alkenyl, heteroaryl-C2-C6-alkynyI, or C3-C6 cycloalkyl,
of which each cyclic moiety may optionally be substituted with one or more substituents independently selected from R14,
R11 and R12 are independently selected from hydrogen, OH, CrC20-alkyl, aryl-CrC6-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R15, and the aryl groups may optionally be substituted one or more substituents independently selected from R1δ; R11 and R12 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R13 is independently selected from halogen, -CN, -CF3, -OCF3, -OR11, -C(0)OR11 , -NR11R12, and -C(0)NR11R12, R14 is independently selected from halogen, -C(0)OR11, -CH2C(0)OR11, -CH2OR11, -CN, - CF3, -OCF3, -N02, -OR11, -NR11R12, -NR11C(0)R11, -S(0)2R11, aryl and C C6-alkyl,
R15 is independently selected from halogen, -CN, -CF3, =0, -0CF3, -OC C6-alkyl, -C(0)OC Cβ-alkyl, -COOH and -NH2,
R16 is independently selected from halogen, -C(0)Od-C6-alkyl, -COOH, -CN, -CF3, -OCF3, - N02, -OH, -Od-Ce-alkyl, -NH2, C(=0) or C C6-alkyl, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
24. A pharmaceutical preparation according to claim 23 wherein X is =0 or =S.
25. A pharmaceutical preparation according to claim 24 wherein X is =0.
26. A pharmaceutical preparation according to claim 24 wherein X is =S.
27. A pharmaceutical preparation according to any one of the claims 23 to 26 wherein Y is -O- or -S-.
28. A pharmaceutical preparation according to claim 27 wherein Y is -0-.
29. A pharmaceutical preparation according to claim 27 wherein Y is -NH-.
30. A pharmaceutical preparation according to claim 27 wherein Y is -S-.
31. A pharmaceutical preparation according to any one of the claims 23 to 30 wherein A is aryl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
32. A pharmaceutical preparation according to claim 31 wherein A is selected from ArG1 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
33. A pharmaceutical preparation according to claim 32 wherein A is phenyl or naphtyl optionally substituted with up to four substituents, R7, R8, R9, and R 0 which may be the same or different.
34. A pharmaceutical preparation according to claim 33 wherein A is
Figure imgf000353_0001
35. A pharmaceutical preparation according to claim 33 wherein A is phenyl.
36. A pharmaceutical preparation according to any one of the claims 23 to 30 wherein A is heteroaryl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
37. A pharmaceutical preparation according to claim 36 wherein A is selected from Het1 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different, and wherein Het1 is furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyra∑olyl, oxazolyl, thia- ∑olyl, imidazolyl, isoxa∑olyl, isothia∑olyl, 1 ,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridyl, pyri- dazinyl, pyrimidinyl, pyrazinyl, 1 ,2,3-tria∑inyl, 1 ,2,4-triazinyl, 1,3,5- triazinyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadia∑olyl, 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, 1 ,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, ben- ∑othienyl, indazolyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl, thiazolidinyl, or 2-thiooxothiazolidinyl.
38. A pharmaceutical preparation according to claim 37 wherein A is selected from Het2 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different, and wherein Het2 is furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thia- zolyl, imidazolyl, isoxazoiyl, isothiazolyl, 1 ,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridyl, pyri- dazinyl, pyrimidinyl, pyrazinyl, 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, 1,3,5- triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, 1 ,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, ben- zothienyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, carbazolyl, thiazolidinyl, or 2-thiooxothiazolidinyl.
39. A pharmaceutical preparation according to claim 38 wherein A is selected from Het3 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different, and wherein Het3 is furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thia- zolyl, imidazolyl, isoxazoiyl, isothiazolyl, 1 ,2,3-triazolyl, 1,2,4-triazolyl, pyridyl, tetrazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, quinolyl, isoquinolyl, quinoxalinyl, carbazolyl, thiazolidinyl, or 2- thiooxothiazolidinyl.
40. A pharmaceutical preparation according to claim 39 wherein A is selected from the group consisting of indolyl, benzofuranyl, quinolyl, furyl, thienyl, or pyrrolyl, wherein each heteroaryl may optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
41. A pharmaceutical preparation according to claim 39 wherein A is benzofuranyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
42. A pharmaceutical preparation according to claim 41 wherein A is
Figure imgf000355_0001
43. A pharmaceutical preparation according to claim 39 wherein A is carbazolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
44. A pharmaceutical preparation according to claim 43 wherein A is
Figure imgf000355_0002
45. A pharmaceutical preparation according to claim 39 wherein A is quinolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
46. A pharmaceutical preparation according to claim 45 wherein A is
Figure imgf000355_0003
47. A pharmaceutical preparation according to claim 39 wherein A is indolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
48. A pharmaceutical preparation according to claim 47 wherein A is
Figure imgf000355_0004
49. A pharmaceutical preparation according to any one of the claims 23 to 48 wherein R is hydrogen.
50. A pharmaceutical preparation according to any one of the claims 23 to 49 wherein R2 is hydrogen.
51. A pharmaceutical preparation according to any one of the claims 23 to 48 wherein R1 and R2 are combined to form a double bond.
52. A pharmaceutical preparation according to any one of the claims 23 to 51 wherein R3 is CrCe-alkyl, halogen, or C(0)NR16R17.
53. A pharmaceutical preparation according to claim 52 wherein R3 is d-Ce-alkyl or C(0)NR16R17.
54. A pharmaceutical preparation according to claim 53 wherein R3 is methyl.
55. A pharmaceutical preparation according to any one of the claims 23 to 30 wherein B is phenyl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
56. A pharmaceutical preparation according to any one of the claims 23 to 30 or 55 wherein R4 is hydrogen.
57. A pharmaceutical preparation according to any one of the claims 23 to 30 or 55 to 56 wherein R5 is hydrogen.
58. A pharmaceutical preparation according to any one of the claims 23 to 57 wherein R6 is aryl.
59. A pharmaceutical preparation according to claim 58 wherein R6 is phenyl.
60. A pharmaceutical preparation according to any one of the claims 23 to 59 wherein R7, R8, R9 and R10 are independently selected from
• hydrogen, halogen, -N02, -OR11, -NR11R12, -SR11, -NR11S(0)2R12, -S(0)2NR11R12, -S(0)NR11R12, -S(0)R11, -S(0)2R11, -OS(0)2 R11, -NR11C(0)R12, -CH2OR11, - CH2OC(0)R11, -CH2NR11R12, -OC(0)R11, -Od-C6-alkyl-C(0)OR11, -OC C6- alkyl-C(0)NR11R12, -Od-C6-alkyl-OR11, -SC C6-alkyl-C(0)OR11, -C2-C6-alkenyl- C(=0)OR11, -C(0)OR11, or-C2-C6-alkeny.-C(=0)R11,
• CrCe-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may each optionally be substituted with one or more substituents independently selected from R13
oaryl, aryloxy, aroyl, arylsulfanyl, aryl-CrC6-alkoxy, aryl-CrC6-alkyl, aryl-C2- Ce-alkenyl, aroyl-C2-C6-alkenyl, aryl-d-Ce-alkynyl, heteroaryl, heteroaryl-CrC6-alkyl, wherein each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14
61. A pharmaceutical preparation according to claim 60 wherein R7, R8, R9 and R10 are independently selected from
ohydrogβn, halogen, -N02, -OR11, -NR11R12, -SR11, -S(0)2R11, -OS(0)2 R11, - CH2OC(0)R11, -OC(0)R11, -OC C6-alkyl-C(0)OR11, -Od-C6-alkyl-OR11, -SC C6- -C(0)OR11, -C(0)OR11, or-C2-C6-alkenyi-C(=0)R11,
o d-Ce-alkyl or Cι-C6-alkenyl which may each optionally be substituted with one or more substituents independently selected from R13
• aryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-CrC6-alkyl, heteroaryl,
of which each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14
62. A pharmaceutical preparation according to claim 61 wherein R7, R8, R9 and R10 are independently selected from
• hydrogen, halogen, -N02, -OR11, -NR11R12, -SR11, -S(0)2R11, -OS(0)2 R11, - CH2OC(0)R11, -OC(0)R11, -Od-C6-alkyl-C(0)OR11, -OC C6-alkyl-OR11, -Sd-C6- alkyl-C(0)OR11, -C(0)OR11, or-C2-C6-alkenyl-C(=0)R11,
• CrCe-alkyl or Ci-Ce- which may each optionally be substituted with one or more substituents independently selected from R13
• aryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-d-C6-alkyl, heteroaryl,
of which each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14.
63. A pharmaceutical preparation according to claim 62 wherein R7, R8, R9 and R10 are independently selected from
-rogen, halogen, -OR11, -0CrC6-alkyl-C(0)0R1\ or -C(0)OR11, • CrCe-alkyl which may each optionally be substituted with one or more substituents independently selected from R13
°aryl, aryloxy, aryl-d-Ce-alkoxy,
of which each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14.
64. A pharmaceutical preparation according to claim 63 wherein R7, R8, R9 and R10 are independently selected from o hydrogen, halogen, -OR11, -OCrC6-alkyl-C(0)OR11, or -C(0)OR11,
• d-d-alky! which may each optionally be substituted with one or more substituents independently selected from R13
• ArG1 , ArGloxy, ArG1-CrC6-alkoxy,
of which each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14.
65. A pharmaceutical preparation according to claim 64 wherein R7, R8, R9 and R10 are independently selected from
• hydrogen, halogen, -OR11, -OC C6-alkyl-C(0)OR11, or -C(0)OR11,
• d-C6-alkyl which may optionally be substituted with one or more substituents independently selected from R13
• phenyl, phenyloxy, phenyl-d-Ce-alkoxy, wherein each of the cyclic moieties optionally may be substituted with one or more substituents independently selected from R14.
66. A pharmaceutical preparation according to any one of the claims 23 to 65 wherein R11 and R12 are independently selected from hydrogen, d-do-alkyl, aryl or aryl-d-Ce-alkyl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R15, and the aryl groups may optionally be substituted one or more substituents independently selected from R16; R and R12 when attached to the same nitro- gen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds.
67. A pharmaceutical preparation according to claim 66 wherein R11 and R12 are independently selected from hydrogen, d-do-alkyl, aryl or aryl-CrC6-alkyl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R15, and the aryl groups may optionally be substituted one or more substituents independently selected from R16.
68. A pharmaceutical preparation according to claim 67 wherein R11 and R12 are independently selected from phenyl or phenyl-d-Ce-alkyl.
69. A pharmaceutical preparation according to claim 67 wherein one or both of R11 and R12 are methyl.
70. A pharmaceutical preparation according to any one of the claims 23 to 69 wherein R13 is independently selected from halogen, CF3, OR11 or NR11R12.
71. A pharmaceutical preparation according to claim 70 wherein R13 is independently selected from halogen or OR11.
72. A pharmaceutical preparation according to claim 71 wherein R13 is OR11.
73. A pharmaceutical preparation according to any one of the claims 23 to 72 wherein R14 is independently selected from halogen, -C(0)OR11, -CN, -CF3, -OR11, S(0)2R11, and C Ce-alkyl.
74. A pharmaceutical preparation according to claim 73 wherein R14 is independently selected from halogen, -C(0)OR11, or -OR11.
75. A pharmaceutical preparation according to any one of the claims 23 to 74 wherein R15 is independently selected from halogen, -CN, -CF3, -C(0)Od-Ce-a!kyl, and -COOH.
76. A pharmaceutical preparation according to claim 75 wherein R15 is independently selected from halogen or -C(0)OC C6-alkyl.
77. A pharmaceutical preparation according to any one of the claims 23 to 76 wherein R16 is independently selected from halogen, -C(0)OCrC6-alkyl, -COOH, -N02, -Od-d-alkyl, -NH2, C(=0) or CrCe-alkyl.
78. A pharmaceutical preparation according to claim 77 wherein R16 is independently selected from halogen, -C(0)OCrC6-alkyl, -COOH, -N02, or d-C6-alkyl.
79. A pharmaceutical preparation according to claim 22 wherein the zinc-binding ligand is
Figure imgf000360_0001
wherein
R19 is hydrogen or d-Ce-alkyl,
R20 is hydrogen or d-d-alky!,
D, D1 and F are a valence bond, d-d-alkylene or CrC6-alkenylene optionally substituted with one or more substituents independently selected from R72,
R is independently selected from hydroxy, d-C6-aIkyl, or aryl,
E is d-Cβ-alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents R2 , R22 and R23,
G and G1 are d-C6-alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents R24, R25 and R26,
R17, R18, R21, R22, R23, R24, R25 and R26 are independently selected from
• hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(0)2CF3, -SCF3, -N02, =0, -OR27, -NR27R28, -SR27, -NR27S(0)2R28, -S(0)2NR27R28, -S(0)NR27R28, -S(0)R27, -S(0)2R27, -C(0)NR27R28, -OC(0)NR27R28, -NR27C(0)R28, -NR27C(0)OR28, -CH2C(0)NR27R28, -OCH2C(0)NR27R28, -CH2OR27, -CH2NR27R28, -OC(0)R27, -Od-C6-alkyl-C(0)OR27, -SCrC6-alkyl-C(0)OR27, -C2-C6- alkenyl-C(=0)OR27, -NR27-C(=0)-CrC6-alkyl-C(=0)OR27, -NR27-C(=0)-d-C6- alkenyl-C(=0)OR27, -C(=0)NR27-CrC6-alkyl-C(=O)OR27, -CrC6-alkyl-C(=0)OR27,or -C(0)OR27,
o d-Cβ-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may optionally be substituted with one or more substituents independently selected from R29,
oaryl, aryloxy, aryloxycarbonyl, aroyl, aryl-d-Ce-alkoxy, aryl-Crd-alkyl, aryl-C2- Ce-alkenyl, aryl-C2-C6-alkyn l, heteroaryl, heteroaryl-Cι-C6-alkyl, heteroaryl-C2-C6- alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30,
R27 and R28 are independently selected from hydrogen, d-Ce-alkyl, aryl-d-Ce-alkyl or aryl, or R27 and R28 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R29 is independently selected from halogen, -CN, -CF3l -OCF3, -OR27, and -NR27R28,
R30 is independently selected from halogen, -C(0)OR27, -CN, -CF3, -OCF3, -N02, -OR27, -NR27R28 and d-Ce-alkyl, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
80. A pharmaceutical preparation according to claim 79 wherein D is a valence bond.
81. A pharmaceutical preparation according to claim 79 wherein D is CrC6-alkylene optionally substituted with one or more hydroxy, d-Cβ-alkyl, or aryl.
82. A pharmaceutical preparation according to any one of the claims 79 to 81 wherein E is aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents independently selected from R21, R22 and R23.
83. A pharmaceutical preparation according to claim 82 wherein E is aryl optionally substituted with up to three substituents independently selected from R21, R22 and R23.
84. A pharmaceutical preparation according to claim 83 wherein E is selected from ArG1 and optionally substituted with up to three substituents independently selected from R21, R22 and R23.
85. A pharmaceutical preparation according to claim 84 wherein E is phenyl optionally substituted with up to three substituents independently selected from R21, R22 and R23.
86. A pharmaceutical preparation according to claim 85 wherein the zinc-binding ligand is
Figure imgf000362_0001
87. A pharmaceutical preparation according to any one of the claims 79 to 86 wherein R21, R22 and R23 are independently selected from
o hydrogen, halogen, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -SCF3, - N02, -OR27, -NR27R28, -SR27, -C(0)NR27R28, -OC(0)NR27R28, -NR27C(0)R28, -NR27C(0)OR28, -CH2C(0)NR27R28, -OCH2C(O)NR27R28, -CH2OR27, -CH2NR27R28, -OC(0)R27, -OC C6-alkyl-C(0)OR27, -Sd-C6-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -NR27-C(=0)-d-C6-alkyl-C(=0)OR27, -NR27-C(=0)-d-C6- alkenyl-C(=0)OR27-, -C(=0)NR27-CrC6-alkyl-C(=0)OR27, -d-C6-alkyl-C(=0)OR27, or -C(0)OR27,
• CrCe-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
which may optionally be substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-Crd-alkoxy, aryl-d-Ce-alkyl, aryl-C2- Ce-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-CrC6-alkyl, heteroaryl-C2-C6- alkenyl or heteroaryl-C2-Ce-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
88. A pharmaceutical preparation according to claim 87 wherein R21, R22 and R23 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(0)R28, -NR27C(0)OR28, -OC(0)R27, -OCrC6-alkyl-C(0)OR27, -Sd-C6-aIkyl-C(0)OR27, -C2-C6- C(=0)OR27, -C(=0)NR27-CrC6-alkyl-C(=0)OR27, -CrC6-alkyf-C(=0)OR27, or -C(0)OR27,
o d-C6-alkyl optionally substituted with one or more substituents independently selected from R29
oaryl, aryloxy, aroyl, aryl-C C6-alkoxy, aryl-d-C6-alkyl, heteroaryl, heteroaryl-CrCβ-
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
89. A pharmaceutical preparation according to claim 88 wherein R21, R22 and R23 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(0)R28, -NR27C(0)OR28, -OC(0)R27, -Od-C6-alkyl-C(0)OR27, -Sd-C6-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -C(=0)NR27-d-C6-alkyl-C(=0)OR27, -CrCθ-alkyl-C(=0)OR27, or -C(0)OR27,
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-d-Ce-alkyl, heteroaryl, heteroaryl-d-C6- alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
90. A pharmaceutical preparation according to claim 89 wherein R21, R22 and R23 are independently selected from
» hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(0)R28, -NR27C(0)OR28, -OC(0)R27, -OCrC6-alkyl-C(0)OR27, -SC C6-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -C(=O)NR27-d-C6-alkyl-C(=0)OR27, -d-C6-alkyl-C(=0)OR27, or -C(0)OR27, • methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
oArG1, ArG1-0-, ArG1-C(0)-, ArG1-CrC6-alkoxy, ArG1-CrC6-alkyl, Het3, Het3-C
Ce-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
91. A pharmaceutical preparation according to claim 90 wherein R21, R22 and R23 are independently selected from
° hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(0)R28, -NR27C(0)OR28, -OC(0)R27, -OCrC6-alkyl-C(0)OR27, -SCrC6-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -C(=0)NR27-CrC6-alkyl-C(=0)OR27, -CrC6-alkyl-C(=0)OR27, or -C(0)OR27,
• C C6-alkyl optionally substituted with one or more substituents independently selected from R29
• phenyl, phenyloxy, phenyl-d-Ce-alkoxy, phenyl-C C6-alkyl, of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
92. A pharmaceutical preparation according to any one of the claims 79 to 91 wherein R19 is hydrogen or methyl.
93. A pharmaceutical preparation according to claim 92 wherein R19 is hydrogen.
94. A pharmaceutical preparation according to any one of the claims 79 to 93 wherein R27 is Hydrogen, d-Ce-alkyl or aryl.
95. A pharmaceutical preparation according to claim 94 wherein R27 is hydrogen or CrCe- alkyl.
96. A pharmaceutical preparation according to any one of the claims 79 to 95 wherein R28 is hydrogen or d-C6-alkyl.
97. A pharmaceutical preparation according to claim 79 wherein F is a valence bond.
98. A pharmaceutical preparation according to claim 79 wherein F is CrC6-alkylene optionally substituted with one or more hydroxy, d-Ce-alkyl, or aryl.
99. A pharmaceutical preparation according to any one of the claims 79 or 97 to 98 wherein G is d-Ce-alkyl or aryl, wherein the aryl is optionally substituted with up to three substituents R24, R25 and R26.
100. A pharmaceutical preparation according to any one of the claims 79 or 97 to 98 wherein G is d-Ce-alkyl or ArG1 , wherein the aryl is optionally substituted with up to three substituents R24, R25 and R26.
101. A pharmaceutical preparation according to claim 99 wherein G is d-Ce-alkyl.
102. A pharmaceutical preparation according to claim 101 wherein G is phenyl optionally substituted with up to three substituents R24, R25 and R26.
103. A pharmaceutical preparation according to any one of the claims 79 to 102 wherein R24, R25 and R26 are independently selected from
• hydrogen, halogen, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2) -SCF3, - N02, -OR27, -NR27R28, -SR27, -C(0)NR27R28, -OC(0)NR27R28, -NR 7C(0)R28, -NR27C(0)OR28, -CH2C(0)NR27R28, -OCH2C(0)NR27R28, -CH2OR27, -CH2NR27R28, -OC(0)R27, -OC C6-alkyl-C(0)OR27, -Sd-C6-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -NR27-C(=0)-d-C6-alkyl-C(=0)OR27, -NR27-C(=0)-d-C6- alkenyl-C(=0)OR27-, -C(=0)NR27-C C6-alkyl-C(=0)OR27, -d-C6-alkyl-C(=0)OR27, or -C(0)OR27,
• CrCe-alkyl, C2-C6-alkenyl or C2-C6-aJkynyJ,
which may optionally be substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-Crd-alkoxy, aryl-CrC6-alkyl, aryl-C2- Ce-alkenyl, aryl-C2-Ce-alkynyl, heteroaryl, heteroaryl-CrC6-alkyl, heteroaryl-C2-C6- alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
104. A pharmaceutical preparation according to claim 103 wherein R24, R25 and R26 are independently selected from • hydrogen, halogen, -OCF3, -OR27, -NR27R2S, -SR27, -NR27C(0)R28, -NR27C(0)OR28, -OC(0)R27, -OCrC6-alkyl-C(0)OR27, -Sd-C6-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -C(=0)NR27-CrC6-alkyl-C(=0)OR27, -d-C6-alkyl-C(=0)OR27, or -C(0)OR27,
o CrC6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
which may optionally be substituted with one or more substituents independently selected from R29
oaryl, aryloxy, aryloxycarbonyl, aroyl, aryl-d-d-alkoxy, aryl-d-C6-alkyl, ary C2- Ce-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-d-Ce-alkyl, heteroaryl-d-Ce- alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
105. A pharmaceutical preparation according to claim 104 wherein R24, R25 and R26 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(0)R28, -NR27C(0)OR28, -OC(0)R27, -Od-C6-alkyl-C(0)OR27, -Sd-Ce-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -C(=0)NR27-d-C6-alkyl-C(=0)OR27, -d-C6-alkyl-C(=0)OR27, or -C(0)OR27,
• CrC6-alkyl optionally substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-Crd-alkyl, heteroaryl, heteroaryl-d-Ce- alkyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
106. A pharmaceutical preparation according to claim 105 wherein R21, R22 and R23 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(0)R28, -NR27C(0)OR28, -OC(0)R27, -0CrC6-alkyl-C(0)0R27, -SC C6-all.yl-C(0)0R27, -C2-C6-alkenyl- C(=0)0R27, -C(=0)NR27-C C6-alkyl-C(=0)0R27, -CrC6-alkyl-C(=0)0R27, or -C(0)OR27,
o methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• ArG1, ArG1-O-, ArG1-C(0)-, ArG1-C C6-alkoxy, ArG1-C C6-alkyl, Het3, Het3-d- Ce-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
107. A pharmaceutical preparation according to claim 106 wherein R21, R22 and R23 are independently selected from
• hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(0)R28, -NR27C(0)OR28, -OC(0)R27, -OCrC6-alkyl-C(0)OR27, -SCrC6-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -C(=0)NR27-CrC6-alkyl-C(=0)OR27, -CrC6-alkyl-C(=0)OR27, or -C(0)OR27,
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• ArG1 , ArG1-0-, ArG1-C(0)-, ArG1-C C6-alkoxy, ArG1-CrC6-alkyl, Het3, Het3-C Ce-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
108. A pharmaceutical preparation according to claim 107 wherein R21, R22 and R23 are independently selected from
o hydrogen, halogen, -OCF3, -OR27, -NR27R28, -SR27, -NR27C(0)R28, -NR27C(0)0R28, -OC(0)R27, -OCrC6-alkyl-C(0)OR27, -Sd-C6-alkyl-C(0)OR27, -C2-C6-alkenyl- C(=0)OR27, -C(=0)NR27-CrC6-alkyl-C(=0)OR27, -C C6-alkyl-C(=0)OR27, or -C(0)OR27,
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
°ArG1, ArG1-0-, ArGI-d-d-alkoxy, ArG1-CrC6-alkyl, of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
109. A pharmaceutical preparation according to any one of the claims 79 or 97 to 108 wherein R20 is hydrogen or methyl.
110. A pharmaceutical preparation according to claim 109 wherein R20 is hydrogen.
111. A pharmaceutical preparation according to any one of the claims 79 or 97 to 110 wherein R27 is hydrogen, d-Ce-alkyl or aryl.
112. A pharmaceutical preparation according to claim 111 wherein R27 is hydrogen or CrCe- alkyl or ArG1.
113. A pharmaceutical preparation according to claim 112 wherein R27 is hydrogen or CrCe- alkyl.
114. A pharmaceutical preparation according to any one of the claims 79 or 97 to 112 wherein R28 is hydrogen or Crd-alkyl.
115. A pharmaceutical preparation according to claim 79 wherein R17and R18 are independently selected from
• hydrogen, halogen, -CN, -CF3, -OCF3, -N02, -OR27, -NR27R28, -SR27, -S(0)R27, -S(0)2R27, -C(O)NR27R28, -CH2OR27, -OC(O)R27, -OCrC6-alkyl-C(O)OR27, -SC C6- alkyl-C(O)OR27, or -C(0)OR27,
• CrCe-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, optionally substituted with one or more substituents independently selected from R29
©aryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-d-Ce-alkyl, heteroaryl, heteroaryl-d-Ce- alkyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
116. A pharmaceutical preparation according to claim 115 wherein R17and R 8 are independently selected from
o hydrogen, halogen, -CN, -CF3, -N02, -OR27, -NR27R28, or -C(0)OR27,
o CrCe-alkyl optionally substituted with one or more substituents independently selected from R29
oaryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-d-C6-alkyl, heteroaryl, hβteroaryl-d-Ce- alkyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
117. A pharmaceutical preparation according to claim 116 wherein R17 and R18 are independently selected from
• hydrogen, halogen, -CN, -CF3, -N02, -OR27, -NR27R28, or -C(0)OR27
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• aryl, aryloxy, aroyl, aryl-d-Ce-alkoxy, aryl-d-Ce-alkyl, heteroaryl, heteroaryl-d-Ce- alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
118. A pharmaceutical preparation according to claim 117 wherein R17and R18 are independently selected from
• hydrogen, halogen, -CN, -CF3, -N02, -OR27, -NR27R28, or -C(0)OR27
• methyl, ethyl propyl optionally substituted with one or more substituents independently selected from R29
• ArG1, ArG1-0-, ArG1-C(0)-, ArG1-C C6-alkoxy, ArG1-d-C6-alkyl, Het3, Het3-C Ce-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
119. A pharmaceutical preparation according to claim 118 wherein R17 and R18 are independently selected from
• hydrogen, halogen, -CN, -CF3, -N02r -OR27, -NR27R28, or -C(0)OR27 • CrCe-alkyl optionally substituted with one or more substituents independently selected from R29
• phenyl, phenyloxy, phenyl-d-d-alkoxy, phenyl-C C6-alkyl, of which the cyclic moieties optionally may be substituted with one or more substituents selected from R30.
120. A pharmaceutical preparation according to any one of the claims 79 to 119 wherein R27 is hydrogen or d-Ce-alkyl.
121. A pharmaceutical preparation according to claim 120 wherein R27 is hydrogen, methyl or ethyl.
122. A pharmaceutical preparation according to any one of the claims 79 to 121 wherein R28 is hydrogen or d-Ce-alkyl.
123. A pharmaceutical preparation according to claim 122 wherein R28 is hydrogen, methyl or ethyl.
124. A pharmaceutical preparation according to any one of the claims 79 to 123 wherein R72 is -OH or phenyl.
125. A pharmaceutical preparation according to claim 79 wherein the zinc-binding ligand is
Figure imgf000370_0001
126. A pharmaceutical preparation according to claim 22 wherein the zinc-binding ligand is of the form H-l-J
wherein H is
Figure imgf000370_0002
wherein the phenyl, naphthalene or benzocarbazole rings are optionally substituted with one or more substituents independently selected from R31
I is selected from • a valence bond,
• -CH2N(R32)- or -S02N(R33)-,
Figure imgf000371_0001
wherein Z1 is S(0)2 or CH2, Z2 is -NH-, -O-or -S-, and n is 1 or 2,
J is o d-Ce-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may each optionally be substituted with one or more substituents selected from R34, oAryl, aryloxy, aryl-oxycarbonyl-, aroyl, aryl-Grd-alkoxy-, aryl-CrC6-alkyl-, aryl-C2- Ce-alkenyl-, aryl-C2-Ce-alkynyl-, heteroaryl, heteroaryl-CrC6-aIkyl-, heteroaryl-C2-Ce- alkenyl- or heteroaryl-C2-C6-alkynyl-, wherein the cyclic moieties are optionally substituted with one or more substituents selected from R37, • Hydrogen,
R31 is independently selected from hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(0)2CF3, -SCF3, -N02, -OR35, -C(0)R35, -NR35R36, -SR35, -NR35S(0)2R36, -S(0)2NR35R36, -S(0)NR35R36, -S(0)R35, -S(0)2R35, -C(0)NR35R36, -OC(0)NR35R36, -NR35C(0)R36, -CH2C(0)NR35R36, -OCH2C(0)NR35R36, -CH2OR35, -CH2NR35R36, -OC(0)R35, -OC Ce-alkyl-C(0)OR35, -SCrC6-alkyl-C(0)OR35 -C2-C6-alkenyl- C(=0)OR35, -NR35-C(=0)-d-C6-alkyl-C(=0)OR35, -NR35-C(=0)-CrC6-alkenyl-C(=0)OR35-, CrC6-alkyl, d-Ce-alkanoyl or -C(0)OR35,
R32 and R33 are independently selected from hydrogen, d-Ce-alkyl or d-Ce-alkanoyl,
R34 is independently selected from halogen, -CN, -CF3, -OCF3f -OR35, and -NR35R36,
R35 and R36 are independently selected from hydrogen, CrCe-alkyl, aryl-d-Ce-alkyl or aryl, or R35 and R36 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds, R37 is independently selected from halogen, -C(0)OR35, -C(0)H, -CN, -CF3, -OCF3, -N02, - OR35, -NR35R36, d-Ce-alkyl or d-Ce-alkanoyl,
or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
127. A pharmaceutical preparation according to claim 126 wherein the zinc-binding ligand is of the form H-l-J, wherein H is
Figure imgf000372_0001
wherein the phenyl, naphthalene or benzocarbazole rings are optionally substituted with one or more substituents independently selected from R31,
I is selected from
• a valence bond,
• -CH2N(R32)- or -S02N(R33)-,
Figure imgf000372_0002
is S(0)2 or CH2, Z2 is N,-0-or -S-, and n is 1 or 2,
J is
• CrCe-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may each optionally be substituted with one or more substituents selected from R34,
• Aryl, aryloxy, aryl-oxycarbonyl-, aroyl, aryl-d-Ce-alkoxy-, aryl-d-Ce-alkyl-, aryl-C2- Ce-alkenyl-, aryl-C2-C6-alkynyl-, heteroaryl, heteroaryl-d-d-alkyl-, heteroaryl-C2-C6- alkenyl- or heteroaryl-C2-C6-alkynyK wherein the cyclic moieties are optionally substituted with one or more substituents selected from R37, o hydrogen,
R31 is independently selected from hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -0CH2CF3, -0CF2CHF2, -S(0)2CF3, -SCF3, -N02, -OR35, -C(0)R35, -NR35R36, -SR35, -NR35S(0)2R36, -S(0)2NR35R36, -SfOjNR^R36, -S(0)R35, -S(0)2R35, -C(0)NR35R36, -OC(0)NR35R36, -NR35C(0)R36, -CH2C(0)NR35R36, -OCH2C(0)NR35R36, -CH2OR35, -CH2NR35R36, -OC(0)R35, -OCrC6-alkyl-C(0)OR35, -SC C6-alkyl-C(0)OR35 -C2-C6-alkenyl- C(=0)OR35, -NR35-C(=0)-d-C6-alkyl-C(=0)OR35, -NR35-C(=0)-C Ce-alkenyl-C(=0)OR35-, CrCe-alkyl, C C6-alkanoyl or -C(0)OR35,
R32 and R33 are independently selected from hydrogen, d-d-alky! or d-d-alkanoyl,
R^ is independently selected from halogen, -CN, -CF3, -OCF3, -OR35, and -NR35R36,
R35 and R36 are independently selected from hydrogen, d-Ce-alkyl, aryl-Crd-alkyl or aryl, or R35 and R36 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R37 is independently selected from halogen, -C(0)OR35, -C(0)H, -CN, -CF3, -OCF3, -N02, - OR35, -NR35R36, CrCe-alkyl or d-C6-alkanoyl,
or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base, With the proviso that R31 and J cannot both be hydrogen.
128. A pharmaceutical preparation according to any one of the claims 126 or 127 wherein H is
Figure imgf000373_0001
129. A pharmaceutical preparation according to claim 128 wherein H is
Figure imgf000373_0002
130. A pharmaceutical preparation according to claim 128 wherein H is
Figure imgf000374_0001
131. A pharmaceutical preparation according to any one of the claims 126 to 130wherein I is a valence bond, -CH2N(R32)-, or -S02N(R33)-.
132. A pharmaceutical preparation according to claim 131 wherein I is a valence bond.
133. A pharmaceutical preparation according to any one of the claims 126 to 132 wherein J is o hydrogen,
• CrCe-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR35, and -NR35R36,
• aryl, or heteroaryl, wherein the cyclic moieties are optionally substituted with one or more substituents independently selected from R37.
134. A pharmaceutical preparation according to claim 133 wherein J is
• hydrogen,
• aryl or heteroaryl, wherein the cyclic moieties are optionally substituted with one or more substituents independently selected from R37.
135. A pharmaceutical preparation according to claim 133 wherein J is
• hydrogen,
• ArG1 or Het3, wherein the cyclic moieties are optionally substituted with one or more substituents independently selected from R37.
136. A pharmaceutical preparation according to claim 135 wherein J is o hydrogen, o phenyl or naphthyl optionally substituted with one or more substituents independently selected from R37.
137. A pharmaceutical preparation according to claim 136 wherein J is hydrogen.
138. A pharmaceutical preparation according to any one of the claims 126 to 137 wherein R32 and R33 are independently selected from hydrogen or Cι-C6-alkyl.
139. A pharmaceutical preparation according to any one of the claims 126 to 138 wherein R34 is hydrogen, halogen, -CN, -CF3, -OCF3, -SCF3, -NO2, -OR35, -C(0)R35, -NR35R36, -SR35, -C(0)NR35R36, -0C(0)NR35R36, -NR35C(0)R36, -0C(0)R35, -0CrC6-alkyl-C(0)0R35, -Sd-C6- alkyl-C(O)OR35 or -C(0)OR35.
140. A pharmaceutical preparation according to claim 139 wherein R34 is hydrogen, halogen, -CF3, -N02, -OR35, -NR35R36, -SR35, -NR35C(0)R36, or -C(0)OR35.
141. A pharmaceutical preparation according to claim 140 wherein R34 is hydrogen, halogen, -CF3, -N02, -OR35, -NR35R36, or -NR35C(0)R3δ.
142. A pharmaceutical preparation according to claim 141 wherein R34 is hydrogen, halogen, or -OR35.
143. A pharmaceutical preparation according to any one of the claims 126 to 142 wherein R35 and R36 are independently selected from hydrogen, d-d-alkyl, or aryl.
144. A pharmaceutical preparation according to claim 143 wherein R35 and R36 are independently selected from hydrogen or d-Ce-alkyl.
145. A pharmaceutical preparation according to any one of the claims 126 to 144 wherein R37 is halogen, -C(0)OR35, -CN, -CF3, OR35, -NR35R36, d-d-alkyl or d-d-alkanoyl.
146. A pharmaceutical preparation according to claim 145 wherein R37 is halogen, - C(0)OR35, -OR35, -NR35R36, d-d-alkyl or d-Ce-alkanoyl.
147. A pharmaceutical preparation according to claim 146 wherein R37 is halogen, -C(0)OR35 or -OR35.
148. A pharmaceutical preparation according to claim 22 wherein the zinc-binding ligand is
Figure imgf000375_0001
wherein K is a valence bond, d-d-alkylene, -NH-C(=0)-U-, -d-d-alkyl-S-, -d-d-alkyl-O-, -C(=0)-, or -C(=0)-NH-, wherein any C C6-alkyl moiety is optionally substituted with R38,
U is a valence bond, d-d-alkenylene, -d-d-alkyl-O- or d-d-alkylene wherein any C C6-alkyl moiety is optionally substituted with d-d-alkyl,
R38 is d-d-alkyl, aryl, wherein the alkyl or aryl moieties are optionally substituted with one or more substituents independently selected from R39,
R39 is independently selected from halogen, cyano, nitro, amino,
M is a valence bond, arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted with one or more substituents independently selected from R40, R40 is selected from
• hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(0)2CF3, -OS(0)2CF3, -SCF3, -N02, -OR41, -NR41R42, -SR41, -NR S(0)2R42, -S(0)2NR41R42, -S(0)NR41R42, -S(0)R41, -S(0)2R41, -0S(0)2 R41, -C(0)NR41R42, -0C(0)NR4V2, -NR41C(G)R42, -CH2C(0)NR41R42, -OC Cβ- alkyl-C(0)NR41R42, -CH2OR41, -CH20C(0)R41 , -CH2NR41R42, -OC(0)R41, -OC C6- alkyl-C(0)OR41, -OC Ce-alkyl-OR41, -S-CrC6-alkyl-C(0)0R41, -C2-C6-alkenyl- C(=0)OR41, -NR41-C(=0)-C C6-alkyl-C(=0)OR41, -NR41-C(=0)-d-Ce- alkenyl-C(=0)OR41 , -C(0)OR41, -C2-C6-alkenyl-C(=0)R41, =0, -NH-C(=0)-0-C Ce-alkyl, or -NH-C(=0)-C(=0)-0-CrC6-alkyl,
• CrCe-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, which may each optionally be substituted with one or more substituents selected from R43,
• aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-d-d-alkoxy, aryl-d-d-alky), aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-d- C6-alkyl, heteroaryl-d-d-alkenyl or heteroaryl-C2-C6-alkynyl, wherein the cyclic moieties optionally may be substituted with one or more substituents selected from R44,
R41 and R42 are independently selected from hydrogen, -OH, d-d-alkyl, d-d-alkenyl, aryl- d-d-alkyl or aryl, wherein the alkyl moieties may optionally be substituted with one or more substituents independently selected from R45, and the aryl moieties may optionally be substituted with one or more substituents independently selected from R46; R41 and R42 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R43 is independently selected from halogen, -CN, -CF3, -OCF3, -OR41, and -NR41R42
R44 is independently selected from halogen, -C(0)OR41, -CH2C(0)OR41, -CH2OR41, -CN, - CF3, -OCF3, -N02, -OR41, -NR41R42 and d-Ce-alkyl,
R45 is independently selected from halogen, -CN, -CF3, -OCF3, -O-d-Ce-alkyl, -C(0)-0-C C6-alkyl, -COOH and -NH2, R46 is independently selected from halogen, -C(0)OCrC6-alkyl, -COOH, -CN, -CF3, -OCF3, N02, -OH, -Od-Ce-alkyl, -NH2, C(=0) or d-d-alkyl,
Q is a valence bond, d-Ge-alkyfene, -C C6-alkyl-0-, -d-C6-alkyl-NH-, -NH-d-d- -NH-C(=0)-, -C(=0)-NH-, -0-CrC6-alkyl, -C(=0)-, or -d-C6-alkyl-C(=0)-N(R47)- wherein the alkyl moieties are optionally substituted with one or more substituents independently selected from R48,
R47 and R48 are independently selected from hydrogen, d-d-alkyl, aryl optionally substituted with one or more R49,
R49 is independently selected from halogen and -COOH,
T is
• hydrogen,
• CrCe-alkyl, C2-C6-alkenyl , C2-C6-alkynyl, d-d-alkyloxy-carbonyl, wherein the alkyl, alkenyl and alkynyl moieties are optionally substituted with one or more substituents independently selected from R50,
• aryl, aryloxy, aryloxy-carbonyl, aryl-Crd-alkyl, aroyl, aryl-d-Ce-alkoxy, aryl-C2- d-alkenyl, aryl-C2-C6-alkyny-, heteroaryl, heteroaryl-Crd-alkyl, heteroaryl-C2- d-alkenyl, heteroaryl-C2-C6-alkynyl,
wherein any alkyl, alkenyl , alkynyl, aryl and heteroaryl moiety is optionally substituted with one or more substituents independently selected from R50,
R50 is d-Ce-alkyl, d-C6-alkoxy, aryl, aryloxy, aryl-d-d-alkoxy, -C(=0)-NH-C C6-alkyl-aryl, -C(=O)-NR50A-CrC6-alkyl, -C(=0)-NH-(CH2CH20)mCrC6-alkyl-C00H, heteroaryl, het- eroaryl-d-Ce-alkoxy, -Ci-Ce-alkyl-COOH, -0-C C6-alkyl-COOH, -S(0)2R51,
-C2-C6-alkenyl-COOH, -OR51, -N02, halogen, -COOH, -CF3, -CN, =0, -N(R51R52), wherein m is 1 , 2, 3 or 4, and wherein the aryl or heteroaryl moieties are optionally substituted with one or more R53, and the alkyl moieties are optionally substituted with one or more R50B. R50A and R50B are independently selected from -C(0)OCrC6-alkyl, -COOH, -d-d-ε C(0)Od-C6-alkyl, -C C6-alkyl-COOH, or d-d-alkyl, R51 and R52 are independently selected from hydrogen and d-d-alkyl, R53 is independently selected from d-d-alkyl, d-d-alkoxy, -d-C6-alkyl-COOH, -C2- Ce-alkenyl-COOH, -OR51, -N02, halogen, -COOH, -CF3, -CN, or -N(R61R52),
or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
149. A pharmaceutical preparation according to claim 148 wherein K is a valence bond, d- d-alkylene, -NH-C(=0)-U-, -d-C6-alkyl-S-, -d-C6-alkyl-0-, or -C(=0)-, wherein any d- d-alkyl moiety is optionally substituted with R38.
150. A pharmaceutical preparation according to claim 149 wherein K is a valence bond, d- Ce-alkylene, -NH-C(=0)-U-, -C C6-alkyl-S-, or -CrC6-alkyl-0, wherein any d-d-alkyl moiety is optionally substituted with R38.
151. A pharmaceutical preparation according to claim 150 wherein K is a valence bond, d- d-alkylene, or -NH-C(=0)-U, wherein any d-d-alkyl moiety is optionally substituted with R38.
152. A pharmaceutical preparation according to claim 151 wherein K is a valence bond or d- d-alkylene, wherein any d-d-alkyl moiety is optionally substituted with R38.
153. A pharmaceutical preparation according to claim 151 wherein K is a valence bond or -NH-C(=0)-U.
154. A pharmaceutical preparation according to claim 152 wherein K is a valence bond.
155. A pharmaceutical preparation according to any one of the claims 148 to 154 wherein U is a valence bond or -d-d-alkyl-0~.
156. A pharmaceutical preparation according to claim 155 wherein U is a valence bond
157. A pharmaceutical preparation according to any one of the claims 148 to 156 wherein M is arylene or heteroarylene, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
158. A pharmaceutical preparation according to claim 157 wherein M is ArG1 or Het1, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
159. A pharmaceutical preparation according to claim 158 wherein M is ArG1 or Het2, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
160. A pharmaceutical preparation according to claim 159 wherein M is ArG1 or Het3, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
161. A pharmaceutical preparation according to claim 160 wherein M is phenylene optionally substituted with one or more substituents independently selected from R40.
162. A pharmaceutical preparation according to claim 160 wherein M is indolylene optionally substituted with one or more substituents independently selected from R40.
163. A pharmaceutical preparation according to claim 162 wherein M is
Figure imgf000379_0001
164. A pharmaceutical preparation according to claim 160 wherein M is carbazolylene optionally substituted with one or more substituents independently selected from R40.
165. A pharmaceutical preparation according to claim 164 wherein M is
Figure imgf000379_0002
166. A pharmaceutical preparation according to any one of the claims 148 to 165 wherein R40 is selected from
• hydrogen, halogen, -CN, -CF3, -OCF3, -N02, -OR41, -NR41R42, -SR41, -S(0)2R41, -NR41C(0)R42, -OCrd-alkyl-C(0)NR41R42, -C2-C6-alkenyl-C(=0)OR41, -C(0)OR41, =0, -NH-C(=0)-0-d-C6-alkyl, or -NH-C(=0)-C(=0)-0-d-C6-alkyl,
d-d-alkyl or C2-C6- alkenyl which may each optionally be substituted with one or more substituents independently selected from R43,
• aryl, aryloxy, aryl-d-Ce-alkoxy, aryl-Crd-alkyl, aryl-C2-C6-alkenyl, heteroaryl, het- eroaryl-d-d-alkyl, or heteroaryl-C2-C6-alkenyl, wherein the cyclic moieties optionally may be substituted with one or more substituents selected from R44.
167. A pharmaceutical preparation according to claim 166 wherein R40 is selected from
• hydrogen, halogen, -CN, -CF3, -OCF3, -N02, -OR41, -NR41R42, -SR41, -S(0)2R41, -NR41C(0)R42, -OC Ce-alkyl-C(0)NR41R42, -C2-C6-alkenyl-C(=0)OR41, -C(0)OR41, =0, -NH-C(=0)-0-C C6-alkyl, or -NH-C(=0)-C(=0)-0-d-C6-alkyl,
d-d-alkyl or C2-C6- alkenyl which may each optionally be substituted with one or more substituents independently selected from R43, • ArG1, ArG1-0-, ArG1-CrC6-aIkoxy, ArG1-d-C6-alkyl, ArG1-C2-C6-alkenyl, Het3, Het3-Cι-C6-alkyl, or Het3-C2-C6-alkenyl, wherein the cyclic moieties optionally may be substituted with one or more substituents selected from R44.
168. A pharmaceutical preparation according to claim 167 wherein R40 is selected from o hydrogen, halogen, -CF3, -N02, -OR41, -NR41R42, -C(0)OR41, =0, or -NR41C(0)R42, o d-Cβ-alkyl. oArGl
169. A pharmaceutical preparation according to claim 168 wherein R40 is hydrogen.
170. A pharmaceutical preparation according to claim 168 wherein R40 is selected from o Halogen, -N02, -OR41, -NR41R42, -C(0)OR41, or -NR41C(0)R42,
• Methyl,
• Phenyl.
171. A pharmaceutical preparation according to any one of the claims 148 to 170 wherein R41 and R42 are independently selected from hydrogen, d-Cβ-alkyl, or aryl, wherein the aryl moieties may optionally be substituted with halogen or -COOH.
172. A pharmaceutical preparation according to claim 171 wherein R41 and R42 are independently selected from hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties may optionally be substituted with halogen or -COOH.
173. A pharmaceutical preparation according to any one of the claims 148 to 172 wherein Q is a valence bond, d-d-alkylene, -d-d-alkyl-O-, -d-d-alkyl-NH-, -NH-d-d-alkyl, -NH-C(=0)-, -C(=0)-NH-, -O-d-d-alkyl, -C(=0)-, or -d-C6-alkyl-C(=0)-N(R47)- wherein the alkyl moieties are optionally substituted with one or more substituents independently selected from R48.
174. A pharmaceutical preparation according to claim 173 wherein Q is a valence bond, -CH2-, -CH2-CH2-, -CH2-0-, -CH2-CH2-0-, -CH2-NH-, -CH2-CH2-NH-, -NH-CH2-, -NH-CH2-CH2-, -NH-C(=0)-, -C(=0)-NH-, -0-CH2-, -0-CH2-CH2-, or -C(=0)-.
175. A pharmaceutical preparation according to claim 174 wherein Q is a valence bond, -CH2-, -CH2-CH2-, -CH2-0-,or -CH2-CH2-0-.
176. A pharmaceutical preparation according to claim 175 wherein Q is a valence bond, -CH2-, or -CH2"CH2-.
177. A pharmaceutical preparation according to claim 176 wherein Q is -CH2-.
178. A pharmaceutical preparation according to any one of the claims 148 to 177 wherein R47 and R48 are independently selected from hydrogen, methyl and phenyl.
179. A pharmaceutical preparation according to any one of the claims 148 to 178 wherein T is
• hydrogen,
° d-d-alkyl optionally substituted with one or more substituents independently selected from R50, oaryl, aryl-d-Ce-alkyl, heteroaryl, wherein the alkyl, aryl and heteroaryl moieties are optionally substituted with one or more substituents independently selected from R50.
180. A pharmaceutical preparation according to claim 179 wherein T is o hydrogen,
• CrCe-alkyl optionally substituted with one or more substituents independently selected from R50,
• ArG1 , ArG1 -d-d-alkyl, Het3, wherein the alkyl, aryl and heteroaryl moieties are optionally substituted with one or more substituents independently selected from R50.
181. A pharmaceutical preparation according to claim 180 wherein T is
• hydrogen,
• CrCe-alkyl, optionally substituted with one or more substituents independently selected from R50,
• phenyl, phenyl-d-d-alkyl, wherein the alkyl and phenyl moieties are optionally substituted with one or more substituents independently selected from R50.
182. A pharmaceutical preparation according to claim 181 wherein T is phenyl substituted with R50.
183. A pharmaceutical preparation according to any one of the claims 148 to 182 wherein R50 is CrCe-alkyl, d-d-alkoxy, aryl, aryloxy, aryl-d-d-alkoxy, -C(=0)-NH-CrC6-alkyl-aryl, -C(=O)-NR50A-Crd-alkyl, -C(=0)-NH-(CH2CH20)mCrC6-alkyl-COOH, heteroaryl, -C Ce-alkyl-COOH, -O-d-d-alkyl-COOH, -S(0)2R51, -C2-C6-alkenyl-COOH, -OR51, -N02, halogen, -COOH, -CF3, -CN, =0, -N(R51R52), wherein the aryl or heteroaryl moieties are optionally substituted with one or more R53.
184. A pharmaceutical preparation according to claim 183 wherein R50 is d-d-alkyl, d- Ce-alkoxy, aryl, aryloxy, -C(=O)-NR50A-CrC6-alkyl, -C(=0)-NH-(CH2CH20)mC C6-alkyl- COOH, aryl-d-d-alkoxy , -OR51, -N02, halogen, -COOH, -CF3, wherein any aryl moiety is optionally substituted with one or more R53.
185. A pharmaceutical preparation according to claim 184 wherein R50 is d-d-alkyl, aryloxy, -C(=O)-NR50A-Crd-alkyl, -C(=0)-NH-(CH2CH20)mCrC6-alkyl-COOH, aryl-d-d-alkoxy , -OR51, halogen, -COOH, -CF3, wherein any aryl moiety is optionally substituted with one or more R53.
186. A pharmaceutical preparation according to claim 185 wherein R50 is d-Ce-alkyl, ArGI-O-, -C(=O)-NR50A-CrC6-alkyl, -C(=0)-NH-(CH2CH20)mCrC6-alkyl-COOH, ArG1-d- d-alkoxy , -OR51, halogen, -COOH, -CF3, wherein any aryl moiety is optionally substituted with one or more R53.
187. A pharmaceutical preparation according to claim 186 wherein R50 is -C(=O)-NR50ACH2, -C(=0)-NH-(CH2CH20)2CH2l-COOH, or -C(=O)-NR50ACH2CH2
188. A pharmaceutical preparation according to claim 186 wherein R50 is phenyl, met ethyl, halogen, or -COOH.
189. A pharmaceutical preparation according to claim 188 wherein R50 is methyl or et
190. A pharmaceutical preparation according to claim 188 wherein R50 is COOH.
191. A pharmaceutical preparation according to any one of the claims 148 to 190 wherein m is 1 or 2.
192. A pharmaceutical preparation according to any one of the claims 148 to 191 wherein R51 is methyl.
193. A pharmaceutical preparation according to any one of the claims 148 to 192 wherein R53 is d-Ce-alkyl, d-d-alkoxy, -OR51, halogen.or -CF3.
194. A pharmaceutical preparation according to any one of the claims 148 to 193 wherein R50A is -C(0)OCH3, -C(0)OCH2CH3 -COOH, -CH2C(0)OCH3, -CH2C(0)OCH2CH3, -CH2CH2C(0)OCH3, -CH2CH2C(0)OCH2CH3, -CH2COOH, methyl, or ethyl.
195. A pharmaceutical preparation according to any one of the claims 148 to 194 wherein R50B is -C(0)OCH3, -C(0)OCH2CH3 -COOH, -CH2C(O)OCH3, -CH2C(O)OCH2CH3, -CH2CH2C(0)OCH3, -CH2CH2C(0)OCH2CH3, -CH2COOH, methyl, or ethyl.
196. A pharmaceutical preparation according to claim 22 wherein the zinc-binding ligand is
Figure imgf000382_0001
wherein V is CrCe-alkyl, aryl, heteroaryl, aryl-Cι-6-alkyl- or aryl-d-e-alkenyl-, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R54, and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R55, R54 is independently selected from halogen, -CN, -CF3, -OCF3, aryl, -COOH and -NH2, R55 is independently selected from
• hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(0)2CF3, -OS(0)2CF3, -SCF3, -N02, -OR56, -NR56R57, -SR56, -NR56S(0)2R57, -S(0)2NRε6R57, -S(0)NR56R57, -S(0)R56, -S(0)2R56, -0S(0)2 R56, -C(0)NR56R57, -OC(0)NR56R57, -NR56C(0)R57, -CH2C(0)NR56R57, -OCrC6- alkyl-C(0)NR56R57, -CH20R56, -CH20C(0)R56, -CH2NR56R57, -OC(0)R56, -OC Cβ- alkyl-C(0)OR56, -OC C6-alkyl-OR56, -SC Ce-alkyl-C(0)OR56, -C2-C6-alkenyl- C(=0)OR56, -NR56-C(=0)-d-C6-alkyl-C(=0)OR56, -NR56-C(=0)-Crd- alkenyl-C(=0)OR56 , -C(0)OR56, or -C2-C6-alkenyl-C(=0)R56,
• CrC6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
which may optionally be substituted with one or more substituents selected from R58,
• aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-d-d-alkoxy, aryl-d-Cβ-alkyl, aryl-d-d-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-d- Ce-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R59,
R5B and R57 are independently selected from hydrogen, OH, CF3, C C12-alkyl, aryl-d-d- alkyl, -C(=0)-CrC6-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents independently selected from R60, and the aryl groups may optionally be substituted with one or more substituents independently selected from R61; R56 and R57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R58 is independently selected from halogen, -CN, -CF3, -OCF3, -OR56, and -NR56R57,
R59 is independently selected from halogen, -C(0)OR56, -CH2C(0)OR56, -CH2OR56, -CN, - CF3, -OCF3, -N02, -OR56, -NR56R57 and d-d-alkyl, R60 is independently selected from halogen, -CN, -CF3, -OCF3, -OCrC6-alkyl, -C(0)Od-C6- alkyl, -C(=0)-R62, -COOH and -NH2,
R61 is independently selected from halogen, -C(0)OCrC6-alkyl, -COOH, -CN, -CF3, -OCF3, - N02, -OH, -Od-Ce-alkyl, -NH2, C(=0) or d-C6-alkyl,
R62 is d-d-alkyl, aryl optionally substituted with one or more substituents independently selected from halogen, or heteroaryl optionally substituted with one or more d-Ce-alkyl independently, or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
197. A pharmaceutical preparation according to claim 196 wherein V is aryl, heteroaryl, or aryl-Ci-6-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected R54, and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R55.
198. A pharmaceutical preparation according to claim 197 wherein V is aryl, Het1, or aryl-d- 6-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from R54, and the aryl or heteroaryl moiety is optionally substituted with one or more substituents independently selected from R55.
199. A pharmaceutical preparation according to claim 198 wherein V is aryl, Het2, or aryl-Cι_ e-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from R54, and the aryl or heteroaryl moiety is optionally substituted with one or more substituents independently selected from R55.
200. A pharmaceutical preparation according to claim 199 wherein V is aryl, Het3, or aryl-d. e-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from R54, and the aryl or heteroaryl moiety is optionally substituted with one or more substituents independently selected from R55.
201. A pharmaceutical preparation according to claim 200 wherein V is aryl optionally substituted with one or more substituents independently selected from R55.
202. A pharmaceutical preparation according to claim 201 wherein V is ArG1 optionally substituted with one or more substituents independently selected from R55.
203. A pharmaceutical preparation according to claim 202 wherein V is phenyl, naphthyl or anthranyl optionally substituted with one or more substituents independently selected from R55.
204. A pharmaceutical preparation according to claim 203 wherein V is phenyl optionally substituted with one or more substituents independently selected from R55.
205. A pharmaceutical preparation according to any one of the claims 196 to 204 wherein R55 is independently selected from o halogen, d-d-alkyl, -CN, -OCF3 ,-CF3, -N02, -OR56, -NR56R57, -NR56C(0)R57 -SR56, -0C d-alkyl-C(0)0R56, or -C(0)OR56,
• d-d-alkyl optionally substituted with one or more substituents independently selected from R58
• aryl, aryl-d-d-alkyl, heteroaryl, or heteroaryl-d-d-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents independently selected from R59.
206. A pharmaceutical preparation according to claim 205 wherein R55 is independently selected from
• halogen, d-d-alkyl, -CN, -OCF3 ,-CF3, -N02, -OR56, -NR56R57, -NR56C(0)R57 -SR56, -Od-d-alkyl-C(0)OR56, or -C(0)OR56
• CrCe-alkyl optionally substituted with one or more substituents independently selected from R58
•ArG1, ArG1 -d-Ce-alkyl, Het3, or Het3-C C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents independently selected from R59.
207. A pharmaceutical preparation according to claim 206 wherein R55 is independently selected from halogen, -OR56, -NR56R57, -C(0)OR56, -Od-C8-alkyl-C(0)OR56, -NR56C(0)R57 or d-Ce-alkyl.
208. A pharmaceutical preparation according to claim 207 wherein R5S is independently selected from halogen, -OR56, -NR56R57, -C(0)OR56, -OCrd-alkyl-C(0)OR56, -NR56C(0)R57, methyl or ethyl.
209. A pharmaceutical preparation according to any one of the claims 196 to 208 wherein R56 and R57 are independently selected from hydrogen, CF3, CrCι2-alkyl, or -C(=0)-C C6-alkyl; R56 and R57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom.
210. A pharmaceutical preparation according to claim 209 wherein R56 and R57 are independently selected from hydrogen or d-C12-alkyl, R56 and R57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom.
211. A pharmaceutical preparation according to claim 210 wherein R56 and R5' are independently selected from hydrogen or methyl, ethyl, propyl butyl, R56 and R57 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom.
212. A pharmaceutical preparation according to claim 22 wherein the zinc-binding ligand is
Figure imgf000386_0001
wherein AA is d-d-alkyl, aryl, heteroaryl, a l-d -6-alkyl- o aryl-C2-6-alkenyl-, wherein the alkyl or alkenyl is optionally substituted with one or more substituents independently selected from R63, and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R64,
R63 is independently selected from halogen, -CN, -CF3, -OCF3, aryl, -COOH and -NH2,
R64 is independently selected from
• hydrogen, halogen, -CN, -CH2CN, -CHF2, -CF3, -OCF3, -OCHF2, -OCH2CF3, -OCF2CHF2, -S(0)2CF3, -OS(0)2CF3, -SCF3, -N02, -OR65, -NR65R66, -SR65, -NR65S(0)2R66, -S(0)2NR65R66, -S(0)NR65R66, -S(0)R65, -S(0)2R65, -OS(0)2 R65, -C(0)NR65R66, -0C(0)NR65R66, -NR65C(0)R66, -CH2C(0)NR65R66, -OC C6- alkyl-C(0)NR65R66, -CH2OR65, -CH2OC(0)R65, -CH2NR65R66, -OC(0)R65, -Od-d- alkyl-C(0)OR65, -OCrC6-alkyl-OR65, -Sd-d-alkyl-C(0)OR65, -C2-C6-alkenyl- C(=0)OR65, -NR65-C(=0)-d-C6-alkyl-C(=0)OR65, -NR65-C(=0)-d-C6- alkenyl-C(=0)OR65 , -C(0)OR65, or -C2-C6-alkenyl-C(=0)R65,
o d-d-alkyl, C2-C6-alkenyl or C2-C6-alkynyl, each of which may optionally be substituted with one or more substituents selected from R67, • aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-CrC6-alkoxy, aryl-d-d-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyI, heteroaryl, heteroaryl-d- Ce-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,
of which the cyclic moieties optionally may be substituted with one or more substituents selected from R68,
R65 and R66 are independently selected from hydrogen, OH, CF3, C C12-alkyl, aryl-d-d- alkyl, -C(=0)-R69, aryl or heteroaryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from R70, and the aryl and heteroaryl groups may optionally be substituted with one or more substituents independently selected from R71; R65 and R66 when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,
R67 is independently selected from halogen, -CN, -CF3, -OCF3, -OR65, and -NR65R66,
R68 is independently selected from halogen, -C(0)OR65, -CH2C(0)OR65, -CH2OR65, -CN, - CF3, -OCF3, -N02, -OR65, -NR65R66 and d-d-alkyl,
R69 is independently selected from d-d-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl optionally substituted with one or more d-d-alkyl,
R70 is independently selected from halogen, -CN, -CF3, -OCF3, -Od-d-alkyl, -C(0)OC d- alkyl, -COOH and -NH2,
R71 is independently selected from halogen, -C(0)OCrC6-aIkyl, -COOH, -CN, -CF3, -OCF3, - N02, -OH, -Od-d-alkyl, -NH2, C(=0) or d-d-alkyl,
or any enantiomer, diastereomer, including a racemic mixture, tautomer as well as a salt thereof with a pharmaceutically acceptable acid or base.
213. A pharmaceutical preparation according to claim 212 wherein AA is aryl, heteroaryl or aryl-d-6-alkyl-, wherein the alkyl is optionally substituted with one or more R63, and the aryl or heteroaryl is optionally substituted with one or more substituents independently selected from R64.
214. A pharmaceutical preparation according to claim 213 wherein AA is aryl or heteroaryl optionally substituted with one or more substituents independently selected from R64.
215. A pharmaceutical preparation according to claim 214 wherein AA is ArG1 or Hef1 optionally substituted with one or more substituents independently selected from R64.
216. A pharmaceutical preparation according to claim 215 wherein AA is ArG1 or Het2 optionally substituted with one or more substituents independently selected from R64.
217. A pharmaceutical preparation according to claim 216 wherein AA is ArG1 or Het3 optionally substituted with one or more substituents independently selected from R64.
218. A pharmaceutical preparation according to claim 217 wherein AA is phenyl, naphtyl, anthryl, carbazolyl, thienyl, pyridyl, or benzodioxyl optionally substituted with one or more substituents independently selected from R64.
219. A pharmaceutical preparation according to claim 218 wherein AA is phenyl or naphtyl optionally substituted with one or more substituents independently selected from R64.
220. A pharmaceutical preparation according to any one of the claims 212 to 219 wherein R64 is independently selected from hydrogen, halogen, -CF3, -OCF3, -OR65, -NR65R66, d-d-alkyl , -OC(0)R65, -Od-C6-alkyl-C(0)OR65, aryl-C2-C6-alkenyl, aryloxy or aryl, wherein d-d-alkyl is optionally substituted with one or more substituents independently selected from R67, and the cyclic moieties optionally are substituted with one or more substituents independently selected from R68.
221. A pharmaceutical preparation according to claim 220 wherein R64 is independently selected from halogen, -CF3, -OCF3, -OR65, -NR65R66, methyl, ethyl, propyl, -OC(0)R65, -OCH2-C(0)OR65, -OCH2-CH2-C(0)OR65, phenoxy optionally substituted with one or more substituents independently selected from R68.
222. A pharmaceutical preparation according to any one of the claims 212 to 221 wherein R65 and R66 are independently selected from hydrogen, CF3, d-C12-alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R71.
223. A pharmaceutical preparation according to claim 222 wherein R65 and R66 are independently hydrogen, C C12-alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R71.
224. A pharmaceutical preparation according to claim 223 wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, ArG1 or Het1 optionally substituted with one or more substituents independently selected from R71.
225. A pharmaceutical preparation according to claim 224 wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, ArG1 or Het2 optionally substituted with one or more substituents independently selected from R71.
226. A pharmaceutical preparation according to claim 225 wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, ArG1 or Hef3 optionally substituted with one or more substituents independently selected from R7'.
227. A pharmaceutical preparation according to claim 226 wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, phenyl, naphtyl, thiadi- a∑olyl optionally substituted with one or more R71 independently; or isoxazoiyl optionally substituted with one or more substituents independently selected from R 1.
228. A pharmaceutical preparation according to any one of the claims 212 to 227 wherein R71 is halogen or d-Ce-alkyl.
229. A pharmaceutical preparation according to claim 228 wherein R71 is halogen or methyl.
230. Method of prolonging the action of an insulin preparation comprising insulin, protamine and zinc ions wherein said method comprises adding a zinc-binding ligand according to any of claims 21 to 229 to the insulin preparation.
231. A method of treating type 1 or type 2 diabetes comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical preparation according to any one of the claims 1 to 229.
232. Use of a preparation according to any one of the claims 1 to 229 for the preparation of a medicament for treatment of type 1 or type 2 diabetes.
233. A method of preparing a pharmaceutical preparation comprising the steps of mixing
• insulin
• a ligand for the His810 Zn2+ site of the insulin hexamer according to any of claims 21 to 229
• zinc ions
• protamine
• optionally further ingredients selected from the group consisting of phenolic preservative, buffer, isotonicity agent, viscosity increasing agent, and a non-ionic surfactant, and allowing the mixture to stand until crystals are formed.
234. A method according to claim 233 wherein the ligand for the His810 Zn2+ site is added to the mixture before crystal growth.
235. A method according to claim 233 wherein the ligand for the His810 Zn2+ site is added to the mixture after completion of crystal growth.
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