Patent Application
20090123563
The present invention discloses insulin preparations comprising branched ligands for the HisB10-Zn2+ sites of the R-state insulin hexamer and insulin, an analogue thereof, a derivative thereof and combinations of any of these, acid-stabilised insulin, fast/rapid acting insulin and long/slow/basal acting insulin. The preparations have a prolonged action designed for flexible injection regimes.
Diabetes is a general term for disorders in man having excessive urine excretion as in diabetes mellitus and diabetes insipidus. Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost.
Since the discovery of insulin in the 1920's, continuous strides have been made to improve the treatment of diabetes mellitus. To help avoid extreme glycaemia levels, diabetic patients often practice multiple injection therapy, whereby insulin is administered with each meal. Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin composition to cover the basal requirement, supplemented by bolus injections of rapid acting insulin to cover the meal-related requirements.
Insulin compositions having a protracted profile of action are well known in the art. Thus, one main type of such insulin compositions comprises injectable aqueous suspensions of insulin crystals or amorphous insulin. Typically, the insulin in these compositions is provided in the form of protamine insulin, zinc insulin or protamine zinc insulin
Soluble, rapid acting insulin compositions usually comprise insulin, insulin analogue or insulin derivative together with zinc ion, phenolic preservative, isotonicity agent, and a buffer substance. In addition, the preparation may optionally contain some salts and/or surfactants. Such preparations contain insulin in the form of an R-state hexamer.
Another approach involves the use of insulin derivatives where the net charge is increased to shift the isoelectric point, and hence the pH of minimum solubility, from about 5.5 to the physiological range. Such preparations may be injected as clear solutions at slightly acidic pH. The subsequent adjustment of the pH to neutral induces crystallization/precipitation in the subcutaneous depot and dissolution again becomes rate-limiting for the absorption. GlyA21ArgB31ArgB32 human insulin belongs to this category of insulin analogues.
Most recently, a series of soluble insulin derivatives with a hydrophobic moiety covalently attached to the side chain of LysB29 have been synthesized. These derivatives may show prolonged action profile due to various mechanisms including albumin binding (e.g. B29-Nε-myristoyl-des(B30) human insulin), extensive protein self-association and/or stickiness (e.g. B29-Nε-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin) induced by the attached hydrophobic group.
WO 0327081 discloses linear ligands for the HisB10-Zn2+ sites of the R-state insulin hexamer, R-state insulin hexamers comprising such ligands, and aqueous insulin preparations comprising such R-state insulin hexamers.
WO 0480480 discloses pharmaceutical preparations comprising linear ligands for the HisB10-Zn2+ sites of the R-state insulin hexamer and acid-stabilised insulin analogues.
The present invention provides insulin preparations comprising branched ligands for the HisB10-Zn2+ sites of the R-state insulin hexamer, zinc ions and insulin.
The resulting branched ligands work to modify the time action profile of insulin formulations. These preparations may be formulated with variable insulin species over a wide range of pH from 3.0 to 8.5 and their time action profiles may be tailored by suitable adjustments of anchor affinity.
The invention also provides a method of preparing branched ligands for the HisB10Zn2+ sites of the R-state insulin hexamer comprising the steps of:
Also provided are methods of treating type 1 or type 2 diabetes comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical preparation of the invention.
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 “C1-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, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl and the like.
The term “C1-C6-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-butadienyl, 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-alkynyl” 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-hexadienyl and the like.
The term “C1-C6-alkoxy” as used herein refers to the radical —O—C1-C6-alkyl, wherein C1-C6-alkyl is as defined above. Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tert-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-cyclooctenyl, 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, phenanthrenyl, fluorenyl, indenyl, azulenyl and the like. Aryl 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-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, benzofuryl, 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, 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 furylene, thienylene, pyrrolylene, oxazolylene, thiazolylene, imidazolylene, isoxazolylene, isothiazolylene, 1,2,3-triazolylene, 1,2,4-triazolylene, pyranylene, pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene, 1,2,3-triazinylene, 1,2,4-triazinylene, 1,3,5-triazinylene, 1,2,3-oxadiazolylene, 1,2,4-oxadiazolylene, 1,2,5-oxadiazolylene, 1,3,4-oxadiazolylene, 1,2,3-thiadiazolylene, 1,2,4-thiadiazolylene, 1,2,5-thiadiazolylene, 1,3,4-thiadiazolylene, tetrazolylene, thiadiazinylene, indolylene, isoindolylene, benzofurylene, benzothienylene, indazolylene, benzimidazolylene, benzthiazolylene, benzisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene, quinazolinylene, quinolizinylene, 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-dihydrobenzofuranylene, 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, anthracenyl, phenanthrenyl, fluorenyl, indenyl, and azulenyl as well as the corresponding 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 corresponding 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-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl, thiazolidinyl, 2-thiooxothiazolidinyl, as well as the corresponding 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-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, benzofuryl, benzothienyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, carbazolyl, thiazolidinyl, 2-thiooxothiazolidinyl, as well as the corresponding divalent radicals.
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, isoquinolyl, quinoxalinyl, carbazolyl, thiazolidinyl, 2-thiooxothiazolidinyl, as well as the corresponding divalent radicals.
“Aryl-C1-C6-alkyl”, “heteroaryl-C1-C6-alkyl”, “aryl-C2-C6-alkenyl” etc. is intended to mean C1-C6-alkyl or C2-C6-alkenyl as defined above, substituted by an aryl or heteroaryl as defined above, for example:
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 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 “a amino acid” as used herein is intended to mean any natural (codable) and non-natural α-aminocarboxylic acid, including D-isomers of these.
The term “amino acid” as used herein is intended to mean any β-aminocarboxylic acid, such as β-alanine, isoserine or the like.
The term “desB30” 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.
When in the specification or claims mention is made of groups of compounds such as carboxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles, 4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thiazolidinediones, 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 all variants of insulin including human insulin, an analogue thereof, a derivative thereof and combinations of any of these, acid-stabilised insulin, fast/rapid acting insulin and long/slow/basal acting insulin.
The term “human insulin” as used herein refers to naturally produced insulin or recombinantly produced insulin. Recombinant human 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.
The term “insulin analogue” as used herein is meant human insulin in which at least one amino acid has been deleted and/or replaced by another amino acid 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 term “insulin derivative” as used herein refers to human insulin or an analogue thereof which has been chemically modified, i.e. at least one organic substituent is bound to one or more of the amino acids, e.g. by introducing a side chain in one or more positions of the insulin backbone or by oxidizing or reducing groups of the amino acid residues in the insulin or by converting a free carboxylic group to an ester group or acylating a free amino group or a hydroxy group.
The term “acid-stabilised insulin” as used herein refers to an insulin analog that does not deamidate or dimerize at pH values below 7. Specifically, the analog cannot have Asn or Asp as a C-terminal residue.
By “fast/rapid acting insulin” as used herein is meant any insulin having an onset of action after injection or any other form of administration faster or equal to that of soluble and neutral formulations of human insulin.
The term “long/slow/basal acting insulin” as used herein is intended to include insulin compounds such as protamine insulin, zinc insulin, protamine zinc insulin.
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.
When an insulin derivative according to the invention is stated to be “soluble at physiological pH values” it means that the insulin derivative can be used for preparing injectable insulin compositions that are fully dissolved at physiological pH values. Such favourable solubility may either be due to the inherent properties of the insulin derivative alone or a result of a favourable interaction between the insulin derivative and one or more ingredients contained in the vehicle.
The term “physiologically relevant pH” as used herein is intended to mean a pH of about 7.1 to 7.9.
4H3N 4-Hydroxy-3-nitrobenzoic acid
DBU 1,8-Diazabicyclo[5,4,0]undec-7-ene
EDAC 1-Ethyl-3-(3′-dimethylamino-propyl)carbodiimide, hydrochloride
Fmoc 9H-Fluorene-9-ylmethoxycarbonyl
HOAt 1-Hydroxy-7-azabenzotriazole
HOAc Acetic acid
AcOH Acetic acid
NMP N-Methyl-2-pyrrolidone
Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl
Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl
TFA Trifluoroacetic acid
Dde 1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl
IvDde 1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl
Eq equivalents
The present invention is based on the discovery that the branched HisB10Zn++ ligand binding sites of the R-state insulin hexamer can be used to obtain an insulin preparation having prolonged action designed for flexible injection regimes including once-daily, based on insulin molecules of any kind.
The basic concept underlying the present invention involves reversible attachment of a branched ligand to the HisB10Zn2+ site of the R-state hexamer. A suitable ligand binds to the hexamer metal site with one end while other moieties are covalently attached to the other end. On this basis, prolonged action via modification of preparation solubility may be obtained in a number of ways. However, all cases involve the same point of protein-ligand attachment and the delivery of human insulin (or analogues or derivatives thereof) as the active species. Use of a acid-stabilized insulin analog allows a stable, clear solution with ligand to be formulated at slightly acidic pH. Following subcutaneous injection, the pH is gradually adjusted towards neutral. As a result the ligand binds to and precipitates insulin in the subcutaneous tissue. The release of insulin analog into the blood stream is then limited by the rate of redissolution of the precipitate. Of particular advantage is the possibility of adjusting the amount of added ligand as well as the charge and affinity of the ligand. Variation of these parameters allows adjustment of the rate of dissolution following precipitation in the subcutis and hence the proportion of slow and fast acting analog in the formulation. Hence formulations covering a wide range of release rates may be prepared by this principle.
The anions currently used in insulin formulations as allosteric ligands for the R-state hexamers (notably chloride ion) bind only weakly to the HisB10 anion site. The present invention, which is based on the discovery of suitable higher affinity ligands for these anion sites, provides ligands which are extended to modify timing via changes in hexamer solubility as outlined above.
Most ligand binding sites in proteins are highly asymmetric. Because the HisB10Zn2+ sites reside on the three-fold symmetry axis, these sites posses a symmetry that is unusual, but not unique. Several other proteins have highly symmetric ligand binding sites.
The HisB10Zn2+ site consists of a tunnel or cavity with a triangular-shaped cross-section that extends ˜12 Å from the surface of the hexamer down to the HisB10Zn2+ ion. The diameter of the tunnel varies along its length and, depending on the nature of the ligand occupying the site, the opening can be capped over by the AsnB3 and PheB1 side chains. The walls of the tunnel are made up of the side chains of the amino acid residues along one face each of the three α-helices. The side chains from each helix that make up the lining of the tunnel are PheB1, AsnB3, and LeuB6. Therefore, except for the zinc ion, which is coordinated to three HisB10 residues and is positioned at the bottom of the tunnel, the site is principally hydrophobic. Depending on the ligand structure, it may be possible for substituents on the ligand to make H-bonding interactions with AsnB3 and with the peptide linkage to CysB7.
The present invention originates from a search for compounds with suitable binding properties by using UV-visible and fluorescence based competition assays described herein which are based on the displacement of chromophoric ligands from the R-state HisB10-Zn2+ site by the incoming ligand in question. These compounds will be referred to as “starter compounds” in the following. These assays are easily transformed into a high-throughput format capable of handling libraries constructed around hits from the initial search of compound databases.
These starter compounds provide the starting point for the task of constructing a chemical handle that allows for attachment of the positively charged fragment, Frg2 (see below).
Thus, from the structure-activity relationship (SAR) information obtained from the binding assay(s) it will be apparent for those skilled in the art to modify the starter compounds in question by introduction of a chemical group that will allow for coupling to a peptide containing e.g. one or more arginine or lysine residues. These chemical groups include carboxylic acid (amide bond formation with the peptide), carbaldehyde (reductive alkylation of the peptide), sulfonyl chloride (sulphonamide formation with the peptide) or the like.
The decision where and how to introduce this chemical group can be made in various ways. For example: From the SAR of a series of closely related starter compounds, a suitable position in the starter compound can be identified and the chemical group can be attached to this position, optionally using a spacer group, using synthesis procedures known to those skilled in the art.
Alternatively, this chemical group can be attached (optionally using a spacer group using and synthesis procedures known to those skilled in the art) to a position on the starter compound remote from the Zn2+-binding functionality.
The invention thus provides pharmaceutical preparation comprising
CGr-Lnk-Frg1-Frg2-X (I)
wherein:
CGr is a chemical group which reversibly binds to a HisB10Zn2+ site of an insulin hexamer;
Lnk is a linker selected from
The present invention also encompasses pharmaceutically acceptable salts of the present compounds. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulphuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, picric, pyruvic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methyl-, dimethyl-, trimethyl-, ethyl-, hydroxyethyl-, diethyl-, n-butyl-, sec-butyl-, tert-butyl-, tetramethylammonium salts and the like.
Also intended as pharmaceutically acceptable acid addition salts are the hydrates, which the present compounds, are able to form.
The acid addition salts may be obtained as the direct products of compound synthesis. In the alternative, the free base may be dissolved in a suitable solvent containing the appropriate acid, and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.
The compounds of the present invention may form solvates with standard low molecular weight solvents using methods well known to the person skilled in the art. Such solvates are also contemplated as being within the scope of the present invention.
In one embodiment CGr is a chemical structure selected from the group consisting of carboxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles, 4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thiazolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, barbiturates, naphthoic acids and salicylic acids.
In another embodiment CGr is a chemical structure selected from the group consisting of benzotriazoles, 3-hydroxy 2-naphthoic acids, salicylic acids, tetrazoles, thiazolidinediones, 5-mercaptotetrazoles, or 4-cyano-1,2,3-triazoles.
In another embodiment CGr is
wherein
R1 and R4 are independently selected from hydrogen or C1-C6-alkyl,
R2 is hydrogen or C1-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, C1-C6-alkyl, or —C(O)NR11R12,
A and B are independently selected from C1-C6-alkylene, arylene, aryl-C1-C6-alkyl-, aryl-C2-C6-alkenyl- or heteroarylene, wherein the alkylene or alkenylene is optionally substituted with one or more substituents independently selected from R6 and the arylene or heteroarylene 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
In another embodiment X is ═O or ═S.
In another embodiment X is ═O.
In another embodiment X is ═S.
In another embodiment Y is —O— or —S—.
In another embodiment Y is —O—.
In another embodiment wherein Y is —S—.
In another embodiment Crg is arylene optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is selected from ArG1 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is phenylene or naphthylene optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is
In another embodiment A is phenylene.
In another embodiment A is heteroarylene optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is selected from Het1 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is selected from Het2 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is selected from Het3 optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is selected from the group consisting of indolylene, benzofuranylidene, quinolylene, furylene, thienylene, or pyrrolylene, wherein each heteroaryl may optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is benzofuranylene optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is
In another embodiment A is carbazolylidene optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is
In another embodiment A is quinolylidene optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is
In another embodiment A is indolylene optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
In another embodiment A is
In another embodiment R1 is hydrogen.
In another embodiment R2 is hydrogen.
In another embodiment R1 and R2 are combined to form a double bond.
In another embodiment R3 is C1-C6-alkyl, halogen, or C(O)NR16R17.
In another embodiment R3 is C1-C6-alkyl or C(O)NR16R17.
In another embodiment R3 is methyl.
In another embodiment B is phenylene optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
In another embodiment R4 is hydrogen.
In another embodiment R5 is hydrogen.
In another embodiment R6 is aryl.
In another embodiment R6 is phenyl.
In another embodiment R7, R8, R9 and R10 are independently selected from
In another embodiment R7, R8, R9 and R10 are independently selected from
In another embodiment R7, R8, R9 and R10 are independently selected from
In another embodiment R7, R8, R9 and R10 are independently selected from
In another embodiment R7, R8, R9 and R10 are independently selected from
In another embodiment R11 and R12 are independently selected from hydrogen, C1-C20-alkyl, aryl or aryl-C1-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.
In another embodiment R11 and R12 are independently selected from hydrogen, C1-C20-alkyl, aryl or aryl-C1-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.
In another embodiment R11 and R12 are independently selected from phenyl or phenyl-C1-C6-alkyl.
In another embodiment R11 and R12 are methyl.
In another embodiment R13 is independently selected from halogen, CF3, OR11 or NR11R12.
In another embodiment R13 is independently selected from halogen or OR11.
In another embodiment R13 is OR11.
In another embodiment R14 is independently selected from halogen, —C(O)OR11, —CN, —CF3, —OR11, S(O)2R11, and C1-C6-alkyl.
In another embodiment R14 is independently selected from halogen, —C(O)OR11, or —OR11.
In another embodiment R15 is independently selected from halogen, —CN, —CF3, —C(O)OC1-C6-alkyl, and —COOH.
In another embodiment R15 is independently selected from halogen or —C(O)OC1-C6-alkyl.
In another embodiment R16 is independently selected from halogen, —C(O)OC1-C6-alkyl, —COOH, —NO2, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl.
In another embodiment R16 is independently selected from halogen, —C(O)OC1-C6-alkyl, —COOH, —NO2, or C1-C6-alkyl.
In another embodiment CGr is
wherein
R19 is hydrogen or C1-C6-alkyl,
R20 is hydrogen or C1-C6-alkyl,
D and F are a valence bond or C1-C6-alkylene optionally substituted with one or more substituents independently selected from R72,
R72 is independently selected from hydroxy, C1-C6-alkyl, or aryl,
E is C1-C6-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R21, R22 and R23,
G is C1-C6-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene 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
In another embodiment D is a valence bond.
In another embodiment D is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl.
In another embodiment E is arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents independently selected from R21, R22 and R23.
In another embodiment E is arylene optionally substituted with up to three substituents independently selected from R21, R22 and R23.
In another embodiment E is selected from ArG1 and optionally substituted with up to three substituents independently selected from R21, R22 and R23.
In another embodiment E is phenylene optionally substituted with up to three substituents independently selected from R21, R22 and R23.
In another embodiment CGr is
In another embodiment R21, R22 and R23 are independently selected from
In another embodiment R21, R22 and R23 are independently selected from
In another embodiment R21, R22 and R23 are independently selected from
In another embodiment R21, R22 and R23 are independently selected from
In another embodiment R21, R22 and R23 are independently selected from
In another embodiment R19 is hydrogen or methyl.
In another embodiment R19 is hydrogen.
In another embodiment R27 is Hydrogen, C1-C6-alkyl or aryl.
In another embodiment R27 is hydrogen or C1-C6-alkyl.
In another embodiment R28 is hydrogen or C1-C6-alkyl.
In another embodiment F is a valence bond.
In another embodiment F is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl.
In another embodiment G is C1-C6-alkylene or arylene, wherein the arylene is optionally substituted with up to three substituents R24, R25 and R26.
In another embodiment G is C1-C6-alkylene or ArG1, wherein the arylene is optionally substituted with up to three substituents R24, R25 and R26.
In another embodiment G is C1-C6-alkylene.
In another embodiment G is phenylene optionally substituted with up to three substituents R24, R25 and R26.
In another embodiment R24, R25 and R26 are independently selected from
In another embodiment R24, R25 and R26 are independently selected from
In another embodiment R24, R25 and R26 are independently selected from
C1-C6-alkyl optionally substituted with one or more substituents independently selected from R29
In another embodiment R24, R25 and R26 are independently selected from
In another embodiment R24, R25 and R26 are independently selected from
In another embodiment R24, R25 and R26 are independently selected from
In another embodiment R20 is hydrogen or methyl.
In another embodiment R20 is hydrogen.
In another embodiment R27 is hydrogen, C1-C6-alkyl or aryl.
In another embodiment R27 is hydrogen or C1-C6-alkyl or ArG1.
In another embodiment R27 is hydrogen or C1-C6-alkyl.
In another embodiment R28 is hydrogen or C1-C6-alkyl.
In another embodiment R17 and R18 are independently selected from
In another embodiment R17 and R18 are independently selected from
In another embodiment R17 and R18 are independently selected from
In another embodiment R17 and R18 are independently selected from
In another embodiment R17 and R18 are independently selected from
In another embodiment R27 is hydrogen or C1-C6-alkyl.
In another embodiment R27 is hydrogen, methyl or ethyl.
In another embodiment R28 is hydrogen or C1-C6-alkyl.
In another embodiment R28 is hydrogen, methyl or ethyl.
In another embodiment R72 is —OH or phenyl.
In another embodiment CGr is
In another embodiment CGr is of the form H—I-J-
wherein H is
wherein the phenyl, naphthalene or benzocarbazole rings are optionally substituted with one or more substituents independently selected from R31
I is selected from
wherein Z1 is S(O)2 or CH2, Z2 is —NH—, —O— or —S—, and n is 1 or 2,
In another embodiment H is
In another embodiment H is
In another embodiment H is
In another embodiment I is a valence bond, —CH2N(R32)—, or —SO2N(R33)—.
In another embodiment I is a valence bond.
In another embodiment J is
In another embodiment J is
In another embodiment J is
In another embodiment J is
In another embodiment R32 and R33 are independently selected from hydrogen or C1-C6-alkyl.
In another embodiment 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, —OC1-C6-alkyl-C(O)OR35, —SC1-C6-alkyl-C(O)OR35 or —C(O)OR35.
In another embodiment R34 is hydrogen, halogen, —CF3, —NO2, —OR35, —NR35R36, —SR35, —NR35C(O)R36, or —C(O)OR35.
In another embodiment R34 is hydrogen, halogen, —CF3, —NO2, —OR35, —NR35R36, or —NR35C(O)R36.
In another embodiment R34 is hydrogen, halogen, or —OR35.
In another embodiment R35 and R36 are independently selected from hydrogen, C1-C6-alkyl, or aryl.
In another embodiment R35 and R36 are independently selected from hydrogen or C1-C6-alkyl.
In another embodiment R37 is halogen, —C(O)OR35, —CN, —CF3, —OR35, —NR35R36, C1-C6-alkyl or C1-C6-alkanoyl.
In another embodiment R37 is halogen, —C(O)OR35, —OR35, —NR35R36, C1-C6-alkyl or C1-C6-alkanoyl.
In another embodiment R37 is halogen, —C(O)OR35 or —OR35.
In another embodiment CGr is
wherein K is a valence bond, C1-C6-alkylene, —NH—C(═O)—U—, —C1-C6-alkyl-S—, —C1-C6-alkyl-O—, —C(═O)—, or —C(═O)—NH—, wherein any C1-C6-alkyl moiety is optionally substituted with R38,
U is a valence bond, C1-C6-alkenylene, —C1-C6-alkyl-O— or C1-C6-alkylene wherein any C1-C6-alkyl moiety is optionally substituted with C1-C6-alkyl,
R38 is C1-C6-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
In another embodiment K is a valence bond, C1-C6-alkylene, —NH—C(═O)—U—, —C1-C6-alkyl-S—, —C1-C6-alkyl-O—, or —C(═O)—, wherein any C1-C6-alkyl moiety is optionally substituted with R38.
In another embodiment K is a valence bond, C1-C6-alkylene, —NH—C(═O)—U—, —C1-C6-alkyl-S—, or —C1-C6-alkyl-O, wherein any C1-C6-alkyl moiety is optionally substituted with R38.
In another embodiment K is a valence bond, C1-C6-alkylene, or —NH—C(═O)—U, wherein any C1-C6-alkyl moiety is optionally substituted with R38.
In another embodiment K is a valence bond or C1-C6-alkylene, wherein any C1-C6-alkyl moiety is optionally substituted with R38.
In another embodiment K is a valence bond or —NH—C(═O)—U.
In another embodiment K is a valence bond.
In another embodiment U is a valence bond or —C1-C6-alkyl-O—.
In another embodiment U is a valence bond
In another embodiment M is arylene or heteroarylene, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
In another embodiment M is ArG1 or Het1, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
In another embodiment M is ArG1 or Het2, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
In another embodiment M is ArG1 or Het3, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
In another embodiment M is phenylene optionally substituted with one or more substituents independently selected from R40.
In another embodiment M is indolylene optionally substituted with one or more substituents independently selected from R40.
In another embodiment M is
In another embodiment M is carbazolylene optionally substituted with one or more substituents independently selected from R40.
In another embodiment M is
In another embodiment R40 is selected from
In another embodiment R40 is selected from
In another embodiment R40 is selected from
In another embodiment R40 is selected from
In another embodiment R41 and R42 are independently selected from hydrogen, C1-C6-alkyl, or aryl, wherein the aryl moieties may optionally be substituted with halogen or —COOH.
In another embodiment R41 and R42 are independently selected from hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties may optionally be substituted with halogen or —COOH.
In another embodiment Q is a valence bond, C1-C6-alkylene, —C1-C6-alkyl-O—, —C1-C6-alkyl-NH—, —NH—C1-C6-alkyl, —NH—C(═O)—, —C(═O)—NH—, —O—C1-C6-alkyl, —C(═O)—, or —C1-C6-alkyl-C(═O)—N(R47)— wherein the alkyl moieties are optionally substituted with one or more substituents independently selected from R48.
In another embodiment Q is a valence bond, —CH2—, —CH2—CH2—, —CH2—O—, —CH2—CH2—O—, —CH2—NH—, —CH2—CH2—NH—, —NH—CH2—, —NH—CH2—CH2—, —NH—C(═O)—, —C(═O)—NH—, —O—CH2—, —O—CH2—CH2—, or —C(═O)—.
In another embodiment R47 and R48 are independently selected from hydrogen, methyl and phenyl.
In another embodiment T is
In another embodiment T is
In another embodiment T is
In another embodiment R50 is C1-C6-alkyl, C1-C6-alkoxy, aryl, aryloxy, aryl-C1-C6-alkoxy, —C(═O)—NH—C1-C6-alkyl-aryl, heteroaryl, —C1-C6-alkyl-COOH, —O—C1-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.
In another embodiment R50 is C1-C6-alkyl, C1-C6-alkoxy, aryl, aryloxy, aryl-C1-C6-alkoxy, —OR51, —NO2, halogen, —COOH, —CF3, wherein any aryl moiety is optionally substituted with one or more R53.
In another embodiment R50 is C1-C6-alkyl, aryloxy, aryl-C1-C6-alkoxy, —OR51, halogen, —COOH, —CF3, wherein any aryl moiety is optionally substituted with one or more R53.
In another embodiment R50 is C1-C6-alkyl, ArG1-O—, ArG1-C1-C6-alkoxy, —OR51, halogen, —COOH, —CF3, wherein any aryl moiety is optionally substituted with one or more R53.
In another embodiment R50 is phenyl, methyl or ethyl.
In another embodiment R50 is methyl or ethyl.
In another embodiment R51 is methyl.
In another embodiment R53 is C1-C6-alkyl, C1-C6-alkoxy, —OR51, halogen, or —CF3.
In another embodiment CGr is
wherein V is C1-C6-alkylene, arylene, heteroarylene, arylene-C1-6-alkylene or arylene-C2-6-alkenylene, wherein the alkylene or alkenylene is optionally substituted with one or more substituents independently selected from R54, and the arylene or heteroarylene 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
In another embodiment V is arylene, heteroarylene, or arylene-C1-C6-alkylene, wherein the alkylene is optionally substituted with one or more substituents independently selected R54, and the arylene or heteroarylene is optionally substituted with one or more substituents independently selected from R55.
In another embodiment V is arylene, Het1, or arylene-C1-C6-alkylene, wherein the alkylene is optionally substituted with one or more substituents independently selected from R54, and the arylene or heteroarylene moiety is optionally substituted with one or more substituents independently selected from R55.
In another embodiment V is arylene, Het2, or arylene-C1-C6-alkylene, wherein the alkylene is optionally substituted with one or more substituents independently selected from R54, and the arylene or heteroarylene moiety is optionally substituted with one or more substituents independently selected from R55.
In another embodiment V is arylene, Het3, or arylene-C1-C6-alkylene, wherein the alkylene is optionally substituted with one or more substituents independently selected from R54, and the arylene or heteroarylene moiety is optionally substituted with one or more substituents independently selected from R55.
In another embodiment V is arylene optionally substituted with one or more substituents independently selected from R55.
In another embodiment V is ArG1 optionally substituted with one or more substituents independently selected from R55.
In another embodiment V is phenylene, naphthylene or anthracylene optionally substituted with one or more substituents independently selected from R55.
In another embodiment V is phenylene optionally substituted with one or more substituents independently selected from R55.
In another embodiment R55 is independently selected from
In another embodiment R55 is independently selected from
In another embodiment R55 is independently selected from halogen, —OR56, —NR56R57, —C(O)OR56, —OC1-C8-alkyl-C(O)OR56, —NR56C(O)R57 or C1-C6-alkyl.
In another embodiment R55 is independently selected from halogen, —OR56, —NR56R57, —C(O)OR56, —OC1-C8-alkyl-C(O)OR56, —NR56C(O)R57, methyl or ethyl.
In another embodiment R56 and R57 are independently selected from hydrogen, CF3, C1-C12-alkyl, or —C(═O)—C1-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.
In another embodiment R56 and R57 are independently selected from hydrogen or C1-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.
In another embodiment 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.
In another embodiment CGr is
wherein AA is C1-C6-alkylene, arylene, heteroarylene, arylene-C1-C6-alkylene or arylene-C2-C6-alkenylene, wherein the alkylene or alkenylene is optionally substituted with one or more substituents independently selected from R63, and the arylene or heteroarylene 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
In another embodiment AA is arylene, heteroarylene or arylene-C1-C6-alkylene, wherein the alkylene is optionally substituted with one or more R63, and the arylene or heteroarylene is optionally substituted with one or more substituents independently selected from R64.
In another embodiment AA is arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with one or more substituents independently selected from R64.
In another embodiment AA is ArG1 or Het1 optionally substituted with one or more substituents independently selected from R64.
In another embodiment AA is ArG1 or Het2 optionally substituted with one or more substituents independently selected from R64.
In another embodiment AA is ArG1 or Het3 optionally substituted with one or more substituents independently selected from R64.
In another embodiment AA is phenylene, naphthylene, anthrylene, carbazolylene, thienylene, pyridylene, or benzodioxylene optionally substituted with one or more substituents independently selected from R64.
In another embodiment AA is phenylene or naphthylene optionally substituted with one or more substituents independently selected from R64.
In another embodiment R64 is independently selected from hydrogen, halogen, —CF3, —OCF3, —OR65, —NR65R66, C1-C6-alkyl, —OC(O)R65, —OC1-C6-alkyl-C(O)OR65, aryl-C2-C6-alkenyl, aryloxy or aryl, wherein C1-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.
In another embodiment 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.
In another embodiment R65 and R66 are independently selected from hydrogen, CF3, C1-C12-alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R71.
In another embodiment R65 and R66 are independently hydrogen, C1-C12-alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R71.
In another embodiment 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.
In another embodiment 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.
In another embodiment 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.
In another embodiment R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, phenyl, naphtyl, thiadiazolyl optionally substituted with one or more R71 independently; or isoxazolyl optionally substituted with one or more substituents independently selected from R71.
In another embodiment R71 is halogen or C1-C6-alkyl.
In another embodiment R71 is halogen or methyl.
In another embodiment Frg1 consists of 0 to 5 neutral amino acids independently selected from the group consisting of Gly, Ala, Thr, and Ser.
In another embodiment Frg1 consists of 0 to 5 Gly.
In another embodiment Frg1 consists of 0 Gly.
In another embodiment Frg1 consists of 1 Gly.
In another embodiment Frg1 consists of 2 Gly.
In another embodiment Frg1 consists of 3 Gly.
In another embodiment Frg1 consists of 4 Gly.
In another embodiment Frg1 consists of 5 Gly.
In another embodiment GB is of the formula B1—B2—C(O)—, B1—B2—SO2— or B1—B2—CH2—.
In another embodiment GB is of the formula B1—B2—C(O)—, B1—B2—SO2— or B1—B2—NH—.
In another embodiment GB is of the formula B1—B2—C(O)—, B1—B2—CH2— or B1—B2—NH—.
In another embodiment GB is of the formula B1—B2—CH2—, B1—B2—SO2— or B1—B2—NH—.
In another embodiment GB is of the formula B1—B2—C(O)— or B1—B2—SO2—.
In another embodiment GB is of the formula B1—B2—C(O)— or B1—B2—CH2—.
In another embodiment GB is of the formula B1—B2—C(O)— or B1—B2—NH—.
In another embodiment GB is of the formula B1—B2—CH2— or B1—B2—SO2—.
In another embodiment GB is of the formula B1—B2—NH— or B1—B2—SO2—.
In another embodiment GB is of the formula B1—B2—CH2— or B1—B2—NH—.
In another embodiment GB is of the formula B1—B2—C(O)—.
In another embodiment GB is of the formula B1—B2—CH2—.
In another embodiment GB is of the formula B1—B2—SO2—.
In another embodiment GB is of the formula B1—B2—NH—.
In another embodiment B1 is a valence bond, —O—, or —S—.
In another embodiment B1 is a valence bond, —O—, or —N(R6)—.
In another embodiment B1 is a valence bond, —S—, or —N(R6)—.
In another embodiment B1 is —O—, —S— or —N(R6)—.
In another embodiment B1 is a valence bond or —O—.
In another embodiment B1 is a valence bond or —S—.
In another embodiment B1 is a valence bond or —N(R6)—.
In another embodiment B1 is —O— or —S—.
In another embodiment B1 is —O— or —N(R6)—.
In another embodiment B1 is —S— or —N(R6)—.
In another embodiment B1 is a valence bond.
In another embodiment B1 is —O—.
In another embodiment B1 is —S—.
In another embodiment B1 is —N(R6)—.
In another embodiment B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-O—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-S—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-NR6—C1-C18-alkyl-C(═O)—; and the alkylene and arylene moieties are optionally substituted as defined in claim 1.
In another embodiment B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-O—C1-C18-alkyl-C(═O)—, and the alkylene and arylene moieties are optionally substituted as defined in claim 1.
In another embodiment B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, and the alkylene and arylene moieties are optionally substituted as defined in claim 1.
In another embodiment B2 is a valence bond, C1-C18-alkylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, and the alkylene and arylene moieties are optionally substituted as defined in claim 1.
In another embodiment B2 is a valence bond, C1-C18-alkylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, and the alkylene and arylene moieties are optionally substituted as defined in claim 1.
In another embodiment B2 is a valence bond, C1-C18-alkylene, arylene, —C1-C18-alkyl-aryl-, and the alkylene and arylene moieties are optionally substituted as defined in claim 1.
In another embodiment B2 is a valence bond or —C1-C18-alkylene, and the alkylene moieties are optionally substituted as defined in claim 1.
In another embodiment Frg2 comprises 1-16 positively charged groups in a branched orientation.
In another embodiment Frg2 comprises 1-12 positively charged groups in a branched orientation.
In another embodiment Frg2 comprises 1-10 positively charged groups in a branched orientation.
In another embodiment Frg2 comprises a branching point comprising Lys, ornithine, Glu, Asp or iminodiacetic acid.
In another embodiment Frg2 is a fragment containing basic amino acids independently selected from the group consisting of Lys and Arg and D-isomers of these.
In another embodiment X is —OH or —NH2.
In another embodiment X is —NH2.
In another embodiment the pharmaceutical preparation further comprises at least 3 phenolic molecules.
In another embodiment the insulin is selected from the group consisting of human insulin, an analogue thereof, a derivative thereof and combinations of any of these.
In another embodiment the insulin is human insulin.
In another embodiment the insulin is an analogue of human insulin.
In another embodiment the insulin is a derivative of human insulin.
In another embodiment the insulin is an analogue of human insulin wherein position B28 is Asp, Glu, Lys, Leu, Val, or Ala.
In another embodiment the insulin is an analogue of human insulin wherein position B28 is Asp, Glu or Lys
In another embodiment the insulin is an analogue of human insulin wherein position B28 is Asp or Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B28 is Asp.
In another embodiment the insulin is an analogue of human insulin wherein position B28 is Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B29 is Pro, Asp or Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B29 is Pro or Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B29 is Pro.
In another embodiment the insulin is an analogue of human insulin wherein position B29 is Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B28 is Asp or Lys, and position B29 is Lys or Pro.
In another embodiment the insulin is an analogue of human insulin wherein position B9 is Asp or Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B10 is Asp or Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B10 is Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B1 is Gly.
In another embodiment the insulin is an analogue of human insulin wherein position B3 is Lys, Thr, Ser, Ala or Gln.
In another embodiment the insulin is an analogue of human insulin wherein position B3 is Lys, Thr, Ser or Ala.
In another embodiment the insulin is an analogue of human insulin wherein position B3 is Lys or Ala.
In another embodiment the insulin is an analogue of human insulin wherein position B3 is Lys.
In another embodiment the insulin is an analogue of human insulin wherein position B3 is Lys and position B29 is Glu.
In another embodiment the insulin is an analogue of human insulin wherein position B25 is deleted.
In another embodiment the insulin is an analogue of human insulin wherein position B27 is deleted.
In another embodiment the insulin is an analogue of human insulin wherein position B30 is deleted.
In another embodiment the insulin is an analogue of human insulin wherein position A18 is Gln.
In another embodiment the insulin is an analogue of human insulin wherein position A21 is Ala, Arg, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Thr, Trp, Tyr, Val or hSer.
In another embodiment the insulin is an analogue of human insulin wherein position A21 is Ala, Arg, Gly, Ile, Leu, Phe, Ser, Thr, Val or hSer.
In another embodiment the insulin is an analogue of human insulin wherein position A21 is Ala or Gly.
In another embodiment the insulin is an analogue of human insulin wherein position A21 is Gly.
In another embodiment the insulin is a derivative of human insulin or an analogue thereof having one or more lipophilic substituents.
In another embodiment the insulin is a derivative of human insulin or an analogue thereof wherein the N68-amino group in position B29Lys is modified by covalent acylation with a hydrophobic moiety such as an fatty acid derivative or an litocholic acid derivative.
In another embodiment 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 LysB28 ProB29 human insulin, B28-Nε-palmitoyl LysB28 ProB29 human insulin, B30-Nε-myristoyl-ThrB29LysB30 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(B30) human insulin, B29-Nε-(ω-carboxyheptadecanoyl)-des(B30) human insulin and B29-Nε-(ω-carboxyheptadecanoyl) human insulin.
In another embodiment, the analogs of human insulin contain any combination of additional stabilizing substitutions.
In another embodiment, the analogs of human insulin contain any combination of the additional stabilizing substitutions in positions B1, B3, A18 and A21.
In another embodiment the insulin is an analogue of human insulin selected from the group consisting of:
In another embodiment the insulin is an analogue of human insulin selected from the group consisting of:
A21G, desB27
A21G, desB25
A21G, desB30
A21G, B28K, B29P, desB30
A21G, B28D, desB30
A21G, B28E, desB30
A21G, B3K, B29E, desB30
A21G, desB27, desB30
A21G, B9E, desB30
A21G, B9D, desB30
A21G, B10E, desB30
A21G, desB25, desB30.
In another embodiment the insulin is an analogue of human insulin selected from the group consisting of:
B1G, A21G, desB27
B1G, A21G, desB25
B1G, A21G, desB30
B1G, A21G, B28K, B29P, desB30
B1G, A21G, B28D, desB30
B1G, A21G, B28E, desB30
B1G, A21G, B3K, B29E, desB30
B1G, A21G, desB27, desB30
B1G, A21G, B9E, desB30
B1G, A21G, B9D, desB30
B1G, A21G, B10E, desB30
B1G, A21G, desB25, desB30.
In another embodiment, the insulin is an analogue of human insulin from above three lists further modified in positions B3 and A18, eg B3T, B3S, B3Q and A18Q.
In another embodiment, the insulin is an analogue of human insulin from the above three lists further modified as follows:
B3T, desB27.
In another embodiment, the insulin is an analogue of human insulin from the above three lists further modified by deletion of B30.
In another embodiment the ratio of the ligand of general formula (I) to zinc ion is 1:20 to 20:1.
In another embodiment the ratio of the ligand of general formula (I) to zinc ion is 1:6 to 10:1.
In another embodiment the amount of zinc ions is 2-6 moles per mole of putative insulin hexamer.
In another embodiment the amount of zinc ions is 2.0-3.5 moles per putative insulin hexamer.
In another embodiment zinc ions are present in an amount corresponding to 10 to 40 μg Zn/100 U insulin.
In another embodiment zinc ions are present in an amount corresponding to 10 to 26 μg Zn/100 U insulin.
In another embodiment the ratio between insulin and the ligand of the invention is in the range from 99:1 to 1:99.
In another embodiment the ratio between insulin and the ligand of the invention is in the range from 95:5 to 5:95.
In another embodiment the ratio between insulin and the ligand of the invention is in the range from 80:20 to 20:80.
In another embodiment the ratio between insulin and the ligand of the invention is in the range from 70:30 to 30:70.
In another aspect the invention relates to a method of preparing a ligand of the invention comprising the steps of:
In another aspect the invention relates to a method of prolonging the action of an insulin preparation which comprises adding the ligand of the invention to the insulin preparation.
In another aspect the invention relates to 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 comprising
In another aspect the invention provides an embodiment 1, which is a pharmaceutical preparation comprising
CGr-Lnk-Frg1-Frg2-X (I)
wherein:
CGr is a chemical group which reversibly binds to a HisB10Zn2+ site of an insulin hexamer;
Lnk is a linker selected from
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein CGr is a chemical structure selected from the group consisting of carboxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles, 4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thiazolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, barbiturates, naphthoic acids and salicylic acids.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein CGr is a chemical structure selected from the group consisting of benzotriazoles, 3-hydroxy 2-naphthoic acids, salicylic acids, tetrazoles, thiazolidinediones, 5-mercaptotetrazoles, or 4-cyano-1,2,3-triazoles.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein CGr is
wherein
R1, R1A and R4 are independently selected from hydrogen or C1-C6-alkyl,
R2 and R2A are hydrogen or C1-C6-alkyl or aryl, R1 and R2 may optionally be combined to form a double bond, R1A and R2A 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, C1-C6-alkyl, or —C(O)NR11R12,
A, A1 and B are independently selected from C1-C6-alkyl, aryl, aryl-C1-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
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein X is ═O or ═S.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein X is ═O.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein X is ═S.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein Y is —O— or —S—.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein Y is —O—.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein Y is —NH—.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein Y is —S—.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein A is aryl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is
16. A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is phenyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein A is heteroaryl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is benzofuranyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is
24. A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is carbazolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is quinolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is indolyl optionally substituted with up to four substituents R7, R8, R9, and R10 which may be the same or different.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein A is
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R1 is hydrogen.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R2 is hydrogen.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R1 and R2 are combined to form a double bond.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R3 is C1-C6-alkyl, halogen, or C(O)NR16R17.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R3 is C1-C6-alkyl or C(O)NR16R17.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R3 is methyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein B is phenyl optionally substituted with up to four substituents, R7, R8, R9, and R10 which may be the same or different.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. or Error! Reference source not found. wherein R4 is hydrogen.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. or Error! Reference source not found. to Error! Reference source not found. wherein R5 is hydrogen.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R6 is aryl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R6 is phenyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R7, R8, R9 and R10 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R7, R8, R9 and R10 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R7, R8, R9 and R10 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R7, R8, R9 and R10 are independently selected from hydrogen, halogen, —OR11, —OC1-C6-alkyl-C(O)OR11, or —C(O)OR11,
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R7, R8, R9 and R10 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R7, R8, R9 and R10 are independently selected from
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R11 and R12 are independently selected from hydrogen, C1-C20-alkyl, aryl or aryl-C1-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R11 and R12 are independently selected from hydrogen, C1-C20-alkyl, aryl or aryl-C1-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R11 and R12 are independently selected from phenyl or phenyl-C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein one or both of R11 and R12 are methyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R13 is independently selected from halogen, CF3, OR11 or NR11R12.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R13 is independently selected from halogen or OR11.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R13 is OR11.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R14 is independently selected from halogen, —C(O)OR11, —CN, —CF3, —OR11, S(O)2R11, and C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R14 is independently selected from halogen, —C(O)OR11, or —OR11.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R15 is independently selected from halogen, —CN, —CF3, —C(O)OC1-C6-alkyl, and —COOH.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R15 is independently selected from halogen or —C(O)OC1-C6-alkyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R16 is independently selected from halogen, —C(O)OC1-C6-alkyl, —COOH, —NO2, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R16 is independently selected from halogen, —C(O)OC1-C6-alkyl, —COOH, —NO2, or C1-C6-alkyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein CGr is
wherein
R19 is hydrogen or C1-C6-alkyl,
R20 is hydrogen or C1-C6-alkyl,
D, D1 and F are a valence bond, C1-C6-alkylene or C1-C6-alkenylene optionally substituted with one or more substituents independently selected from R72,
R72 is independently selected from hydroxy, C1-C6-alkyl, or aryl,
E is C1-C6-alkyl, aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with up to three substituents R21, R22 and R23,
G and G1 are C1-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
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein D is a valence bond.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein D is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein E is aryl optionally substituted with up to three substituents independently selected from R21, R22 and R23.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein E is selected from ArG1 and optionally substituted with up to three substituents independently selected from R21, R22 and R23.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein E is phenyl optionally substituted with up to three substituents independently selected from R21, R22 and R23.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein CGr is
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R21, R22 and R23 are independently selected from
A pharmaceutical composition according to embodiment 68 wherein R21, R22 and R23 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R21, R22 and R23 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R21, R22 and R23 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R21, R22 and R23 are independently selected from
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R19 is hydrogen or methyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R19 is hydrogen.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R27 is Hydrogen, C1-C6-alkyl or aryl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R27 is hydrogen or C1-C6-alkyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R28 is hydrogen or C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein F is a valence bond.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein F is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. to Error! Reference source not found. wherein G is C1-C6-alkyl or aryl, wherein the aryl is optionally substituted with up to three substituents R24, R25 and R26.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. to Error! Reference source not found. wherein G is C1-C6-alkyl or ArG1, wherein the aryl is optionally substituted with up to three substituents R24, R25 and R26.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein G is C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein G is phenyl optionally substituted with up to three substituents R24, R25 and R26
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R24, R25 and R26 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R24, R25 and R26 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R24, R25 and R26 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R21, R22 and R23 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R21, R22 and R23 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R21, R22 and R23 are independently selected from
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. to Error! Reference source not found. wherein R20 is hydrogen or methyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R20 is hydrogen.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. to Error! Reference source not found. wherein R27 is hydrogen, C1-C6-alkyl or aryl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R27 is hydrogen or C1-C6-alkyl or ArG1.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R27 is hydrogen or C1-C6-alkyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. to Error! Reference source not found. wherein R28 is hydrogen or C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R17 and R18 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R17 and R18 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R17 and R18 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R17 and R18 are independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R17 and R18 are independently selected from
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R27 is hydrogen or C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R27 is hydrogen, methyl or ethyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R28 is hydrogen or C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R28 is hydrogen, methyl or ethyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R72 is —OH or phenyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein CGr is
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein CGr is of the form H—I-J-
wherein H is
wherein the phenyl, naphthalene or benzocarbazole rings are optionally substituted with one or more substituents independently selected from R31
I is selected from
wherein Z1 is S(O)2 or CH2, Z2 is —NH—, —O— or —S—, and n is 1 or 2,
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein CGr is of the form H—I-J, wherein H is
wherein the phenyl, naphthalene or benzocarbazole rings are optionally substituted with one or more substituents independently selected from R31,
I is selected from
wherein Z1 is S(O)2 or CH2, Z2 is N, —O— or —S—, and n is 1 or 2,
With the proviso that R31 and J cannot both be hydrogen.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein H is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein H is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein H is
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found wherein I is a valence bond, —CH2N(R32)—, or —SO2N(R33)—.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein I is a valence bond.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein J is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein J is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein J is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein J is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein J is hydrogen.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R32 and R33 are independently selected from hydrogen or C1-C6-alkyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. 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, —OC1-C6-alkyl-C(O)OR35, —SC1-C6-alkyl-C(O)OR35 or —C(O)OR35.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R34 is hydrogen, halogen, —CF3, —NO2, —OR35, —NR35R36, —SR35, —NR35C(O)R36, or —C(O)OR35.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R34 is hydrogen, halogen, —CF3, —NO2, —OR35, —NR35R36, or —NR35C(O)R36.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R34 is hydrogen, halogen, or —OR35.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R35 and R36 are independently selected from hydrogen, C1-C6-alkyl, or aryl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R35 and R36 are independently selected from hydrogen or C1-C6-alkyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R37 is halogen, —C(O)OR35, —CN, —CF3, —OR35, —NR35R36, C1-C6-alkyl or C1-C6-alkanoyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R37 is halogen, —C(O)OR35, —OR35, —NR35R36, C1-C6-alkyl or C1-C6-alkanoyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R37 is halogen, —C(O)OR35 or —OR35.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein CGr is
wherein K is a valence bond, C1-C6-alkylene, —NH—C(═O)—U—, —C1-C6-alkyl-S—, —C1-C6-alkyl-O—, —C(═O)—, or —C(═O)—NH—, wherein any C1-C6-alkyl moiety is optionally substituted with R38,
U is a valence bond, C1-C6-alkenylene, —C1-C6-alkyl-O— or C1-C6-alkylene wherein any C1-C6-alkyl moiety is optionally substituted with C1-C6-alkyl,
R38 is C1-C6-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
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein K is a valence bond, C1-C6-alkylene, —NH—C(═O)—U—, —C1-C6-alkyl-S—, —C1-C6-alkyl-O—, or —C(═O)—, wherein any C1-C6-alkyl moiety is optionally substituted with R38.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein K is a valence bond, C1-C6-alkylene, —NH—C(═O)—U—, —C1-C6-alkyl-S—, or —C1-C6-alkyl-0, wherein any C1-C6-alkyl moiety is optionally substituted with R38.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein K is a valence bond, C1-C6-alkylene, or —NH—C(═O)—U, wherein any C1-C6-alkyl moiety is optionally substituted with R38.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein K is a valence bond or C1-C6-alkylene, wherein any C1-C6-alkyl moiety is optionally substituted with R38.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein K is a valence bond or —NH—C(═O)—U.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein K is a valence bond.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein U is a valence bond or —C1-C6-alkyl-O—.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein U is a valence bond.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein M is arylene or heteroarylene, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein M is ArG1 or Het1, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein M is ArG1 or Het2, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein M is ArG1 or Het3, wherein the arylene or heteroarylene moieties are optionally substituted with one or more substituents independently selected from R40.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein M is phenylene optionally substituted with one or more substituents independently selected from R40.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein M is indolylene optionally substituted with one or more substituents independently selected from R40.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein M is
b 145. A pharmaceutical composition according to embodiment Error! Reference source not found. wherein M is carbazolylene optionally substituted with one or more substituents independently selected from R40.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein M is
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R40 is selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R40 is selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R40 is selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R40 is hydrogen.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R40 is selected from
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R41 and R42 are independently selected from hydrogen, C1-C6-alkyl, or aryl, wherein the aryl moieties may optionally be substituted with halogen or —COOH.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein Q is a valence bond, C1-C6-alkylene, —C1-C6-alkyl-O—, —C1-C6-alkyl-NH—, —NH—C1-C6-alkyl, —NH—C(═O)—, —C(═O)—NH—, —O—C1-C6-alkyl, —C(═O)—, or —C1-C6-alkyl-C(═O)—N(R47)— wherein the alkyl moieties are optionally substituted with one or more substituents independently selected from R48.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein Q is a valence bond, —CH2—, —CH2—CH2—, —CH2—O—, —CH2—CH2—O—, —CH2—NH—, —CH2—CH2—NH—, —NH—CH2—, —NH—CH2—CH2—, —NH—C(═O)—, —C(═O)—NH—, —O—CH2—, —O—CH2—CH2—, or —C(═O)—.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R47 and R48 are independently selected from hydrogen, methyl and phenyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein T is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein T is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein T is
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein T is phenyl substituted with R50.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R50 is C1-C6-alkyl, C1-C6-alkoxy, aryl, aryloxy, aryl-C1-C6-alkoxy, —C(═O)—NH—C1-C6-alkyl-aryl, —C(═O)—NR50A—C1-C6-alkyl, —C(═O)—NH—(CH2CH2O)mC1-C6-alkyl-COOH, heteroaryl, —C1-C6-alkyl-COOH, —O—C1-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R50 is C1-C6-alkyl, C1-C6-alkoxy, aryl, aryloxy, —C(═O)—NR50A—C1-C6-alkyl, —C(═O)—NH—(CH2CH2O)mC1-C6-alkyl-COOH, aryl-C1-C6-alkoxy, —OR51, —NO2, halogen, —COOH, —CF3, wherein any aryl moiety is optionally substituted with one or more R53.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R50 is C1-C6-alkyl, aryloxy, —C(═O)—NR50A—C1-C6-alkyl, —C(═O)—NH—(CH2CH2O)mC1-C6-alkyl-COOH, aryl-C1-C6-alkoxy, —OR51, halogen, —COOH, —CF3, wherein any aryl moiety is optionally substituted with one or more R53.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R50 is C1-C6-alkyl, ArG1-O—, —C(═O)—NR50A—C1-C6-alkyl, —C(═O)—NH—(CH2CH2O)mC1-C6-alkyl-COOH, ArG1-C1-C6-alkoxy, —OR51, halogen, —COOH, —CF3, wherein any aryl moiety is optionally substituted with one or more R53.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R50 is —C(═O)—NR50ACH2, —C(═O)—NH—(CH2CH2O)2CH2I—COOH, or —C(═O)—NR50ACH2CH2.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R50 is phenyl, methyl or ethyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R50 is methyl or ethyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein m is 1 or 2.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R51 is methyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R53 is C1-C6-alkyl, C1-C6-alkoxy, —OR51, halogen, or —CF3.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R50A is —C(O)OCH3, —C(O)OCH2CH3—COOH, —CH2C(O)OCH3, —CH2C(O)OCH2CH3, —CH2CH2C(O)OCH3, —CH2CH2C(O)OCH2CH3, —CH2COOH, methyl, or ethyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R50B is —C(O)OCH3, —C(O)OCH2CH3—COOH, —CH2C(O)OCH3, —CH2C(O)OCH2CH3, —CH2CH2C(O)OCH3, —CH2CH2C(O)OCH2CH3, —CH2COOH, methyl, or ethyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein CGr is
wherein V is C1-C6-alkyl, aryl, heteroaryl, aryl-C1-6-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
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein V is aryl, heteroaryl, or aryl-C1-6-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein V is aryl, Het1, or aryl-C1-6-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein V is aryl, Het2, or aryl-C1-6-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein V is aryl, Het3, or aryl-C1-6-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein V is aryl optionally substituted with one or more substituents independently selected from R55.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein V is ArG1 optionally substituted with one or more substituents independently selected from R55.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein V is phenyl, naphthyl or anthranyl optionally substituted with one or more substituents independently selected from R55.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein V is phenyl optionally substituted with one or more substituents independently selected from R55.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R55 is independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R55 is independently selected from
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R55 is independently selected from halogen, —OR56, —NR56R57, —C(O)OR56, —OC1-C8-alkyl-C(O)OR56, —NR56C(O)R57 or C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R55 is independently selected from halogen, —OR56, —NR56R57, —C(O)OR56, —OC1-C8-alkyl-C(O)OR56, —NR56C(O)R57, methyl or ethyl.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R56 and R57 are independently selected from hydrogen, CF3, C1-C12-alkyl, or —C(═O)—C1-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R56 and R57 are independently selected from hydrogen or C1-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein CGr is
wherein AA is C1-C6-alkyl, aryl, heteroaryl, aryl-C1-6-alkyl- or 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
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein AA is aryl, heteroaryl or aryl-C1-6-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein AA is aryl or heteroaryl optionally substituted with one or more substituents independently selected from R64.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein AA is ArG1 or Het1 optionally substituted with one or more substituents independently selected from R64.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein AA is ArG1 or Het2 optionally substituted with one or more substituents independently selected from R64.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein AA is ArG1 or Het3 optionally substituted with one or more substituents independently selected from R64.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein AA is phenyl, naphtyl, anthryl, carbazolyl, thienyl, pyridyl, or benzodioxoyl optionally substituted with one or more substituents independently selected from R64.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein AA is phenyl or naphtyl optionally substituted with one or more substituents independently selected from R64.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R64 is independently selected from hydrogen, halogen, —CF3, —OCF3, —OR65, —NR65R66, C1-C6-alkyl, —OC(O)R65, —OC1-C6-alkyl-C(O)OR65, aryl-C2-C6-alkenyl, aryloxy or aryl, wherein C1-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.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R65 and R66 are independently selected from hydrogen, CF3, C1-C12-alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R71.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R65 and R66 are independently hydrogen, C1-C12-alkyl, aryl, or heteroaryl optionally substituted with one or more substituents independently selected from R71.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. 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.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R65 and R66 are independently hydrogen, methyl, ethyl, propyl, butyl, 2,2-dimethyl-propyl, phenyl, naphtyl, thiadiazolyl optionally substituted with one or more R71 independently; or isoxazolyl optionally substituted with one or more substituents independently selected from R71.
A pharmaceutical composition according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein R71 is halogen or C1-C6-alkyl.
A pharmaceutical composition according to embodiment Error! Reference source not found. wherein R71 is halogen or methyl.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein Frg1 consists of 0 to 5 neutral amino acids independently selected from the group consisting of Gly, Ala, Thr, and Ser.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg1 consists of 0 to 5 Gly.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg1 consists of 0 Gly.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg1 consists of 1 Gly.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg1 consists of 2 Gly.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg1 consists of 3 Gly.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg1 consists of 4 Gly.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg1 consists of 5 Gly.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein GB is of the formula B1—B2—C(O)—, B1—B2—SO2— or B1—B2—CH2—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein GB is of the formula B1—B2—C(O)—, B1—B2—SO2— or B1—B2—NH—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein GB is of the formula B1—B2—C(O)—, B1—B2—CH2— or B1—B2—NH—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein GB is of the formula B1—B2—CH2—, B1—B2—SO2— or B1—B2—NH—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—C(O)— or B1—B2—SO2—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—C(O)— or B1—B2—CH2—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—C(O)— or B1—B2—NH—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—CH2— or B1—B2—SO2—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—NH— or B1—B2—SO2—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—CH2— or B1—B2—NH—, wherein B1 and B2 are as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—C(O)—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—CH2—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—SO2—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. Error! Reference source not found. or Error! Reference source not found. wherein GB is of the formula B1—B2—NH—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein B1 is a valence bond, —O—, or —S—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein B1 is a valence bond, —O—, or —N(R6B)—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein B1 is a valence bond, —S—, or —N(R6B)—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein B1 is —O—, —S— or —N(R6B)—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein B1 is a valence bond or —O—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein B1 is a valence bond or —S—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein B1 is a valence bond or —N(R6B)—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein B1 is —O— or —S—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein B1 is —O— or —N(R6B)—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. or Error! Reference source not found. wherein B1 is —S— or —N(R6B)—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. Error! Reference source not found. or Error! Reference source not found. wherein B1 is a valence bond.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. Error! Reference source not found. or Error! Reference source not found. wherein B1 is —O—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. Error! Reference source not found. or Error! Reference source not found. wherein B1 is —S—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. Error! Reference source not found. or Error! Reference source not found. wherein B1 is —N(R6B)—.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-O—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-S—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-NR6—C1-C18-alkyl-C(═O)—; and the alkylene and arylene moieties are optionally substituted as defined in embodiment 1.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-O—C1-C18-alkyl-C(═O)—, and the alkylene and arylene moieties are optionally substituted as defined in embodiment 1.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, and the alkylene and arylene moieties are optionally substituted as defined in embodiment 1.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein B2 is a valence bond, C1-C18-alkylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, and the alkylene and arylene moieties are optionally substituted as defined in embodiment 1.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein B2 is a valence bond, C1-C18-alkylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, and the alkylene and arylene moieties are optionally substituted as defined in embodiment 1.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein B2 is a valence bond, C1-C18-alkylene, arylene, —C1-C18-alkyl-aryl- and the alkylene and arylene moieties are optionally substituted as defined in embodiment 1.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein B2 is a valence bond or —C1-C18-alkylene, and the alkylene moieties are optionally substituted as defined in embodiment 1.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein Frg2 comprises 1 to 16 positively charged groups in a branched orientation.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg2 comprises 1 to 12 positively charged groups in a branched orientation.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg2 comprises 1 to 10 positively charged groups in a branched orientation.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein Frg2 comprises 10 to 20 positively charged groups in a branched orientation.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg2 comprises 12 to 20 positively charged groups in a branched orientation.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein Frg2 comprises 16 to 20 positively charged groups in a branched orientation.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. wherein the positively charged groups of Frg2 are basic amino acids independently selected from the group consisting of Lys and Arg and D-isomers of these.
A pharmaceutical preparation according to embodiment Error! Reference source not found. wherein the basic amino acids are Lys or Arg, except for the branching point which comprises Lys, Glu or Asp.
A pharmaceutical preparation according to embodiment 257 wherein the basic amino acids are all Lys, except for the branching point which comprises Lys, Glu or Asp.
A pharmaceutical preparation according to embodiment 257 wherein the basic amino acids are all Arg, except for the branching point which comprises Lys, Glu or Asp.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to 259, wherein Frg2 comprises one or more neutral amino acids independently selected from the group consisting of Gly, Ala, Thr, and Ser.
A pharmaceutical preparation according to embodiment 260, wherein Frg2 comprises one or more Gly.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to 261 wherein X is —OH or —NH2.
A pharmaceutical preparation according to embodiment 262 wherein X is —NH2.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to 263 which further comprises at least 3 phenolic molecules per putative insulin hexamer.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to 264 wherein the insulin is selected from the group consisting of human insulin, an analogue thereof, a derivative thereof, and combinations of any of these.
A pharmaceutical preparation according to embodiment 265 wherein the insulin is human insulin.
A pharmaceutical preparation according to embodiment 265 wherein the insulin is an analogue of human insulin wherein position B28 is Asp, Glu, Lys, Leu, Val or Ala.
A pharmaceutical preparation according to embodiment 267 wherein position B28 is Asp, Glu or Lys.
A pharmaceutical preparation according to embodiment 268 wherein position B28 is Asp or Glu.
A pharmaceutical preparation according to embodiment 269 wherein position B28 is Asp.
A pharmaceutical preparation according to embodiment 269 wherein position B28 is Glu.
A pharmaceutical preparation according to any one of the embodiments 265 to 271 wherein the insulin is an analogue of human insulin wherein position B29 is Pro, Asp or Glu.
A pharmaceutical preparation according to embodiment 272 wherein position B29 is Pro or Glu.
A pharmaceutical preparation according to embodiment 273 wherein position B29 is Pro.
A pharmaceutical preparation according to embodiment 273 wherein position B29 is Glu.
A pharmaceutical preparation according to any one of the embodiments 265 to 275 wherein the insulin is an analogue of human insulin wherein position B9 is Asp or Glu.
A pharmaceutical preparation according to any one of the embodiments 265 to 276 wherein the insulin is an analogue of human insulin wherein position B10 is Asp or Glu.
A pharmaceutical preparation according to embodiment 277 wherein position B10 is Glu.
A pharmaceutical preparation according to any one of the embodiments 265 to 278 wherein the insulin is an analogue of human insulin wherein position B1 is Gly.
A pharmaceutical preparation according to any one of the embodiments 265 to 279 wherein the insulin is an analogue of human insulin wherein position B3 is Lys, Thr, Ser, Ala or Gln.
A pharmaceutical preparation according to embodiment 280 wherein position B3 is Lys, Thr, Ser or Ala.
A pharmaceutical preparation according to embodiment 281 wherein position B3 is Lys or Ala.
A pharmaceutical preparation according to embodiment 282 wherein position B3 is Lys.
A pharmaceutical preparation according to any one of the embodiments 265 to 283 wherein the insulin is an analogue of human insulin wherein position B25 is deleted.
A pharmaceutical preparation according to any one of the embodiments 265 to 284 wherein the insulin is an analogue of human insulin wherein position B27 is deleted.
A pharmaceutical preparation according to any one of the embodiments 265 to 285 wherein the insulin is an analogue of human insulin wherein position B30 is deleted.
A pharmaceutical preparation according to any one of the embodiments 265 to 286 wherein the insulin is an analogue of human insulin wherein position A18 is Gln.
A pharmaceutical preparation according to any one of the embodiments 265 to 287 wherein insulin is an analogue of human insulin wherein position A21 is Ala, Arg, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Thr, Trp, Tyr, Val or hSer.
A pharmaceutical preparation according to embodiment 288 wherein position A21 is Ala, Arg, Gly, Ile, Leu, Phe, Ser, Thr, Val or hSer.
A pharmaceutical preparation according to embodiment 289 wherein position A21 is Ala or Gly.
A pharmaceutical preparation according to embodiment 290 wherein position A21 is Gly.
A pharmaceutical preparation according to any one of the embodiments 265 to 291 wherein the insulin is a derivative of human insulin or an analogue thereof having one or more lipophilic substituents.
A pharmaceutical preparation according to embodiment 292 wherein the Nε-amino group in position B29Lys is modified by covalent acylation with a hydrophobic moiety such as an fatty acid derivative or an litocholic acid derivative.
A pharmaceutical preparation according to embodiment 292 or 293 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 LysB28 ProB29 human insulin, B28-Nε-palmitoyl LysB28 ProB29 human insulin, B30-Nε-myristoyl-Thr B29LysB3 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(B30) human insulin, B29-Nε-(ω-carboxyheptadecanoyl)-des(B30) human insulin and B29-Nε-(ω-carboxyheptadecanoyl) human insulin.
A pharmaceutical preparation according to any one of the embodiments 265 to 294 wherein the insulin contain any combination of additional stabilizing substitutions.
A pharmaceutical preparation according to embodiment 295 wherein the insulin contain any combination of the additional stabilizing substitutions in positions B1, B3, A18 and A21.
A pharmaceutical preparation according to embodiment 265 wherein the insulin is an analogue of human insulin selected from the group:
desB27
desB25
A pharmaceutical preparation according to embodiment 265 wherein the insulin is an analogue of human insulin selected from the group:
A21G, desB27
A21G, desB25
A21G, desB30
A21G, B28K, B29P, desB30
A21G, B28D, desB30
A21G, B28E, desB30
A21G, B3K, B29E, desB30
A21G, desB27, desB30
A21G, B9E, desB30
A21G, B9D, desB30
A21G, B10E, desB30
A21G, desB25, desB30.
A pharmaceutical preparation according to embodiment 265 wherein the insulin is an analogue of human insulin selected from the group:
B1G, A21G, desB27
B1G, A21G, desB25
B1G, A21G, desB30
B1G, A21G, B28K, B29P, desB30
B1G, A21G, B28D, desB30
B1G, A21G, B28E, desB30
B1G, A21G, B3K, B29E, desB30
B1G, A21G, desB27, desB30
B1G, A21G, B9E, desB30
B1G, A21G, B9D, desB30
B1G, A21G, B10E, desB30
B1G, A21G, desB25, desB30.
A pharmaceutical preparation according to any one of the embodiments 297 to 299 wherein the insulin is an analogue of human insulin further modified in positions
B3 and A18 as follows:
A pharmaceutical preparation according to any one of the embodiments 297 to 299 wherein the insulin is an analogue of human insulin further modified as follows:
B3T, desB27.
A pharmaceutical preparation according to any one of the embodiments 297 to 301 wherein the insulin is an analogue of human insulin further modified by deletion of B30.
A pharmaceutical preparation according to embodiments Error! Reference source not found. to 302 wherein the ratio of the branched ligand of general formula (I) to zinc ion is 1:20 to 20:1.
A pharmaceutical preparation according to embodiment 303 wherein the ratio of the branched ligand of general formula (I) to zinc ion is 1:6 to 10:1.
A pharmaceutical preparation according to embodiments Error! Reference source not found. to 304 wherein the amount of zinc ions is 2-6 moles per mole of putative insulin hexamer.
A pharmaceutical preparation according to embodiment 305 wherein the amount of zinc ions is 2.0-3.5 moles per putative insulin hexamer.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to 306 wherein zinc ions are present in an amount corresponding to 10 to 40 μg Zn/100 U insulin.
A pharmaceutical preparation according to embodiment 307 wherein zinc ions are present in an amount corresponding to 10 to 26 μg Zn/100 U insulin.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to 308 wherein the ratio between insulin and the branched ligand according to any one of the embodiments Error! Reference source not found. to 249 is in the range from 99:1 to 1:99.
A pharmaceutical preparation according to embodiment 309 wherein the ratio between insulin and the branched ligand according to any one of the embodiments Error! Reference source not found. to 249 is in the range from 95:5 to 5:95.
A pharmaceutical preparation according to embodiment 310 wherein the ratio between insulin and the branched ligand according to any one of the embodiments Error! Reference source not found. to 249 is in the range from 80:20 to 20:80.
A pharmaceutical preparation according to embodiment 311 wherein the ratio between insulin and the branched ligand according to any one of the embodiments Error! Reference source not found. to 249 is in the range from 70:30 to 30:70.
A pharmaceutical preparation according to any one of the embodiments Error! Reference source not found. to 312 wherein the concentration of insulin is 60 to 3000 nmol/ml.
A pharmaceutical preparation according to embodiment 313 wherein the concentration of insulin is 240 to 1200 nmol/ml.
A pharmaceutical preparation according to embodiment 314 wherein the concentration of insulin is about 600 nmol/ml.
A method of preparing a branched ligand according to embodiment Error! Reference source not found. comprising the steps of
Method of prolonging the action of an insulin preparation which comprises adding a branched ligand according to any one of the embodiments Error! Reference source not found. to Error! Reference source not found. to the insulin preparation.
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 Error! Reference source not found. to 315.
Use of a preparation according to any one of the embodiments Error! Reference source not found. to 315 for the preparation of a medicament for treatment of type 1 or type 2 diabetes.
The present invention also relates to a pharmaceutical preparation for the treatment of diabetes in a patient in need of such a treatment comprising an R-state hexamer of insulin according to the invention together with a pharmaceutically acceptable carrier.
In one embodiment of the invention the insulin preparation comprises 60 to 3000 nmol/ml of insulin.
In another embodiment of the invention the insulin preparation comprises 300-2400 nmol/ml of insulin.
In another embodiment of the invention the insulin preparation comprises 240 to 1200 nmol/ml of insulin.
In another embodiment of the invention the insulin preparation comprises about 600 nmol/ml of insulin.
Zinc ions may be present in an amount corresponding to 10 to 40 μg Zn/100 U insulin, more preferably 10 to 26 μg Zn/100 U insulin.
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, 7-hydroxyindole or any mixture thereof.
In one embodiment the invention provides a pharmaceutical preparation further comprising at least 3 molecules of a phenolic compound per insulin hexamer.
In another embodiment of the invention 0.5 to 4.0 mg/ml of phenolic compound may be employed.
In another embodiment of the invention 0.6 to 4.0 mg/ml of m-cresol may be employed.
In another embodiment of the invention 0.5 to 4.0 mg/ml of phenol may be employed.
In another embodiment of the invention 1.4 to 4.0 mg/ml of phenol may be employed.
In another embodiment of the invention 0.5 to 4.0 mg/ml of a mixture of m-cresol or phenol may be employed.
In another embodiment of the invention 1.4 to 4.0 mg/ml of a mixture of m-cresol or phenol may be employed.
In another embodiment the invention provides a pharmaceutical preparation which may optionally contain a preservative such as e.g. phenol, m-cresol or mixtures thereof.
In another embodiment the invention provides a pharmaceutical preparation which may optionally contain an isotonicity agent such as e.g. 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.
In another embodiment the invention provides a pharmaceutical preparation which may optionally contain a buffer substance, such as a TRIS, phosphate, glycine or glycylglycine (or another zwitterionic substance) buffer
In another embodiment the invention provides a pharmaceutical preparation which optionally comprises between 0.001% by weight and 1% by weight of a non-ionic surfactant, for example tween 20 or Polox 188. A nonionic detergent can be added to stabilise insulin against fibrillation during storage and handling.
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 one embodiment the pharmaceutical preparation of the present invention may have a pH value in the range of 2.5 to 5.5, e.g. pH 2.5 to 4.5, pH 3 to 5.5, pH 3 to 4.
In another embodiment insulin preparation of the present invention may have a pH value in the range of 3.5 to 8.5, e.g. pH 5.0 to 8.5, pH 5.5 to 8.5, pH 7.4 to 7.9.
For pharmaceutical preparations of the present invention intended for formulation in the pH-range about 5.0-8.5, stabilizing mutations may include B1Gly, des(B1), B3 may be Thr, Ser, or Gln, and A18 may be Gln.
For pharmaceutical preparations of the present invention intended for formulation in the pH-range 3.0-5.0 these substitutions may be combined with the A21Gly stabilizing substitution.
In one embodiment the preparations of the invention are used in connection with insulin pumps. The insulin pumps may be prefilled and disposable, or the insulin preparations may be supplied from a reservoir which is removable. Insulin pumps may be skin-mounted or carried, and the path of the insulin preparation from the storage compartment of the pump to the patient may be more or less tortuous. Non-limiting examples of insulin pumps are disclosed in U.S. Pat. No. 5,957,895, U.S. Pat. No. 5,858,001, U.S. Pat. No. 4,468,221, U.S. Pat. No. 4,468,221, U.S. Pat. No. 5,957,895, U.S. Pat. No. 5,858,001, U.S. Pat. No. 6,074,369, U.S. Pat. No. 5,858,001, U.S. Pat. No. 5,527,288, and U.S. Pat. No. 6,074,369.
In another embodiment the preparations of the 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 preparations of the invention are used in connection with devices for pulmonary administration of aqueous insulin preparations, a non-limiting example of which is the AerX® device.
The invention furthermore relates to treatment of a patient in which the pharmaceutical preparation of the invention, i.e. comprising zinc ions, insulin, eg human insulin, an analogue thereof, a derivative thereof or combinations of any of these analogue, acid-stabilised insulin, fast/rapid acting insulin and long/slow/basal acting insulin, and a ligand for the R-state HisB10Zn2+ 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, MC4 (melanocortin 4) agonists, MC3 (melanocortin 3) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin agonists, β3 adrenergic agonists such as CL-316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140, MSH (melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists, serotonin re-uptake inhibitors such as fluoxetine, seroxat or citalopram, 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 neurotrophic 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 peptidase-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, chlorpropamide, 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. troglitazone, 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-102, 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, emiglitate, 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 repaglinide.
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, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
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.
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.
The following instrumentation was used:
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.
The following instrumentation was used:
Sciex API 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:
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. 75μ fused silica capillary to the API interface of API 1100 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.
HPLC-MS (Method C) The following instrumentation is used:
The instrument is controlled by HP Chemstation software.
The HPLC pump is connected to two eluent reservoirs containing:
The analysis is performed at 40° C. by injecting an appropriate volume of the sample (preferably 1 μl) 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.
After the DAD the flow is divided yielding approximately 1 ml/min to the ELS and 0.5 ml/min to the MS.
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 75μ fused silica capillary to the API interface of API 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 LC conditions, detector settings and mass spectrometer settings used for the different methods are given in the following table.
4-[(1H-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 1N hydrochloric acid (45 mL) was added. The mixture was added water (200 mL) and extracted with ethyl acetate (2×500 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-[(1H-benzotriazole-5-carbonyl)amino]benzoic acid was isolated (0.52 g).
1H-NMR (DMSO-d6): 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.
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 1H-benzotriazole-5-carboxylic acid is activated, ie the OH functionality is converted into a leaving group L (selected from eg fluorine, chlorine, bromine, iodine, 1-imidazolyl, 1,2,4-triazolyl, 1-benzotriazolyloxy, 1-(4-aza benzotriazolyl)oxy, pentafluorophenoxy, 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 such as THF, dioxane, 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:
Benzotriazole-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 aliquot of this mixture was added to aniline (13.7 μL, 0.15 mmol) and the resulting mixture was vigorously shaken at room temperature for 16 hours. 1N hydrochloric acid (2 mL) and ethyl acetate (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.
HPLC-MS (Method B): m/z: 239 (M+1); Rt=3.93 min.
The compounds in the following examples were similarly made. Optionally, the compounds may be isolated by filtration or by chromatography.
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.
HPLC-MS (Method B): m/z: 354 (M+1); Rt=4.58 min.
HPLC-MS (Method B): m/z: 296 (M+1); Rt=3.32 min.
HPLC-MS (Method B): m/z: 257 (M+1); Rt=4.33 min.
HPLC-MS (Method B): m/z: 273 (M+1); Rt=4.18 min.
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.
HPLC-MS (Method B): m/z: 295 (M+1); Rt=5.80 min.
HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.08 min.
HPLC-MS (Method B): m/z: 253 (M+1); Rt=3.88 min.
HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.40 min.
HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.25 min.
HPLC-MS (Method B): m/z: 283 (M+1); Rt=3.93 min.
HPLC-MS (Method B): m/z: 283 (M+1); Rt=3.97 min.
HPLC-MS (Method B): m/z: 343 (M+1); Rt=5.05 min.
HPLC-MS (Method B): m/z: 331 (M+1); Rt=4.45 min.
HPLC-MS (Method B): m/z: 297 (M+1); Rt=3.35 min.
HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.08 min.
HPLC-MS (Method B): m/z: 301 (M+1); Rt=4.50 min.
HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.15 min.
HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.13 min.
HPLC-MS (Method B): m/z: 301 (M+1); Rt=4.55 min.
HPLC-MS (Method B): m/z: 343 (M+1); Rt=5.00 min.
HPLC-MS (Method B): m/z: 321 (M+1); Rt=4.67 min.
HPLC-MS (Method B): m/z: 253 (M+1); Rt=3.82 min.
HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.05 min.
HPLC-MS (Method B): m/z: 345 (M+1); Rt=4.37 min.
HPLC-MS (Method B): m/z: 281 (M+1); Rt=4.15 min.
HPLC-MS (Method B): m/z: 341 (M+1); Rt=3.78 min;
HPLC-MS (Method B): m/z: 297 (M+1); Rt=3.48 min.
HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.19 min.
HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.18 min.
HPLC-MS (Method A): m/z: 340 (M+1); Rt=1.71 min.
HPLC-MS (Method A): m/z: 297 (M+1); Rt=2.02 min.
HPLC-MS (Method A): m/z: 309 (M+1); Rt=3.19 min.
HPLC-MS (Method A): m/z: 297 (M+1); Rt=2.10 min.
HPLC-MS (Method A): m/z: 341 (M+1); Rt=2.42 min.
HPLC-MS (Method A): m/z: 354 (M+1); Rt=1.78 min.
HPLC-MS (Method A): m/z: 311 (M+1); Rt=2.20 min.
HPLC-MS (Method A): m/z: 345 (M+1); Rt=3.60 min.
HPLC-MS (Method A): m/z: 303 (M+1); Rt=2.88 min.
HPLC-MS (Method A): m/z: 331 (M+1); Rt=3.62 min.
HPLC-MS (Method A): m/z: 311 (M+1); Rt=3.59 min.
HPLC-MS (Method A): m/z: 402 (M+1); Rt=3.93 min.
HPLC-MS (Method A): m/z: 323 (M+1); Rt=2.57 min.
HPLC-MS (Method A): m/z: 297 (M+1); Rt=1.86 min.
HPLC-MS (Method A): m/z: 329 (M+1); Rt=2.34 min.
HPLC-MS (Method A): m/z: 177 (M+1); Rt=0.84 min.
The following compound is prepared according to general procedure (N) as described below:
HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.40 min.
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., 1999, 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), rhodamine (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:
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.
HPLC-MS (Method C): m/z: 249 (M+1); Rt=4.90 min
HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.16 min.
HPLC-MS (Method A): m/z: 206 (M+1); Rt=4.87 min.
HPLC-MS (Method A): m/z: 277 (M+1); Rt=4.73 min.
HPLC-MS (Method A): m/z: 263 (M+1); Rt=4.90 min.
HPLC-MS (Method A): m/z: 240 (M+1); Rt=5.53 min.
HPLC-MS (Method A): m/z: 251 (M+1); Rt=4.87 min.
HPLC-MS (Method A): m/z: 252 (M+1); Rt=4.07 min.
HPLC-MS (Method A): m/z: 252 (M+1); Rt=5.43 min.
HPLC-MS (Method C): m/z: 292 (M+1); Rt=4.75 min.
1H NMR (DMSO-d6): δ=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).
HPLC-MS (Method A): m/z: 354 (M+1); Rt=4.97 min.
HPLC-MS (Method A): m/z: 262 (M+1); Rt=6.70 min.
HPLC-MS (Method A): m/z: 263 (M+1); Rt=3.90 min.
HPLC-MS (Method A): m/z: 282 (M+1); Rt=4.52 min.
HPLC-MS (Method A): m/z: 298 (M+1); Rt=6.50 min.
HPLC-MS (Method A): m/z: 312 (M+1); Rt=6.37 min.
HPLC-MS (Method A): m/z: 312 (M+1); Rt=6.87 min.
HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.15 min.
HPLC-MS (Method A): m/z: 250 (M+1), Rt=3.18 min.
HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.51 min.
HPLC-MS (Method A): m/z: 265 (M+1); Rt=5.66 min.
HPLC-MS (Method A): m/z: 267 (M+1); Rt=3.94 min.
HPLC-MS (Method A): m/z: 268 (M+1); Rt=6.39 min.
HPLC-MS (Method A): m/z: 270 (M+1); Rt=5.52 min.
HPLC-MS (Method A): m/z: 272 (M+1); Rt=6.75 min.
HPLC-MS (Method A): m/z: 293 (M+1); Rt=5.99 min.
HPLC-MS (Method A): m/z: 298 (M+1); Rt=7.03 min.
HPLC-MS (Method A): m/z: 314 (M+1); Rt=6.89 min.
HPLC-MS (Method A): m/z: 328 (M+1); Rt=6.95 min.
HPLC-MS (Method A): m/z: 328 (M+1); RT=6.89 min.
HPLC-MS (Method A): m/z: 272 (M+1); Rt=6.43 min.
HPLC-MS (Method A): m/z: 236 (M+1); Rt=3.05 min.
HPLC-MS (Method A): m/z: 392 (M+23), Rt=4.32 min.
HPLC-MS (Method A): m/z: 410 (M+23); Rt=3.35 min.
HPLC-MS (Method A): m/z: 285 (M+1); Rt=4.01 min.
HPLC-MS (Method A): m/z: 285 (M+1); Rt=4.05 min.
HPLC-MS (Method A): m/z: 240 (M+1); Rt=3.91 min.
HPLC-MS (Method A): m/z: 212 (M+1); Rt=3.09 min.
HPLC-MS (Method A): m/z: 291 (M+1); Rt=3.85 min.
HPLC-MS (Method A): m/z: 274 (M+1); Rt=4.52 min.
HPLC-MS (Method A): m/z: 259 (M+1); Rt=3.55 min.
HPLC-MS (Method A): m/z: 245 (M+1); Rt=2.73 min.
HPLC-MS (Method A): m/z: 280 (M+1); Rt=4.34 min.
HPLC-MS (Method A): m/z: 220 (M+1); Rt=3.38 min.
HPLC-MS (Method A): m/z: 250 (M+1); Rt=3.55 min.
HPLC-MS (Method A): m/z: 270 (M+1); Rt=4.30 min.
HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.18 min.
HPLC-MS (Method A): m/z: 296 (M+1); Rt=4.49 min.
HPLC-MS (Method A): m/z: 250 (M+1); Rt=3.60 min.
HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.26 min.
HPLC-MS (Method A): m/z: 312 (M+1); Rt=4.68 min.
HPLC-MS (Method A): m/z: 268 (M+1); Rt=3.58 min.
HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.13 min.
HPLC-MS (Method A): m/z: 306 (M+1); Rt=4.64 min.
HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.02 min.
HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.31 min.
HPLC-MS (Method A): m/z: 299 (M+1); Rt=4.22 min.
HPLC-MS (Method A): m/z: 270 (M+1); Rt=4.47 min.
5-Pyridin-2-ylmethylene-thiazolidine-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 16 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)
HPLC-MS (Method A): m/z: 6.43 min; 99% (2A)
HPLC-MS (Method A): m/z: 236 (M+1); Rt=4.97 min
HPLC-MS (Method A): m/z: 219 (M+1); Rt=2.43 min.
HPLC-MS (Method A): m/z: 219 (M+1); Rt=2.38 min.
HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.57 min.
HPLC-MS (Method C): m/z: 250 (M+1); Rt=4.00 min.
HPLC-MS (Method C): m/z: 264 (M+1); Rt=5.05 min.
HPLC-MS (Method C): m/z: 342 (M+1); Rt=5.14 min.
HPLC-MS (Method C): m/z: 222 (M+1); Rt=3.67 min.
1H-NMR (DMSO-d6): 7.60 (2H, “s”), 7.78 (1H, s), 7.82 (1H, s).
1H-NMR (DMSO-d6): 7.40 (1H, t), 7.46 (1H, t), 7.57 (1H, d), 7.62 (1H, d), 7.74 (1H, s).
1H-NMR (DMSO-d6): 7.33 (1H, t), 7.52 (1H, t), 7.60 (1H, d), 7.71 (1H, s), 7.77 (1H, d).
HPLC-MS (Method C): m/z: 266 (M+1) Rt=4.40 min.
HPLC-MS (Method C): m/z: 236 (M+1); Rt=4.17 min.
HPLC-MS (Method C): m/z: 242 (M+1); Rt=4.30 min.
HPLC-MS (Method C): m/z: 234 (M+1); Rt=5.00 min.
HPLC-MS (Method C): m/z: 296 (M+1); Rt=4.27 min.
HPLC-MS (Method C): m/z: 252 (M+1); Rt=3.64 min.
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.
HPLC-MS (Method C): m/z: 290 (M+1); Rt=4.94 min.
HPLC-MS (Method C): m/z: 282 (M+1); Rt=5.17 min.
HPLC-MS (Method C): m/z: 312 (M+1); Rt=5.40 min.
HPLC-MS (Method A): m/z: 250 (M+1); Rt=4.30 min.
HPLC-MS (Method C): m/z: 277 (M+1); Rt=3.63 min.
HPLC-MS (Method C): m/z: 262 (M+1); Rt=3.81 min.
HPLC-MS (Method C): m/z: 262 (M+1); Rt=3.67 min.
HPLC-MS (Method C): m/z=260 (M+1) Rt=2.16 min.
HPLC-MS (Method C): m/z=334 (M+1); Rt=3.55 min.
HPLC-MS (Method C): m/z=356 (M+1); Rt=5.75 min.
HPLC-MS (Method C): m/z=412 (M+1); Rt=6.44 min.
HPLC-MS (Method C): m/z=315 (M+1); Rt=3.24 min.
HPLC-MS (Method C): m/z=334 (M+1); Rt=3.14 min.
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:
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-d6): 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.
Mp=256° C.; 1H NMR (DMSO-d6) 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-d6) 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.
HPLC-MS (Method C): m/z: 250 (M+1); Rt.=2.45 min.
HPLC-MS (Method C): m/z: 506 (M+23); Rt.=4.27 min.
HPLC-MS (Method C): m/z: 354 (M+1); Rt.=4.36 min.
Mp 310-314° C., 1H NMR (DMSO-d6): 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-d6): δC=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); Rt=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 H2SO4 (10% solution, 15 mL) was added followed by water until all salt was dissolved. The resulting solution was extracted with ethyl acetate (3×), the combined organic phases were dried with MgSO4, evaporated to dryness to afford 0.89 g of 6-hydroxy-2-naphtalenecarbaldehyde.
Mp.: 153.5-156.5° C.; HPLC-MS (Method A): m/z: 173 (M+1); Rt=2.67 min; 1H NMR (DMSO-d6): 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-BuLi (2.5 M, 100 mL, 0.250 mol) dropwise. The mixture turned yellow and the temperature rose to −64° C. 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 Na2SO4 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.
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); 13C NMR (DMSO-d6): 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 aqueous sodium thiosulfate solution (1 M) until the colour disappeared. Subsequent extraction with dichloromethane (3×20 mL) followed by drying with MgSO4 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., H NMR (CDCl3): δH=8.80 (s, 1H), 8.31 (d, 1H), 7.85 (dd, 1H) 6.92 (d, 1H), 3.99 (s, 3H).
HPLC-MS (Method A): m/z:=336 (M+1); Rt=4.46 min.
1H NMR (DMSO-d6): δH=7.88 (s, 1H), 7.78 (s, 1H), 4.10 (q, 2H), 4.0-3.8 (m, 2H), 3.40-3.18 (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.
1H NMR (DMSO-d6): δH=12.6 (s, broad, 1H), 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.
HPLC-MS (Method A): m/z: 257 (M+1); Rt=2.40 min.
1H NMR (DMSO-d6): δH=12.35 (s, broad, 1H), 7.82 (t, 1H), 7.78 (s, 1H), 7.65 (d, 1H), 7.18 (d, 1H), 2.52 (s, 3H); HPLC-MS (Method A): m/z: 221 (M+1); Rt=3.03 min.
1H NMR (DMSO-d6): 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.
HPLC-MS (Method A): m/z: 276 (M+1); Rt=0.98 min.
HPLC-MS (Method A): m/z: 352 (M+1); Rt=3.01 min.
HPLC-MS (Method A): m/z: 257 (M+1); Rt=3.40 min.
HPLC-MS (Method A): m/z: 256 (M+1); Rt=1.96 min.
HPLC-MS (Method A): m/z: 272 (M+1); Rt=2.89 min.
HPLC-MS (Method A): m/z: 272 (M+1); Rt=1.38 min.
HPLC-MS (Method A): m/z: 323 (M+1); Rt=4.52 min.
HPLC-MS (Method A): m/z: 323 (M+1); Rt=4.35 min.
HPLC-MS (Method A): m/z: 259 (M+1); Rt=3.24 min.
2-Methylindole (1.0 g, 7.6 mmol) dissolved in diethyl ether (100 mL) under nitrogen was treated with n-Butyl lithium (2 M in pentane, 22.8 mmol) and potassium tert-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 1N hydrochloric acid and the mixture was extracted with diethyl ether. The organic phase was dried with Na2SO4 and evaporated to dryness. The residue was purified by column chromatography on silica gel using heptane/ether (4/1) as eluent. This afforded 720 mg (69%) of 2-ethylindole.
1H NMR (DMSO-d6): δ=10.85 (1H, s); 7.39 (1H, d); 7.25 (1H, d); 6.98 (1H, t); 6.90 (1H, t); 6.10 (1H, s); 2.71 (2H, q); 1.28 (3H, t).
2-Ethylindole (0.5 g, 3.4 mmol) 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 MgSO4 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.
HPLC-MS (Method A): m/z: 447 (M+1); Rt=5.25 min.
HPLC-MS (Method A): (anyone 1) m/z: 430 (M+1); Rt=5.47 min.
HPLC-MS (Method A): m/z: 416 (M+1); Rt=5.02 min.
HPLC-MS (Method A): m/z: 283 (M+1), Rt=2.97 min.
HPLC-MS (Method A): m/z: 418 (M+1); Rt=5.13 min.
HPLC-MS (Method A): m/z: 357 (M+1); Rt=4.45 min.
HPLC-MS (Method A): m/z: 321 (M+1); Rt=3.93 min.
HPLC-MS (Method A): m/z: 351 (M+1); Rt=4.18 min.
HPLC-MS (Method A): m/z: 222 (M+1); Rt=2.42 min.
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); HPLC-MS (Method A): m/z: 259 (M+1); Rt=4.00 min.
Mp 330-333° C., 1H NMR (DMSO-d6): 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.
HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.27 min.
HPLC-MS (Method A): m/z: 314 (M+1), Rt=3.96 min.
HPLC-MS (Method A): m/z: 327 (M+1); Rt=2.90 min.
HPLC-MS (Method A): m/z: 303 (M+1); Rt=3.22-3-90 min.
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methyl ester (example 203, 59 mg; 0.195 mmol) 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.
HPLC-MS (Method A): m/z: 289 (M+1); Rt=2.67 min.
HPLC-MS (Method A): m/z: 335 (M+1); Rt=4.55 min.
HPLC-MS (Method A): m/z:=385 (M+1); Rt=4.59 min.
HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.45 min.
HPLC-MS (Method A): m/z: 357 (M+1); Rt=4.42 min.
HPLC-MS (Method A): m/z: 317 (M+1); Rt=4.35 min.
HPLC-MS (Method A): m/z: 420 (M+1); Rt=5.92 min.
HPLC-MS (Method A): (Anyone 1) m/z: 398 (M+1); Rt=4.42 min.
HPLC-MS (Method A): m/z: 351 (M+1); Rt=3.95 min.
HPLC-MS (Method A): m/z: 262 (M+1); Rt=4.97 min.
HPLC-MS (Method A): m/z: 404 (M+1); Rt=4.96 min.
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 (2×250 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.
HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.14 min.
1H NMR (DMSO-d6): δH=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.
HPLC-MS (Method A): m/z: 385 (M+1); Rt=5.08 min.
Indole-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.
HPLC-MS (Method A): m/z: 393 (M+1); Rt=4.60 min.
HPLC-MS (Method A): m/z: 465 (M+1); Rt=5.02 min.
HPLC-MS (Method A): m/z: 458 (M+23); Rt=4.81 min.
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.
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.
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.
HPLC-MS (Method A): m/z: 293 (M+1); Rt=4.10 min.
HPLC-MS (Method A): m/z: 474 (M+1); Rt=6.61 min.
HPLC-MS (Method C): m/z: 348 (M+1); Rt.=3.13 min
1H-NMR: (DMSO-d6): 11.5 (1H, broad); 7.95 (1H, d); 7.65 (1H, s); 7.45 (1H, dd); 7.01 (1H, dd); 3.4 (1H, broad).
HPLC-MS (Method C): m/z: 309 (M+1); Rt.=4.07 min
Mp. 152-154° C.
HPLC-MS (Method C): m/z: 274 (M+1), Rt.=3.70 min
1H-NMR: (DMSO-d6): 12.8 (1H, broad); 7.72 (1H, s); 7.60 (2H, d); 7.50 (1H, t).
HPLC-MS (Method C): m/z: 436 (M+1); Rt. 4.81 min
HPLC-MS (Method C): m/z: 508 (M+1); Rt.=4.31 min
HPLC-MS (Method C): m/z: 499 (M+1); Rt.=3.70 min
HPLC-MS (Method C): m/z: 342 (M+1); Rt.=3.19 min
HPLC-MS (Method C): m/z: 282 (M+1); Rt.=2.56, mp=331-333° C.
M.p: 104-105° C.
HPLC-MS (Method C): m/z: 234 (M+1); Rt.=3.58 min,
Mp: 241-242° C.
HPLC-MS (Method C): m/z: 266 (M+1); Rt.=3.25 min;
Mp: 255-256° C.
HPLC-MS (Method C): m/z: 435 (M+1), Rt 4.13 min,
HPLC-MS (Method C): m/z: 246 (M+1); Rt.=3.65 min, mp=265-266° C.
HPLC-MS (Method C): m/z: 276 (M+1); Rt.=3.63, mp=259-263° C.
1H-NMR: (DMSO-d6) δ=12.3 (1H, 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).
Mp: 203-210° C.
HPLC-MS (Method C): m/z: 246 (M+1); Rt=3.79 min.
Mp: 251-254° C.
HPLC-MS (Method C): m/z: 266 (M+1; Rt=3.90 min
Mp: 338-347° C.
HPLC-MS (Method C): m/z: 273 (M+1); Rt.=2.59 min.
HPLC-MS (Method C): m/z: 459 (M+1); Rt.=3.65 min.
HPLC-MS (Method C): m/z: 339 (M+1); Rt=3.37 min.
HPLC-MS (Method C): m/z: 319 (M+1); Rt=3.48 min.
HPLC-MS (Method C): m/z: 325 (M+1); Rt=3.54 min.
HPLC-MS (Method C): m/z: 287 (M+1); Rt=2.86 min.
HPLC-MS (Method C): m/z: 303 (M+1); Rt=2.65 min.
HPLC-MS (Method C): m/z: 257 (M+1); Rt=2.77 min.
HPLC-MS (Method C): m/z: 273 (M+1); Rt=3.44 min.
HPLC-MS (Method C): m/z: 257 (M+1); Rt=3.15 min.
HPLC-MS (Method C): m/z: 366 (M+1); Rt=4.44 min.
HPLC-MS (Method C): m/z: 287 (M+1); Rt.=2.89 min.
HPLC-MS (Method C): m/z: 469 (M+1); Rt=5.35 min.
HPLC-MS (Method C): m/z: 320 (M+1); Rt=2.71 min.
Preparation of the Intermediate, 7-formyl-4-methoxybenzofuran-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-methoxybenzofuran-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 mL). 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 Na2SO4 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, 1H); 7.17 (d, 1H); 4.08 (s, 3H).
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 Na2SO4 and concentrated in vacuo to afford 1.5 g crude product. Column chromatography SiO2, EtOAc/heptanes=1/4 gave 1.1 g (46%) of 4-methoxybenzofuran-7-carbaldehyde as a solid.
1H NMR (CDCl3): 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.
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 hydrochloride (4.8 g, 41.7 mmol) 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 Na2SO4 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-hydroxybenzofuran-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.
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) afforded 23 g of 5-bromo-2,3-dihydrobenzofuran-7-carbaldehyde as a solid.
1H NMR (CDCl3): δ: 10.18 (s, 1H); 7.75 (d, 1H); 7.55 (d, 1H); 4.80 (t, 2H); 3.28 (t, 2H).
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%).
1H NMR (CDCl3): δ9.96 (s, 1H), 7.80 (d, 2H), 7.35 (d, 2H), 2.58 (m, 1H), 1.94-1.70 (m, 5H), 1.51-1.17 (m, 5H)
Other ligands of the invention include
HPLC-MS (Method C): m/z=350 (M+1); Rt.=3.45 min.
HPLC-MS (Method C): m/z=380 (M+1); Rt=3.52 min.
HPLC-MS (Method C): m/z=304 (M+1); Rt=2.95 min.
HPLC-MS (Method C): m/z=339 (M+1); Rt.=4.498 min.
HPLC-MS (Method C): m/z=369 (M+1); Rt.=4.456 min.
HPLC-MS (Method C): m/z=322 (M+1); Rt.=2.307 min.
HPLC-MS (Method C): m/z=306 (M+1); Rt.=3.60 min.
HPLC-MS (Method C): m/z=300 (M+1); Rt.=3.063 min.
HPLC-MS (Method C): m/z=378 (M+1); Rt=3.90 min.
HPLC-MS (Method C): m/z=355 (M+1); Rt 3.33 min.
HPLC-MS (Method C): m/z=322 (M+1); Rt.=2.78 min.
HPLC-MS (Method C): m/z=372 (M+1); Rt.=2.78 min.
HPLC-MS (Method C): m/z=431 (M+1); Rt.=3.30 min.
HPLC-MS (Method C): m/z=310 (M+1); Rt.=4.97 min.
HPLC-MS (Method C): m/z=330 (M+1); Rt.=5.33 min.
HPLC-MS (Method C): m/z=396 (M+1); Rt.=3.82 min.
HPLC-MS (Method C): m/z=324 (M+1); Rt.=3.82 min.
HPLC-MS (Method C): m/z=457 (M+1); Rt=4.23 min.
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)benzoic acid.
HPLC-MS (Method C): m/z=358 (M+1), RT.=4.15 min.
Starting aldehyde commercially available (Syncom BV, NL)
HPLC-MS (Method C): m/z=366 (M+1); Rt.=3.37 min.
HPLC-MS (Method C): m/z=401 (M+1); Rt.=4.08 min.
Starting aldehyde commercially available (Syncom BV, NL)
HPLC-MS (Method C): m/z=394 (M+1); Rt.=3.71 min.
HPLC-MS (Method C): m/z=232 (M+1); Rt.=3.6 min.
5-(2-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione (prepared as described in example 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 hydrochloric acid to pH 1. The aqeuous layer was extracted 3 times with ethyl acetate, dried with MgSO4 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.
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 (3×500 mL) and dried. Recrystallization from acetonitrile (500 mL) afforded 2.5 g of the title compound.
1H-NMR (DMSO-d6, selected peaks): δ=3.42 (1H, dd), 3.90 (1H, dd), 4.16 (2H, “It”), 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 (1H, bs).
HPLC-MS (Method C): m/z=382 (M+23); Rt=3.23 min.
5-Naphthalen-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 (3×100 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.
1H-NMR (DMSO-d6): δ=3.54 (1H, dd), 3.98 (1H, dd), 5.00 (1H, 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:
The following compounds are commercially available and may be prepared using general procedures (B) and/or (C).
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 C1-C6-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:
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 N,N-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 (1H, s). HPLC-MS (Method A): m/z=237 (M+1); Rt=3.46 min.
4-(4-Formylphenoxy)butyric acid ethyl ester (19.6 g, 75 mmol) was dissolved in methanol (250 mL) and 1N 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 1N 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 (1H, s), 12.2 (1H, bs). HPLC-MS (Method A): m/z=209 (M+1); Rt=2.19 min.
Thiazolidine-2,4-dione (3.55 g, 27.6 mmol), 4-(4-formylphenoxy)butyric acid (5.74 g, 27.6 mmol), anhydrous sodium acetate (11.3 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-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid as a solid.
1H-NMR (DMSO-d6): δ 1.97 (2H, p), 2.40 (2H, t), 4.07 (2H, t), 7.08 (2H, d), 7.56 (2H, d), 7.77 (1H, s), 12.2 (1H, bs), 12.5 (1H, bs); HPLC-MS (Method A): m/z: 308 (M+1); Rt=2.89 min.
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.
1H-NMR (DMSO-d6): δ6.63 (1H, d), 7.59-7.64 (3H, m), 7.77 (1H, s), 7.83 (2H, m).
Triethylamine salt: 1H-NMR (DMSO-d6): δ 4.27 (2H, s), 6.90 (2H, d), 7.26 (1H, s), 7.40 (2H, d).
1H-NMR (DMSO-d6): δ 7.57 (1H, s), 7.60 (1H, t), 7.79 (1H, dt), 7.92 (1H, dt), 8.14 (1H, t).
1H-NMR (DMSO-d6): δ 2.00 (2H, p), 2.45 (2H, t), 4.17 (2H, t), 7.31 (1H, d), 7.54 (1H, dd), 7.69 (1H, d), 7.74 (1H, s), 12.2 (1H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 364 (M+23); Rt=3.19 min.
1H-NMR (DMSO-d6): δ 1.99 (2H, p), 2.46 (2H, t), 4.17 (2H, t), 7.28 (1H, d), 7.57 (1H, dd), 7.25 (1H, s), 7.85 (1H, d), 12.2 (1H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 410 (M+23); Rt=3.35 min.
1H-NMR (DMSO-d6): δ 1.99 (2H, p), 2.45 (2H, t), 4.18 (2H, t), 7.28 (1H, d), 7.55 (1H, dd), 7.60 (1H, s), 7.86 (1H, 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
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.
HPLC-MS (Method A): m/z: 394 (M+23); Rt=3.62 min.
1H-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 (1H, bs).
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 brominated 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
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.
1H-NMR (DMSO-d6): 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.
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.
HPLC-MS (Method B): m/z: 310 (M+1); Rt=3.43 min.
HPLC-MS (Method A): m/z: 330 (M+1); Rt=3.25 min.
HPLC-MS (Method A): m/z: 299 (M+1); Rt=2.49 min.
HPLC-MS (Method A): m/z: 303 (M+1); Rt=2.90 min.
3-Formylindol (10 g, 69 mmol) was dissolved in N,N-dimethylformamide (100 mL) and under an atmosphere of nitrogen and 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 N,N-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 (2×150 mL). The combined organic extracts were washed with a saturated aqueous solution of ammonium chloride (100 mL), dried (MgSO4) and concentrated in vacuo to afford 15.9 g (quant.) of (3-formylindol-1-yl)acetic acid ethyl ester as an oil.
1H-NMR (CDCl3): δH=1.30 (3H, t), 4.23 (2H, q), 4.90 (2H, s), 7.3 (3H, m), 7.77 (1H, s), 8.32 (1H, d), 10.0 (1H, s).
(3-Formylindol-1-yl)acetic acid ethyl ester (15.9 g 69 mmol) was dissolved in 1,4-dioxane (100 mL) and 1N 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 (MgSO4) 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).
HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.08 min.
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 reflux 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-d6): δH=1.10 (3H, t), 2.94 (2H, t), 4.02 (2H, q), 4.55 (2H, t), 7.3 (2H, m), 7.67 (1H, d), 8.12 (1H, d), 8.30 (1H, s), 9.90 (1H, 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-d6): 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 (1H, s), 12.5 (1H, bs).
HPLC-MS (Method A): m/z: 429 (M+23); Rt=3.89 min.
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.
HPLC-MS (Method C): m/z: 492 (M+23); Rt.=5.3 min.
The intermediate aldehyde was prepared similarly as described in example 481.
HPLC-MS (Method C): m/z: 478 (M+23); Rt.=5.17 min.
The intermediate aldehyde was prepared similarly as described in example 481.
HPLC-MS (Method C): m/z: 534 (M+23); Rt.=6.07 min.
The intermediate aldehyde was prepared similarly as described in example 481.
HPLC-MS (Method C): m/z: 408 (M+23); Rt.=3.71 min.
HPLC-MS (Method C): m/z: 380 (M+23); Rt.=3.23 min.
HPLC-MS (Method C): m/z: 436 (M+23); Rt.=4.64 min.
HPLC-MS (Method C): m/z: 408 (M+23); Rt.=4.28 min.
HPLC-MS (Method C): m/z=444 (M+1); Rt=3.84 min.
HPLC-MS (Method C): m/z=500 (M+1); Rt=5.18 min.
HPLC-MS (Method C): m/z=369 (M+1); Rt=2.68 min.
To a mixture of 4-[4-(2,4-dioxothiazolidin-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. N-(3-aminopropylcarbamic acid tert-butyl ester (3.45 g, 19.8 mmol) was added and the resulting mixture was stirred at 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-ylidenemethyl)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:
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, 6H), 1.39 (m, 2H), 1.55 (m, 2H), 1.87 (m, 4H), 3.48 (t, 1H), 4.13 (m, 4H), 4.27 (t, 2H), 7.17 (d, 1H), 7.64 (t, 1H), 7.75 (t, 1H), 8.13 (d, 1H), 8.29 (d, 1H), 9.24 (d, 1H), 10.19 (s, 1H).
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 until 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, 2H), 1.55 (m, 2H), 1.80 (m, 2H), 1.90 (m, 2H), 3.24 (t, 1H), 4.29 (t, 2H), 7.19 (d, 1H), 7.64 (t, 1H), 7.75 (t, 1H), 8.14 (d, 1H), 8.30 (d, 1H), 9.23 (d, 1H), 10.18 (s, 1H), 12.69 (s, 2H).
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 precipitate 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-CO2+Na); Rt=4.08 min. 1H-NMR (DMSO-d6): δ=1.41 (m, 2H), 1.55 (m, 4H), 1.88 (m, 2H), 2.23 (t, 1H), 4.24 (t, 2H), 7.61-7.74 (m, 3H), 8.12 (d, 1H), 8.28 (d, 1H), 8.38 (s, 1H), 12.00 (s, 1H), 12.59 (s, 2H).
The following compounds are commercially available and may be prepared according to general procedure (D):
The following salicylic acid derivatives do all bind to the HisB10Zn2+ site of the insulin hexamer:
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.
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.
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. N,N-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 (3×100 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-d6): δ11.95 (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.
The following compounds were prepared as described below:
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.
1H-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.
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-d6): 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.
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-methylpyrrolidone (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 acetonitrile 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 (1H, d).
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 (2×100 mL). Drying (MgSO4) 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 (1H, d), 6.77 (2H, d), 6.96 (2H, d), 7.40 (1H, dd), 8.05 (1H, d), 9.6 (1H, bs).
wherein Lea is a leaving group such as Cl, Br, I or OSO2CF3, R is hydrogen or C1-C6-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:
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(II) 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 (3×40 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): δ12.60 (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.
HPLC-MS (Method (A)): m/z: 295 (M+1); Rt=4.60 min.
HPLC-MS (Method (A)): m/z: 265 (M+1); Rt=4.6 min.
HPLC-MS (Method (A)): m/z: 279 (M+1); Rt=4.95 min.
HPLC-MS (Method (A)): m/z: 293 (M+1); Rt=4.4 min.
HPLC-MS (Method (A)): m/z: 315 (M+1); Rt=5.17 min.
HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=3.60 min.
HPLC-MS (Method (A)): m/z: 305 (M+1); Rt=4.97 min.
HPLC-MS (Method (A)): m/z: 295 (M+1); Rt=4.68 min.
HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=4.89 min.
HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=5.61 min.
HPLC-MS (Method (A)): m/z: 341 (M+1); Rt=5.45 min.
wherein R30 is hydrogen or C1-C6-alkyl and T is as defined above This general procedure (F) is further illustrated in the following example:
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 mmol) 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 (3×2 mL) to yield the title compound (40 mg) as its hydrochloride salt. No further purification was necessary.
1H-NMR (DMSO-d6): δ 10.95 (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).
HPLC-MS (Method C): m/z: 372 (M+1); Rt=4.31 min.
HPLC-MS (Method C): m/z: 362 (M+1); Rt=4.75 min.
HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.43 min.
HPLC-MS (Method C): m/z: 345 (M+1); Rt=2.26 min.
HPLC-MS (Method C): m/z: 324 (M+1); Rt=2.57 min.
HPLC-MS (Method C): m/z: 350 (M+1); Rt=2.22 min.
HPLC-MS (Method C): m/z: 342 (M+1); Rt=2.45 min.
HPLC-MS (Method C): m/z: 357 (M+1); Rt=2.63 min.
HPLC-MS (Method C): m/z: 384 (M+1); Rt=2.90 min.
HPLC-MS (Method C): m/z: 400 (M+1); Rt=3.15 min.
HPLC-MS (Method C): m/z: 338 (M+1); Rt=2.32 min.
wherein J is as defined above and the moiety (C1-C6-alkanoyl)2O is an anhydride.
The general procedure (G) is illustrated by the following example:
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 hydrogencarbonate (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 (3×1 mL) to yield the title compound (21 mg). No further purification was necessary.
1H-NMR (DMSO-d6): δ10.96 (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.
HPLC-MS (Method C): m/z: 414 (M+1); Rt=3.76 min.
HPLC-MS (Method C): m/z: 392 (M+1); Rt=3.26 min.
HPLC-MS (Method C): m/z: 384 (M+1); Rt=3.67 min.
Compounds of the invention may also include tetrazoles:
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 MgSO4 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 C18H13NO:
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-yloxymethyl)-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 C18H14N4O:
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, 1H), 7.74 (d, 1H), 7.84 (m, 3H), 8.02 (d, 1H), 8.22 (s, 1H).
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 evaporated in vacuo. The resulting mixture was filtered and the filter cake was washed with water followed by heptane (2×25 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-dimethylformamide (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 l) 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 be further purified by treatment with ethanol at reflux temperature.
1H 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 C18H13N5O, 0.75H2O:
C, 65.74%; H, 4.44%; N, 21.30%. Found:
C, 65.58%; H, 4.50%; N, 21.05%.
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 (3×50 mL), ethanol (2×50 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, CDCl3) 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)-benzonitrile (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 1N hydrochloric acid (60 mL) and ice water (500 mL). The precipitate was filtered off and washed with water (3×100 mL), 50% ethanol (3×100 mL), ethanol (50 mL), 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).
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 (2×200 mL). The combined organic extracts were washed with water (2×100 mL), dried (MgSO4) 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-dimethylformamide (100 mL) 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 aqueous 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, CDCl3+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).
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 (3×100 mL), n-hexane (2×80 mL) and dried in vacuo 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-ylmethoxy)-benzonitrile (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 (2×50 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).
This compound was made similarly as described in example 576.
This compound was prepared similarly as described in example 572, step 2.
This compound was prepared similarly as described in example 572, step 2.
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,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 collected 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 C20H16N4O, 2H2O:
C, 65.92%; H, 5.53%; N, 15.37%. Found:
C, 65.65%; H, 5.01%; N, 14.92%.
This compound was prepared similarly as described in example 576.
This compound was prepared similarly as described in example 572, step 2.
This compound was made similarly as described in example 576.
This compound was prepared similarly as described in example 572, step 2.
This compound was prepared similarly as described in example 572, step 2.
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 1N 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), sodium 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 1N hydrochloric acid and the mixture was extracted with diethyl ether. The combined organic layers were dried over Na2SO4 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-cyanomethyloxybenzoate (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 Na2SO4 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-d6): 4.70 (2H, s), 7.48 (2H, d), 7.73 (2H, d), 13 (1H, bs).
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 aqueous 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 ammonium chloride (9.7 g, 0.18 mol) in DMF (1000 mL) was refluxed overnight under N2. The mixture was concentrated in vacuo. The residue was suspended in cold water and extracted with diethyl ether. The combined organic phases were washed with brine, dried over Na2SO4 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-d6): 5.58 (2H, s), 7.15 (2H, d), 7.93 (2H, d), 12.7 (1H, bs).
3-Bromo-9H-carbazole was prepared as described by Smith et 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 N-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 (3×50 ml). The filtrate was extracted with ethyl acetate (3×250 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 recrystallised 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-carbazole (5.77 g, 30 mmol) was dissolved in N,N-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 (3×200 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 (2×200 ml) and dried in vacuo at 50° C. affording 5.79 g (82%) of the title compound as a solid.
1H-NMR (DMSO-d6): δ11.78 (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 HisB10Zn2+ site of the insulin hexamer:
wherein K, M, and T are as defined above.
The reaction is generally known as a reductive alkylation reaction and is generally performed by stirring 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:
A solution of 5-(3-aminophenyl)-2H-tetrazole (example 874, 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.
HPLC-MS (Method D): m/z: 252 (M+1); Rt=3.74 min.
HPLC-MS (Method D): m/z: 282.2 (M+1); Rt=3.57 min.
HPLC-MS (Method D): m/z: 268.4 (M+1); Rt=2.64 min.
HPLC-MS (Method D): m/z: 297.4 (M+1); Rt=3.94 min.
HPLC-MS (Method D): m/z: 287.2 (M+1); Rt=4.30 min.
HPLC-MS (Method D): m/z: 286 (M+1); Rt=4.40 min.
HPLC-MS (Method D): m/z: 332 (M+1); Rt=4.50 min.
HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.94 min.
HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.
HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.60 min.
HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.24 min.
HPLC-MS (Method D): m/z: 412 (M+1); Rt=5.54 min.
HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.04 min.
HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.00 min.
HPLC-MS (Method D): m/z: 326 (M+1); Rt=3.10 min.
HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.97 min.
HPLC-MS (Method D): m/z: 322 (M+1); Rt=3.60 min.
HPLC-MS (Method D): m/z: 345 (M+1); Rt=3.07 min.
HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.97 min.
HPLC-MS (Method D): m/z: 362 (M+1); Rt=5.27 min.
For preparation of starting material, see example 875.
HPLC-MS (Method D): m/z: 252 (M+1); Rt=3.97 min.
HPLC-MS (Method D): m/z: 282 (M+1); Rt=3.94 min.
HPLC-MS (Method D): m/z: 268 (M+1); Rt=3.14 min.
HPLC-MS (Method D): m/z: (M+1); Rt=3.94 min.
HPLC-MS (Method D): m/z: (M+1); Rt=4.47 min.
HPLC-MS (Method D): m/z: 286 (M+1); Rt=4.37 min.
HPLC-MS (Method D): m/z: 331 (M+1); Rt=4.57 min.
HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.07 min.
HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.
HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.
HPLC-MS (Method D): m/z: 328 (M+1); Rt=5.07 min.
HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.34 min.
HPLC-MS (Method D): m/z: 412 (M+1); Rt=5.54 min.
HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.07 min.
HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.03 min.
HPLC-MS (Method D): m/z: 286 (M+1); Rt=3.47 min.
HPLC-MS (Method D): m/z: 326 (M+1); Rt=3.40 min.
HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.14 min.
HPLC-MS (Method D): m/z: 322 (M+1); Rt=3.66 min.
HPLC-MS (Method D): m/z: 345 (M+1); Rt=3.10 min.
HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.04 min.
HPLC-MS (Method D): m/z: 362 (M+1); Rt=5.30 min.
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:
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 hydrochloride (4.2 mmol) and the resulting mixture was stirred at room temperature for 1 hour. An aliquot 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.
HPLC-MS (Method D): m/z: 310 (M+1); Rt=2.89 min.
HPLC-MS (Method D): m/z: 336 (M+1); Rt=3.10 min.
HPLC-MS (Method D): m/z: 338 (M+1); Rt=2.97 min.
HPLC-MS (Method D): m/z: 340 (M+1); Rt=3.03 min.
HPLC-MS (Method D): m/z: 372 (M+1); Rt=4.47 min.
HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.50 min.
HPLC-MS (Method D): m/z: 354 (M+1); Rt=4.60 min.
HPLC-MS (Method D): m/z: 383 (M+1); Rt=4.60 min.
HPLC-MS (Method D): m/z: 266 (M+1); Rt=3.23 min.
The starting material was prepared as described in example 592.
HPLC-MS (Method D): m/z: 340 (M+1); Rt=2.83 min.
HPLC-MS (Method D): m/z: 340 (M+1); Rt=2.90 min.
HPLC-MS (Method D): m/z: 366 (M+1); Rt=3.07 min.
HPLC-MS (Method D): m/z: 368 (M+1); Rt=2.97 min.
HPLC-MS (Method D): m/z: 370 (M+1); Rt=3.07 min.
HPLC-MS (Method D): m/z: 402 (M+1); Rt=4.43 min.
HPLC-MS (Method D): m/z: 388 (M+1); Rt=4.50 min.
HPLC-MS (Method D): m/z: 384 (M+1); Rt=4.57 min.
HPLC-MS (Method D): m/z: 413 (M+1); Rt=4.57 min.
HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.23 min.
The starting material was prepared as described in example 593.
HPLC-MS (Method D): m/z: 356 (M+1); Rt=2.93 min.
HPLC-MS (Method D): m/z: 356 (M+1); Rt=3.00 min.
HPLC-MS (Method D): m/z: 382 (M+1); Rt=3.26 min.
HPLC-MS (Method D): m/z: 384 (M+1); Rt=3.10 min.
HPLC-MS (Method D): m/z: 386 (M+1); Rt=3.20 min.
HPLC-MS (Method D): m/z: 418 (M+1); Rt=4.57 min.
HPLC-MS (Method D): m/z: 404 (M+1); Rt=4.60 min.
HPLC-MS (Method D): m/z: 400 (M+1); Rt=4.67 min.
HPLC-MS (Method D): m/z: 429 (M+1); Rt=4.67 min.
HPLC-MS (Method D): m/z: 312 (M+1); Rt=3.40 min.
wherein T is as defined above.
This general procedure (J) is further illustrated in the following example.
3-(2H-Tetrazol-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 (2×250 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-tetrazol-5-yl]-9H-carbazole (31.5 g) which was used without further purification.
1H-NMR (CDCl3): δ8.87 (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/z: 243 (triphenylmethyl); Rt=5.72 min. 3-[2-(Triphenylmethyl)-2H-tetrazol-5-yl]-9H-carbazole (200 mg, 0.42 mmol) was dissolved in methyl sulfoxide (1.5 mL). 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 (3×10 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 (3×10 mL) and dichloromethane (3×10 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 (1H, t), 7.36-7.27 (4H, m), 7.08 (1H, 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.
HPLC-MS (Method C): m/z: 360 (M+1); Rt=4.31 min.
HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.26 min.
HPLC-MS (Method C): m/z: 394 (M+1); Rt=4.40 min.
HPLC-MS (Method C): m/z: 432 (M+1); Rt=4.70 min.
HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.25 min.
1H-NMR (DMSO-d6): δ8.91 (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).
1H-NMR (DMSO-d6): δ8.94 (1H, s), 8.33 (1H, d), 8.17 (1H, dd), 7.95 (1H, d), 7.77 (1H, d), 7.61-7.27 (11H, m), 5.82 (2H, s).
HPLC-MS (Method C): m/z: 356 (M+1); Rt=3.99 min.
HPLC-MS (Method C): m/z: 376 (M+1); Rt=4.48 min.
HPLC-MS (Method C): m/z: 404 (M+1); Rt=4.33 min.
HPLC-MS (Method C): m/z: 402 (M+1); Rt=4.80 min.
1H-NMR (DMSO-d6): δ8.91 (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.
HPLC-MS (Method C): m/z: 452 (M+1); Rt=4.37 min.
HPLC-MS (Method C): m/z: 462 (M+1); Rt=4.70 min.
1H-NMR (DMSO-d6): δ8.89 (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.
HPLC-MS (Method C): m/z: 426 (M+1); Rt=4.78 min.
3.6 fold excess sodium hydride was used.
1H-NMR (DMSO-d6): δ12.89 (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.
Alternative mode of preparation of 9-(4-Carboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole:
Carbazole (52.26 g, 0.30 mol) was dissolved in dichloromethane (3 L) and silicagel (60 mesh, 600 g) was added to the mixture and the mixture was cooled to 10° C. A mixture of N-bromosuccinimide (NBS, 55 g, 0.30 mol) in dichloromethane (400 mL) was added at 10° C. After addition, the mixture was allowed to reach room temperature. After standing for 42 hours, the mixture was filtered, and the solid was washed with dichloromethane (4×200 mL), the combined filtrates were washed with water (300 mL) and dried over Na2SO4. Evaporation in vacuo to dryness afforded 77 g of crude product. Recrystallization from 2-propanol (800 mL) afforded 71% 3-bromocarbazole.
To a stirred solution of 3-bromocarbazole (63 g, 0.256 mol) in N-methylpyrrolidone (900 mL) was added cuprous cyanide (CuCN, 25.22 g, 0.28 mol) and the mixture was heated to 190° C. After 9 hours of heating, the mixture was cooled to room temperature. The mixture was concentrated by bulb-to-bulb distillation (100° C., 0.1 mm Hg). The residue was treated with NH4OH (25%, 300 mL) and subsequently extracted with ethyl acetate (10%) in toluene. The organic layer was dried over Na2SO4 and concentrated by bulb-to-bulb distillation (100° C., 0.1 mm Hg) to give 34 g (70%) of 3-cyanocarbazole.
Sodium hydride 55-60% in mineral oil (3.7 g, 0.093 mol) was added in portions to a stirred, cooled (5° C.) mixture of 3-cyanocarbazole (17.5 g, 0.091 mol) in N,N-dimethylformamide (200 mL). After 0.5 hours, a solution of methyl 4-bromomethylbenzoate (22.9 g, 100 mmol) in N,N-dimethylformamide (80 mL) was added dropwise to the cooled mixture. The mixture was subsequently slowly warmed to room temperature and stirred overnight. The mixture was poured into ice water and extracted with dichloromethane (2×200 mL), the organic layer was washed several times with water, dried over Na2SO4 and concentrated in vacuo. A mixture of ethyl acetate and heptane (1/1, 50 mL) was added to the concentrate and the solid was product filtered off. Yield 24 g (78%) of 4-(3-cyanocarbazol-9-ylmethyl)benzoic acid methyl ester.
Sodium azide (7.8 g, 0.12 mol) and ammonium chloride (6.42 g, 0.12 mol) were added to a stirred mixture of 4-(3-cyanocarbazol-9-ylmethyl)benzoic acid methyl ester (24.8 g, 0.073 mol) in N,N-dimethylformamide (130 mL) and the mixture was heated to 110° C. After 48 hours, the mixture was cooled to room temperature and poured into water (500 mL) and cooled to 5° C. Hydrochloric acid (10 N) was then added to pH=2. After stirring for 1 hour at 5° C. the precipitate was filtered off and washed with water. The solid obtained was air dried. Yield 27.9 g of 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid methyl ester. 31.1 g of 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid methyl ester was added to a solution of sodium hydroxide (8.76 g, 0.219 mol) in water (150 mL) and the mixture was heated to 80° C., after 0.5 h activated carbon (0.5 g) was added and the mixture was filtered through celite. The filtrate was treated with hydrochloric acid (10 N) to pH=1 and the formed precipitate was filtered off and air dried. This procedure was repeated as the first treatment did not give complete hydrolysis of the ester. Finally the product was dissolved in 2-propanol, the filtered the mother liquor was concentrated to approximately 100 mL and the product was isolated by filtration to afford 19 g of the title compound. After evaporation of the mother liquor to dryness and re-treatment with 2-propanol further 8 g of product was isolated resulting in a yield of 90%.
HPLC-MS (Method B): m/z: 360 (M+1); Rt=5.30 min.
1H-NMR (DMSO-d6): δ8.88 (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.
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 (1H, t), 7.37-7.27 (2H, m), 7.12-7.02 (2H, m), 6.97 (1H, d), 5.78 (2H, s); HPLC-MS (Method C): m/z: 344 (M+1); Rt=4.10 min.
HPLC-MS (Method C): m/z: 452 (M+1); Rt=4.58 min.
3.6 fold excess sodium hydride was used.
1H-NMR (DMSO-d6): δ12.97 (1H, bs), 8.90 (1H, bs), 8.30 (1H, d), 8.12 (1H, bd), 7.89 (1H, d), 7.82 (1H, m), 7.77 (1H, bs), 7.71 (1H, d), 7.53 (1H, t), 7.46-7.41 (2H, m), 7.32 (1H, t), 5.84 (2H, s); HPLC-MS (Method C): m/z: 370 (M+1); Rt=3.35 min.
1H-NMR (DMSO-d6): δ8.87 (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.
HPLC-MS (Method C): m/z: 386 (M+1); Rt=4.03 min.
HPLC-MS (Method B): m/z: 380 (M+1); Rt=5.00 min.
HPLC-MS (Method B): m/z: 383 (M+1); Rt=4.30 min.
1H-NMR (DMSO-d6): δ8.86 (1H, d), 8.26 (1H, d), 8.10 (1H, dd), 7.90 (1H, d), 7.73 (1H, d), 7.51 (1H, t), 7.30 (1H, 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.
1H-NMR (DMSO-d6): δ8.87 (1H, d), 8.27 (1H, d), 8.09 (1H, dd), 7.77 (1H, d), 7.60 (1H, d), 7.49 (1H, t), 7.29 (1H, t), 7.23 (1H, bt), 7.07 (1H, bd), 6.74 (1H, bt), 6.61 (1H, bd), 5.65 (2H, s), 3.88 (3H, s); HPLC-MS (Method B): m/z: 356 (M+1); Rt=4.97 min.
HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.74 min.
HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.73 min.
1H-NMR (DMSO-d6): δ8.87 (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.
1H-NMR (DMSO-d6): δ10.54 (1H, s), 8.87 (1H, bs), 8.27 (1H, d), 8.12 (1H, bd), 7.83 (1H, 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/z: 369 (M+1); Rt=3.44 min.
1H-NMR (DMSO-d6): δ8.85 (1H, d), 8.31 (1H, t), 8.25 (1H, d), 8.10 (1H, 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.
1H-NMR (DMSO-d6): δ8.92 (1H, d), 8.32 (1H, d), 8.09 (1H, dd), 7.76 (1H, 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.
1H-NMR (DMSO-d6): δ8.92 (1H, d), 8.32 (1H, d), 8.08 (1H, dd), 7.72 (1H, d), 7.55 (1H, d), 7.48 (1H, t), 7.32 (1H, t), 7.26 (1H, d), 7.12 (1H, t), 6.92 (1H, 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.
HPLC-MS (Method C): m/z: 371 (M+1); Rt=3.78 min.
HPLC-MS (Method B): m/z: 394 (M+1); Rt=5.62 min.
1H-NMR (DMSO-d6): δ8.89 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.88 (1H, d), 7.69 (1H, d), 7.52 (1H, 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.
1H-NMR (DMSO-d6): δ8.90 (1H, bs), 8.31 (1H, d), 8.13 (1H, bd), 7.90 (1H, d), 7.73 (1H, d), 7.54 (1H, t), 7.34 (1H, t), 7.14 (1H, t), 6.87 (2H, bd), 5.80 (2H, s); HPLC-MS (Method B): m/z: 362 (M+1); Rt=5.17 min.
1H-NMR (DMSO-d6): δ8.89 (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.
HPLC-MS (Method B): m/z: 452 (M+1); Rt=5.50 min.
1H-NMR (DMSO-d6): δ8.89 (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.
3.6 fold excess sodium hydride was used.
HPLC-MS (Method B): m/z: 413 (M+1); Rt=3.92 min.
HPLC-MS (Method B): m/z: 335 (M+1); Rt=3.70 min.
HPLC-MS (Method B): m/z: 459 (M+1); Rt=5.37 min.
HPLC-MS (Method B): m/z: 425 (M+1); Rt=5.35 min.
HPLC-MS (Method C): m/z: 397 (M+1); Rt=3.43 min.
HPLC-MS (Method C): m/z: 394 (M+1); Rt=4.44 min.
HPLC-MS (Method C): m/z: 412 (M+1); Rt=4.21 min.
HPLC-MS (Method C): m/z: 462 (M+1); Rt=4.82 min.
HPLC-MS (Method C): m/z: 368 (M+1); Rt=4.59 min.
HPLC-MS (Method C): m/z: 382 (M+1); Rt=4.47 min.
HPLC-MS (Method C): m/z: 376 (M+1); Rt=4.43 min.
HPLC-MS (Method C): m/z: 438 (M+1); Rt=4.60 min.
HPLC-MS (Method C): m/z: 404 (M+1); Rt=4.50 min.
HPLC-MS (Method C): m/z: 344 (M+1); Rt=4.09 min.
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.
HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.08 min.
HPLC-MS (Method C): m/z: 412 (M+1); Rt=4.34 min.
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 (2×10 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.
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 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: 492 (M+1); Rt=4.27 min.
HPLC-MS (Method C): m/z=362 (M+1); Rt=4.13 min.
HPLC-MS (Method C): m/z=362 (M+1); Rt=4.08 min.
HPLC-MS (Method C): m/z=416 (M+1); Rt=4.32 min.
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:
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-tert-butylphosphine)palladium (0).
wherein T is as defined above.
The general procedure (K) is further illustrated by the following example:
5-Cyanoindole (1.0 g, 7.0 mmol) was dissolved in N,N-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 mL, 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 (3×25 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-1H-indole-5-carbonitrile.
HPLC-MS (Method C): m/z: 233 (M+1); Rt=4.17 min.
1-Benzyl-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.
HPLC-MS (Method C): m/z: 354 (M+1); Rt=3.80 min.
1H-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 (1H, dd), 8.35 (1H, d).
Calculated for C22H17N5, H2O:
73.32% C, 5.03% H, 19.43% N. Found:
73.81% C, 4.90% H, 19.31% N.
5-(2H-Tetrazol-5-yl)-1H-indole (Syncom BV, Groningen, NL) (1.66 g, 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)-1H-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 CCl4 (100 mL) and under an atmosphere of nitrogen, the slurry was added N-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-Trityl-2H-tetrazol-5-yl)-1H-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 (100 mL) and the precipitate was isolated by filtration and treated with THF/6N HCl (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%) of the title compound.
HPLC-MS (Method C): m/z=396 (M+1); Rt.=3.51 min.
5-(2H-Tetrazol-5-yl)-1H-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.
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-tetrazol-5-yl)-1H-indole was prepared from 1-benzyl-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.
wherein T is as defined above and
Pol- is a polystyrene resin loaded with a 2-chlorotrityl linker, graphically shown below:
This general procedure (L) is further illustrated by the following example:
2-Chlorotritylchloride 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)-1H-indole (example 810) (63 mg, 0.34 mmol) in a mixture of N,N-dimethylformamide, dichloromethane and N,N-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 N,N-dimethylformamide (2×4 mL), dichloromethane (6×4 mL) and methyl sulfoxide (2×4 mL). Methyl sulfoxide (1 mL) was added, followed by the addition of a solution of lithium bis(trimethylsilyl)amide 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 (2×4 mL), dichloromethane (2×4 mL), methanol (2×4 mL), dichloromethane (2×4 mL) and tetrahydrofuran (4 mL). 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.
1H-NMR (DMSO-d6): δ8.29 (1H, s), 7.80 (1H, dd), 7.72 (2H, m), 7.64 (2H, bs), 7.56 (1H, t), 7.48 (1H, d), 6.70 (1H, 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.
HPLC-MS (Method B): m/z: 310 (M+1); Rt=4.11 min.
HPLC-MS (Method B): m/z: 310 (M+1); Rt=4.05 min.
HPLC-MS (Method B): m/z: 306 (M+1); Rt=3.68 min.
HPLC-MS (Method B): m/z: 290 (M+1); Rt=3.98 min.
HPLC-MS (Method B): m/z: 344 (M+1); Rt=4.18 min.
HPLC-MS (Method B): m/z: 310 (M+1); Rt=4.01 min.
HPLC-MS (Method B): m/z: 290 (M+1); Rt=3.98 min.
HPLC-MS (Method B): m/z: 344 (M+1); Rt=4.41 min.
HPLC-MS (Method B): m/z: 306 (M+1); Rt=3.64 min.
HPLC-MS (Method B): m/z: 294 (M+1); Rt=3.71 min.
HPLC-MS (Method B): m/z: 294 (M+1); Rt=3.68 min.
HPLC-MS (Method B): m/z: 402 (M+1); Rt=4.11 min.
HPLC-MS (Method B): m/z: 326 (M+1); Rt=4.18 min.
HPLC-MS (Method B): m/z: 354 (M+1); Rt=4.08 min.
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.
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=2.91 min.
HPLC-MS (Method B): m/z: 312 (M+1); Rt=3.78 min.
HPLC-MS (Method B): m/z: 312 (M+1); Rt=3.78 min.
HPLC-MS (Method B): m/z: 312 (M+1); Rt=3.81 min.
HPLC-MS (Method B): m/z: 318 (M+1); Rt=4.61 min.
HPLC-MS (Method B): m/z: 336 (M+1); Rt=3.68 min.
HPLC-MS (Method B): m/z: 377 (M+1); Rt=4.11 min.
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:
The following compounds of the invention may be prepared eg. from 1-(4-bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole (example 807) or from the analogue 1-(3-bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole 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-tert-butylphosphine)palladium (0).
wherein T is as defined above.
The general procedure (M) is further illustrated by the following example:
To a solution of 5-cyanoindole (1.0 g, 7.0 mmol) in dichloromethane (8 mL) was added 4-(dimethylamino)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 mL) 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-1H-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.
1-Benzoyl-1H-indole-4-carbonitrile was prepared from 4-cyanoindole according to the method described in example 865.
HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.24 min.
1-Benzoyl-4-(2H-tetrazol-5-yl)-1H-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.
HPLC-MS (Method B): m/z=376 (M+1); Rt=4.32 min.
HPLC-MS (Method B): m/z=320 (M+1); Rt=3.70 min.
HPLC-MS (Method B): m/z=335 (M+1); Rt=3.72 min.
HPLC-MS (Method B): m/z=335 (M+1); Rt=3.71 min.
HPLC-MS (Method C): m/z=340 (M+1); Rt=4.25 min.
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 Zn2+ site of the insulin hexamer:
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-tetrazole.
1H NMR DMSO-d6): δ=5.7 (3H, bs), 6.69 (2H, d), 7.69 (2H, d).
HPLC-MS (Method C): m/z: 162 (M+1); Rt=0.55 min.
wherein Frag is any fragment carrying a carboxylic acid group, R is hydrogen, optionally substituted aryl or C1-8-alkyl and R′ is hydrogen or C1-4-alkyl.
Frag-CO2H may be prepared eg by general procedure (D) or by other similar procedures described herein, or may be commercially available.
The procedure is further illustrated in the following example 878:
[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]acetic 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-dimethylaminopropyl)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 2×10 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.
HPLC-MS (Method A): m/z: 465 (M+1); Rt=4.35 min.
HPLC-MS (Method A): m/z: 431 (M+1); Rt=3.68 min.
HPLC-MS (Method A): m/z: 483 (M+1); Rt=4.06 min.
HPLC-MS (Method A): m/z: 403 (M+1); Rt=4.03 min.
HPLC-MS (Method A): m/z: 399 (M+1); Rt=3.82.
HPLC-MS (Method A): m/z: 431 (M+1); Rt=3.84 min.
HPLC-MS (Method A): m/z: 511 (M+1); Rt=4.05 min.
HPLC-MS (Method A): m/z: 527 (M+1); Rt=4.77 min.
HPLC-MS (Method C): m/z: 431 (M+1); Rt.=4.03 min.
HPLC-MS (Method C): m/z: 440 (M+1); Rt.=3.57 min.
HPLC-MS (Method C): m/z: 481 (M+1); Rt=4.08 min.
HPLC-MS (Method C): m/z: 441 (M+1); Rt=4.31 min.
HPLC-MS (Method C): m/z: 436 (M+1); Rt.=3.55 min.
HPLC-MS (Method C): m/z: 493 (M+1); Rt=4.19 min.
HPLC-MS (Method C): m/z: 493 (M+1); Rt=4.20 min.
HPLC-MS (Method C): m/z: 507 (M+1); Rt=4.37 min.
HPLC-MS (Method C): m/z=521 (M+1); Rt.=4.57 min.
HPLC-MS (Method C): m/z=515 (M+23); Rt.=3.09 min.
HPLC-MS (Method C): m/z=536 (M+1); Rt=3.58 min.
4-[3-(1H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid (2.00 g, 5.41 mmol), 1-hydroxybenzotriazole (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 (2×25 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).
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 (2×25 mL) and diethyl ether (2×25 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.
HPLC-MS (Method C): m/z=513 (M+1); Rt=3.65 min.
HPLC-MS (Method C): m/z=527 (M+1); Rt=3.57 min.
HPLC-MS (Method C): m/z=513 (M+1); Rt=3.55 min.
HPLC-MS (Method C): m/z=499 (M+1); Rt=2.87 min.
HPLC-MS (Method C): m/z=541 (M+1); Rt=3.91 min.
HPLC-MS (Method C: m/z=585 (M+1); Rt=2.81 min.
HPLC-MS (Method C): m/z=554 (M-3); Rt=3.19 min.
HPLC-MS (Method C): m/z=485 (M+1); Rt=3.04 min.
HPLC-MS (Method C): m/z=612 (M+1); Rt=3.24 min.
HPLC-MS (Method C): m/z=527 (M+1); Rt=3.65 min.
HPLC-MS (Method C): m/z=527 (M+1); Rt=3.65 min.
HPLC-MS (Method C): m/z=527 (M+1); Rt=3.65 min.
HPLC-MS (Method C): m/z=499 (M+1); Rt=3.00 min.
1H-NMR (DMSO-d6): 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, m), 5.79 (2H, s), 4.17 (2H, s), 4.02 (2H, s), 3.62 (3H, s), 3.49 (3H, s).
HPLC-MS (Method C): m/z=513 (M+1); Rt=3.70 min.
HPLC-MS (Method C): m/z=485 (M+1); Rt=2.96 min.
HPLC-MS (Method C): m/z=485 (M+1); Rt=2.87 min.
The title compound was prepared by coupling of (S)-2-{4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]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-ylmethyl]benzoic acid 2,5-dioxopyrrolidin-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-ylmethyl]-benzoylamino}butyric acid
HPLC-MS (Method C): m/z: 669 (M+1); Rt=2.84 min.
HPLC-MS (Method C): m/z: 515 (M+1); Rt=3.10 min.
HPLC-MS (Method C): m/z=611 (M+1); Rt=4.64 min.
HPLC-MS (Method C): m/z: 455 (M+1); Rt=3.13 min.
The title compound was prepared by coupling of 4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic 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/z: 515 (M+1); Rt=3.10 min.
The commercially available compounds in the following examples do all bind to the HisB10 Zn2+ site:
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:
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. SnCl2, 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, CSCl2, 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:
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-benzenesulfonyl-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 (0.47 g, 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, 1H). HPLC-MS (Method C) m/z:=187 (M+1); Rt.=1.93 min
wherein
AA is as defined above,
Steps 1 and 2 are described in the literature (eg Beck & Gûnther, Chem. Ber., 106, 2758-66 (1973))
Step 1 is a Knoevenagel condensation of the aldehyde AA-CHO with phenylsulfonylacetonitrile 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 949:
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 mL) 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-1H-[1,2,3]triazol-4-yl)phenoxy]acetic acid.
HPLC-MS (Method C): m/z: 245 (M+1) Rt. 2.19 min.
HPLC-MS (Method C): m/z: 221 (M+1); Rt. 3.43 min.
HPLC-MS (Method C): m/z: 221 (M+1); Rt=3.66 min.
HPLC-MS (Method C): m/z=422 (M+1); Rt=3.85 min.
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)benzoic acid.
HPLC-MS (Method C): m/z=358 (M+1), RT.=4.15 min.
HPLC-MS (Method C): m/z: 271 (M+1); Rt=3.87 min.
HPLC-MS (Method C): m/z: 251 (M+1); Rt=3.57 min.
HPLC-MS (Method C): m/z: 288 (M+1); Rt=3.67 min.
HPLC-MS (Method C): m/z=277 (M+1); Rt=3.60 min.
HPLC-MS (Method C): m/z=273 (M+1); Rt=4.12 min.
HPLC-MS (Method C): m/z=434 (M+1); Rt=4.64 min.
HPLC-MS (Method C: m/z=300 (M+1); Rt.=3.79 min.
This compound is commercially available (MENAI).
This compound is commercially available (MENAI).
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:
The following cyanotriazoles are also compounds of the invention:
wherein
n is 1 or 3-20,
AA is as defined above,
R″ is a standard carboxylic acid protecting group, such as C1-C6-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 & Gûnther, Chem. Ber., 106, 2758-66 (1973))
Step 3 is a Knoevenagel condensation of the aldehyde obtained in step 2 with phenylsulfonylacetonitrile 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
6-Hydroxynaphthalene-2-carbaldehyde (Syncom BV. NL, 15.5 g, 90 mmol) and K2CO3 (62.2 g, 450 mmol) were mixed in DMF (300 mL) and stirred at room temperature for 1 hour. 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-yloxy)pentanoic acid ethyl ester (28.4 g, 94.5 mmol), phenylsulfonylacetonitrile (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)naphthalen-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.
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 aqeuous layer was acidified with hydrochloric acid (12N). The precipitate was isolated by filtration, dissolved in THF/diethyl ether, the solution was treated with MgSO4 and activated carbon, filtrated and evaporated in vacuo to almost dryness followed by precipitation by addition of pentane (1 L). 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.
HPLC-MS (Method C): m/z=351 (M+1); Rt=3.68 min.
HPLC-MS (Method C): m/z=443 (M+23); Rt=4.92 min.
HPLC-MS (Method C): m/z=465 (M+1); Rt.=4.95 min.
HPLC-MS (Method C): m/z=465 (M+1); Rt.=4.95 min.
HPLC-MS (Method C): m/z=381 (M+1); Rt.=3.12 min.
HPLC-MS (Method C): m/z 0 409 (M+1); Rt.=3.51 min.
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):
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:
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-carbazole (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 (2×4 mL) and dichloromethane (6×4 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 (2×4 mL), methanol (2×4 mL) and tetrahydrofuran (4 mL). 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-d6): δ 8.91 (1H, s), 8.34 (1H, d), 8.05 (1H, d), 7.78 (3H, m), 7.63 (3H, m), 7.46 (2H, m), 7.33 (1H, dd).
The compounds in the following examples were prepared in a similar fashion.
HPLC-MS (Method C): m/z: 290 (M+1); Rt=3.04 min.
1H-NMR (DMSO-d6): 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).
HPLC-MS (Method B): m/z=326 (M+1); Rt=3.85 min.
HPLC-MS (Method B): m/z=376 (M+1); Rt=4.32 min.
HPLC-MS (Method B): m/z=335 (M+1); Rt=3.72 min.
HPLC-MS (Method B): m/z=335 (M+1); Rt=3.71 min.
HPLC-MS (Method C): m/z=340 (M+1); Rt=4.25 min.
HPLC-MS (Method C): m/z: 354 (M+1); Rt=3.91 min.
HPLC-MS (Method C): m/z: 418 (M+1); Rt=4.39 min.
HPLC-MS (Method C): m/z: 370 (M+1); Rt=4.01 min.
HPLC-MS (Method C): m/z: 374 (M+1); Rt=4.28 min.
HPLC-MS (Method C): m/z: 416 (M+1); Rt=4.55 min.
HPLC-MS (Method C): m/z: 354 (M+1); Rt=4.22 min.
HPLC-MS (Method C): m/z: 358 (M+1); Rt=3.91 min.
HPLC-MS (Method C): m/z: 390 (M+1); Rt=4.38 min.
HPLC-MS (Method C): m/z: 418 (M+1); Rt=4.36 min.
HPLC-MS (Method C): m/z: 304 (M+1); Rt=3.32 min.
HPLC-MS (Method C): m/z: 368 (M+1); Rt=3.84 min.
HPLC-MS (Method C): m/z: 320 (M+1); Rt=3.44 min.
HPLC-MS (Method C): m/z: 324 (M+1); Rt=3.73 min.
HPLC-MS (Method C): m/z: 304 (M+1); Rt=3.64 min.
HPLC-MS (Method A): m/z: 308 (M+1); Rt=3.61 min.
HPLC-MS (Method C): m/z: 368 (M+1); Rt=3.77 min.
HPLC-MS (Method A): (sciex) m/z: 326 (M+1); Rt=3.73 min.
HPLC-MS (Method C): m/z: 326 (M+1); Rt=3.37 min.
HPLC-MS (Method C): m/z: 374 (M+1); Rt=4.03 min.
The branched compounds of the invention can be prepared by using standard solid phase peptide synthesis using standard procedures known to the person skilled in the art. Such procedures for straight chain peptides can be found in WO 0327081 and WO 0480480, which references are hereby incorporated by reference.
Branching points can be obtained using, optionally orthogonally protected, trivalent residues, such as lysine, ornithine, glutamic acid, aspartic acid, iminodipropionic acid or the like.
Strategies for preparing the branched compounds of the invention can be for example attachment of an orthogonally protected lysine to resin-bound oligo-arginine, followed by either further arginine chain elongation and lastly attachment of the zink-binding motif (CGr) or vice verca. Examples of these procedures can eg. be found in the following examples 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1024 and 1025. Branching points can be placed after one and another, giving rise to dendrimer-like assemblies like in the following following examples 1010, 1011, 1012, 1013, and 1026.
Alternatively, the zink-binding motif (CGr) can be extended with a dicarboxylic acid, such as glutamic acid or aspartic acid eg. described in examples 903, 915, 916, 917 and 896. The resulting dicarboxylic acids can be coupled to resin-bound oligo-arginines. If stoichiometry is correct, the dicarboxylic acid cross-links two arginine chains and upon cleavage from the resin, a branched compound of the invention can be isolated eg. as described in the following examples 1021, 1022 and 1023.
10 gram of resin (Rink amid, Novabiochem 0.43 mmol/g) was swelled in NMP for >30 min. Then Fmoc-Lys(Alloc)-OH (Neosystems 15 mmol), dissolved in 0.5M HOAt in NMP (30 mL) and 15 mmol DIC was added.
After 1 hour the coupling was complete and the resin was deprotected with 25% piperidine in NMP for ca. 20 min, washed, and coupled with Fmoc-Lys(IvDde)-OH (Novabiochem, 5.4 gram, 10 mmol)+10 mmol HOAt+10 mmol DIC in 20 mL NMP. The coupling was carried out overnight.
The resin was subsequently washed with NMP, then deprotected with 25% piperidine in NMP for 20 min and coupled with 15 mmol Fmoc-Lys(Fmoc)-OH (Novabiochem) for 2 h. The resin was capped with a mixture of 10 mmol HOAc, 10 mmol HOBt, and 10 mmol DIC for 1 h and then washed with NMP. The resulting wet resin was used for further syntheses without further purification.
1 mmol of the above mentioned resin (Fmoc-Lys(Fmoc)-Lys(IvDde)-Lys(Alloc)Resin) was deprotected with 25% piperidine, 3% hydrazine, and 2% allyl alcohol for 15 min. This removed the IvDde and Fmoc groups. 10 mmol Fmoc-Lys(Fmoc)-OH+10 mmol HOBt+10 mmol DIC in NMP was subsequently coupled to resin overnight. The resulting resin was washed with NMP and deprotected with 25% piperidine in NMP for 20 min. followed by NMP wash.
The resin was then coupled with 20 mmol Fmoc-Arg(Pbf)-OH (Multisyntech), 20 mmol HOAt, and 20 mmol DIC. After 1 hour, the resin was coupled with another portion of 10 mmol Fmoc-Arg(Pbf)-OH, 10 mmol HOAt, and 10 mmol DIC by adding the coupling mixture to the resin mixture without draining.
The resin was washed with NMP then with CHCl3 and deprotected with 10% AcOH+5% NMM in CHCl3 with 0.5 mmol tetrakis(triphenylphosphine)palladium (0) and 0.5 mmol triphenylphosphine for 4 hours while bubbling with argon. The mixture was washed with CHCl3 and then with NMP and coupled with a mixture of 3 mmol 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid (prepared as described in example 738), 3 mmol HOAt, and 3 mmol DIC overnight. After coupling, the mixture was washed with ethanol and dried.
The dry resin was deprotected with a mixture of 5% thioanisol and 5% ethanol in TFA for 2 h. The mixture was concentrated be stream of argon then precipitated with diethyl ether, washed five times with diethyl ether and lyophilized in 5% AcOH.
Yield 1.5 g of H-Arg-Lys(Arg-yl)-Lys(Arg-Lys(Arg-yl)-yl)-Lys(Arg-Lys(Arg-yl)-yl)Lys(4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl)-NH2.
MALDI-TOF MS analysis: found: m/z=2074.05; calculated: m/z=2072.
2 mmol of the above mentioned Fmoc-Lys(Fmoc)-Lys(IvDde)-Lys(Alloc)-Resin was deprotected with 25% piperidine, 3% hydrazine, and 2% allyl alcohol for 20 min.
The resin was subsequently reacted with a mixture of 20 mmol Fmoc-Lys(Fmoc)OH, 20 mmol HOBt, and 20 mmol DIC in NMP overnight.
The resin was washed with NMP then with CHCl3 and deprotected with 10% AcOH+5% NMM in CHCl3 with 0.5 mmol tetrakis(triphenylphosphine)palladium(0) and 0.5 mmol triphenylphosphine for 5 h while bubbling with argon. The resin was washed with CHCl3 and then with NMP and subsequently coupled with a mixture of 4 mmol 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid, 4 mmol HOAt, and 4 mmol DIC for 2 days. After coupling, the resin washed with ethanol and dried.
The dry resin was treated with a mixture of 5% thioanisol and 5% ethanol in TFA for 2 h. The TFA solution was concentrated be stream of argon and precipitated in diethyl ether, washed four times with diethyl ether and lyophilized in 5% AcOH.
Yield 2.2 g. MALDI-TOF MS analysis: found: m/z=1221.4; calculated: m/z=1221.
2 gram of Fmoc-Rink amide AM resin (Novabiochem) (1.14 mmol) was coupled with Fmoc-Lys(alloc)-OH/HOBt/HOAt/DIC (each 3 mmol) in NMP and subsequently capped with 4 mmol AcOH/HOBt/960 μl DIC for 1 h using the method described above.
A mixture of Fmoc-Lys(IvDde)-OH, HOAt, and DIC (each 2 mmol) was coupled to the resin and allowed to stand overnight. The resin was then capped with 4 mmol AcOH/HOBt/DIC for 30 min, washed/deprotected as described above and coupled with Fmoc-Lys(Fmoc-)—OH (3 mmol) activated with HOBt, and DIC (each 3 mmol) for ca. 3 h. The resin was then capped with 4 mmol AcOH, 3 mmol HOBt+3 mmol DIC for ca. 30 min.
Then the resin was subsequently deprotected with 25% piperidine, 4% hydrazine, 2% allyl alcohol for 20 min. The resin was washed and coupled with a mixture of Fmoc-Lys(Fmoc)-OH (Novabiochem), HOBt, and DIC (each 5 mmol) overnight, followed by deprotection with 25% piperidine in NMP for 30 min. Subsequently, the resin was coupled with Fmoc-Lys(Fmoc)-OH, HOBt, and DIC (each 10 mmol) overnight followed by capping with AcOH, HOBt, and DIC (each 6 mmol) in NMP for 30 min. The resin was washed with NMP followed by wash with CHCl3 and then the Alloc group was removed with 0.3 mmol tetrakis(triphenylphosphine)palladium(0) and 0.5 mmol triphenylphosphine for 4 h, followed by extensive washing with CHCl3 and NMP.
4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid (2 mmol) was subsequently coupled to the resin using 2 mmol HOAt and 2 mmol DIC and allowed to stand overnight.
The resin was washed with NMP then with ethanol and dried. The resin was treated with 30 mL TFA containing 5% thioanisol, and 5% ethanol. TFA was reduced in volume and the peptide was precipitated with diethyl ether and washed four times with diethyl ether.
The resulting peptide was suspended in 5% AcOH and lyophilized resulting in 2.1 g of the trifluoroacetate salt.
MALDI-TOF MS analysis: found m/z=1903, calculated: m/z=1904.
4 gram of a Fmoc-(Arg(Pbf))4-Rink amid (1.12 mmol) resin was swelled in NMP for 1 h, then deprotected with 25% piperidine in NMP for 30 min followed by NMP wash. The resin was coupled with Fmoc-Glu(OH)—OH (0.6 mmol, Bachem), HOAt (1.2 mmol), and DIC (1.2 mmol), for 2 h in NMP. Then another portion of Fmoc-Glu(OH)—OH (0.6 mmol, Bachem), HOAt (1.2 mmol), and DIC (1.2 mmol) was added and allowed to stand overnight. The resin was subsequently capped with AcOH, HOBt, and DIC (each 2 mmol) for 1 h. Then Fmoc was removed with 25% piperidine in NMP for 30 min and 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid, HOAt, and DIC (each 1 mmol) was coupled to the resin overnight. The resin was washed with NMP and ethanol and dried for 3 days.
The resin was treated with 90% TFA, 5% thioanisol, and 5% ethanol for 2 h. After filtration, TFA was concentrated by a stream of argon and the peptide precipitated with diethyl ether. The precipitate was washed five times with diethyl ether and dried. The peptide was dissolved in 5% AcOH and lyophilized, resulting in 87 mg of the title compound.
7 gram Fmoc-Arg6-Rink Amide AM resin (0.22 mmol/g) was swelled in warm NMP (50° C.) then deprotected with 25% piperidine in NMP for 20 min. followed by NMP wash.
Subsequently the resin was coupled with Fmoc-Lys(IvDde)-OH, HOBt, and DIC (each 4 mmol) for 3 days. The resin was then capped with AcOH/HOBt/DIC and washed with NMP.
6 Arginines were subsequently coupled using the standard protocol (I) with the modification that double couplings for 2-4 h were employed. Then the resin was washed and deprotected with 3% hydrazine, 5% piperidine in NMP for 20 min. Coupling with 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid, HOAt, and DIC (each 4 mmol) for 3 days afforded the resin-bound title compound.
Cleavage from the resin was performed with 5% thioanisol and 5% ethanol in TFA. After filtration, the TFA was concentrated to minimum volume and subsequently the peptide was precipitated with diethyl ether, washed three times with diethyl ether and then solubilised in 5% aqueous AcOH, washed twice with diethyl ether and then lyophilized.
Yield of crude product 3.5 g; MS (MALDI-TOF): m/z: 2407 g/mol; calculated: 2412 g/mol.
Dde-Lys(Fmoc)-Arg6-Rink Amide AM resin (0.82 mmol) was swelled in NMP overnight. Deprotection of the Fmoc group was performed with 2% DBU in NMP (20 mL) by shaking for 2 min 4 times followed by NMP wash after the 2nd and 4th treatment.
Subsequently the resin was treated with a mixture of 4-fold excess of Fmoc-Arg(Pbf)-OH; HOAt, and DIC in NMP for 3 h followed by a double coupling with the same mixture overnight. After removal of the coupling mixture, a capping was performed for 1 h using 20 times excess of HOAc/HOBt/DIC in NMP. The resin was then deprotected by use of the standard procedure using NMP/Piperidine/DBU (80/20/2) for 15 min followed by a new deprotection for 3 h. In the same way, 4 more Arg residues were coupled to the resin. Finally an Arg residue was coupled to the resin using Boc-Arg(Pbf)-OH instead of the usual Fmoc protected arginine. After the final coupling, the dde group was removed by use of 2% hydrazine in NMP (20 mL) for 10 min and then for 2 h followed by wash with NMP. Then the resin was coupled with 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid/HOAt/DIC in 4 molar excess for 3 h and an extra coupling for 16 hours followed by capping as described above. The resin was then washed with NMP (×2), DCM (×3), and diethyl ether (×4), and dried overnight. The resulting resin was treated with TFA/thioanisol/ethanol 90/5/5 (100 mL) overnight. The mixture was filtered and the resin was washed with TFA (×2). The resulting combined TFA filtrates were concentrated to 20 mL in vacuo and was slowly poured into cold diethyl ether resulting in a precipitate. This was washed three times with diethyl ether and dried in vacuo and lyophilised, Yield 2.2 g crude product.
MS (MALDI-TOF): m/z: 2371 g/mol; calculated: 2368 g/mol.
Fmoc-protected Rink amide AM resin (NovaBiochem, 0.70 mmol/g, 8.4 g, 5.9 mmol) was used to prepare resin bound Fmoc-(Arg(Pbf))6 by the solid phase peptide synthesis protocol (I). The following amounts were used for each coupling: Fmoc-Arg(Pbf)-OH 15.3 g, HOBt 2.38 g, HOAt 800 mg and DIC 3.61 mL in NMP (30 mL). Capping: AcOH 3.36 mL, HOBt 7.93 g and DIC 12.0 mL in NMP (25 mL). De-Fmoc conditions: As in protocol (I) (below). 1/10 of the resin bound Fmoc-(Arg(Pbf))6 (0.59 mmol) was withdrawn for further synthesis. After Fmoc-removal, it was washed with NMP and drained. A mixture of Fmoc-Lys(Dde)-OH (3 eq, 940 mg)+HOBt (2 eq, 159 mg)+HOAt (1 eq, 80 mg) in NMP was added followed by DIC (270 μl) and the mixture was shaken at room temperature for 16 hours. Synthesis protocol (I) was then used to couple six arginines with the following modification: coupling was repeated (double coupling, second coupling 2-3 h) before capping was performed and Fmoc-removal was carried out as a double deprotection using 10 min+60 min as reaction times. Amounts: Fmoc-Arg(Pbf)-OH 1.53 g, HOBt 238 mg, HOAt 80 mg and DIC 361 μl. Capping: AcOH 336 μl, HOBt 793 mg and DIC (1.20 mL). Fmoc was removed, the resin was washed and the free amine was acylated with Boc2O (5 eq., 654 mg) and DIPEA (5 eq., 510 μl) in NMP. After washing, the Dde group was removed by treatment with 2% hydrazine hydrate in NMP, 3 times, 3 min each. The resin was washed and acylated for 72 h using 5-[6-(5-Cyano-1H-[1,2,3]triazol-4-yl)naphthalen-2-yloxy]pentanoic acid (4 eq, 0.8 g) (prepared as described in example 977)+HOAt (4 eq, 0.32 g)+DIC (360 (I). After washing with NMP and DCM, the resin was dried in a stream of N2.
The product was cleaved from the resin by treatment with 15 mL of a mixture of TFA:water:thioanisol (18:1:1) for 2.5 h. The cleavage mixture was filtered into stirred diethyl ether (75 mL), whereby the product precipitated. After filtration and washing with diethyl ether, the solid was dried in vacuo overnight to obtain the crude title compound as trifluoroacetate salt. Yield 1.51 g.
The crude product (400 mg) was dissolved in 2 mL 0.25 M HCl and purified by reversed phase HPLC using an Agilent Technologies Zorbax 250×21.2 mm column (7 μm, 300 Å particles), a set of two buffers A (0.1% TFA in water) and B (0.1% TFA in acetonitrile:water 9:1), and a gradient of 0.5% B-buffer/min (flow rate 9.5 mL/min). Yield 198 mg of the purified title compound. Analysed on reversed phase HPLC using an Agilent Technologies Zorbax 50×4.6 mm column (3.5 μm, 300 Å) and a set of two buffers A (0.1% TFA in water) and B (0.1% TFA in acetonitrile:water 9:1). Using a gradient of 5% B-buffer/min (flow rate 1 mL/min), the product eluted at 7.73 min. MALDI-TOF MS analysis: found 2336.0 (M+H), calculated 2335.
This compound was prepared, purified and analysed as described in the above example 1016 except that Dde-Lys(Fmoc)-OH was used in place of Fmoc-Lys(Dde)-OH.
RP HPLC analysis: the product eluted at 7.58 min. MALDI-TOF MS analysis: found m/z=2336.2 (M+H), calculated 2335.
Resin bound Fmoc-(Arg(Pbf))6 (3.5 mmol) prepared by the solid phase peptide synthesis protocol (I)) and after Fmoc-removal was coupled with Fmoc-Lys-(IvDde)OH/HOBt/DIC (3 eq each) by double coupling as described above. After deprotection with NMP/Piperidine/DBU (80/20/2) for 1 h, repeated once, the resin was coupled with a mixture of 4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid (prepared as described in example 469) HOAt/DIC (3 eq of each) overnight and a double coupling was carried out for 3 h. Capping was performed as described above. The resin was the deprotected using 3% hydrazine in NMP (40 mL), repeated once. After washing, the synthesis protocol (I) was used to couple six arginines with the following modification: coupling was repeated (double coupling, second coupling 2-3 h) before capping, and Fmoc-removal was carried out using 60 min+60 min as reaction times. After coupling of the last arginine unit, it was deprotected as done in the protocol (I). After wash with DCM and diethyl ether the product was cleaved from the resin by treatment with 280 mL of a mixture of TFA/ethanol/thioanisol (28/1/1) for 2 days. The cleavage mixture was filtered, concentrated to 20 mL in vacuo and with stirring slowly poured into cold diethyl ether (500 mL), whereby the product precipitated. After filtration and washing with diethyl ether, the solid was dried in vacuo overnight to obtain the crude title compound as trifluoroacetate salt. Yield 12.3 g of crude product.
MS (MALDI-TOF): m/z: 2359 g/mol; calculated: 2356 g/mol.
Resin bound Fmoc-(Arg(Pbf))6 (3.5 mmol), prepared by the solid phase peptide synthesis protocol (I), was coupled with Dde-Lys-(Fmoc)OH/HOBt/DIC (5 g/1.6 g/1.65 mL) in NMP (40 mL) by double coupling as described above for 16 h and 3.5 h followed by capping. After deprotection with NMP/Piperidine/DBU (80/20/2) for 10 min, repeated once, the resin was coupled with a mixture of 4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid/HOAt/DIC (5 g/1.9 g/2.2 mL) for 4 h and then repeated with a new coupling mixture overnight. Capping was performed as described above. The resin was the deprotected using NMP/piperidine/hydrazine (160/5/5, 40 mL), repeated once. After washing, the synthesis protocol (I) was used to couple six arginines. double couplings were performed one coupling overnight, the other 3 h followed by one capping, Fmoc-removal was carried out using 60 min+60 min as reaction times as described in protocol (I). The last arginine residue to be coupled was Boc protected instead of Fmoc protected. After the last coupling, the resin was washed with NMP then with DCM and diethyl ether and dried. The product was cleaved from the resin by treatment with a mixture of TFA/ethanol/thioanisol (450/25/25) 500 mL for a period of 1 day. The cleavage mixture was filtered and evaporated to 50 mL in vacuo and with stirring poured slowly into cold diethyl ether (200 mL), whereby the product precipitated. After filtration and washing with diethyl ether, the solid was dried in vacuo overnight to obtain the crude title compound as trifluoroacetate salt. Yield 16.5 g of crude wet product. 1.2 g of this was purified by HPLC resulting in 150 mg pure compound. MS (MALDI-TOF): m/z: 2359 g/mol; calculated: 2356 g/mol.
Resin bound Fmoc-(Arg(Pbf))6 prepared by the solid phase peptide synthesis protocol (I) (2.3 mmol), was coupled with Fmoc-Lys(IvDde)OH/HOBt/DIC (3 eq) by double coupling as described above. After deprotection and washing, the synthesis protocol (I) was used to couple six arginine residues, double couplings were performed, one coupling overnight, the other 3 h followed by one capping. Fmoc-removal was carried out using 60 min+60 min as reaction times. The last arginine residue coupled was Boc protected instead of Fmoc. After deprotectioned using NMP/hydrazine hydrate/piperidine (31/1/1 mL) for 30 min, repeated once for 1 h, the resin was coupled with a mixture of 4-[4-(2,4-dioxothiazolidin-5-ylmethyl)naphthalen-1-yloxy]butyric acid (prepared as described in example 283) HOAt/DIC (3 eq each) overnight and a double coupling was carried out for 3 h. Capping was performed as described above. After wash with DCM and diethyl ether followed by drying overnight giving 12 g of dry resin, the product was cleaved from the resin by treatment with 335 mL of a mixture of TFA/ethanol/thioanisol (300:17:17) overnight. The cleavage mixture was filtered and concentrated in vacuo to 50 mL and with stirring poured into cold diethyl ether (200 mL), whereby the product precipitated. After filtration and washing with diethyl ether, the solid was dried in vacuo overnight to obtain the crude title compound. MS (MALDI-TOF): m/z: 2357.7 g/mol; calculated: 2358 g/mol.
A small portion of the crude product was purified by HPLC resulting in 70 mg title compound as the trifluoroacetate.
This compound was prepared by a slight modification of the general method: 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid (1 equivalent) was coupled to PS—NH(Arg)6-NH2 (prepared by the general method). Purification by HPLC afforded a yield of 10% of the title compound.
MS (MALDI-TOF): m/z: 2369 g/mol; calculated: 2370 g/mol.
This compound was prepared by a slight modification of the general method: 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid (1 equivalent) was coupled to PS—NH(Arg)6-NH2 (prepared by the general method) followed by coupling with Fmoc-ethylendiamine hydrochloride/collidine (3 equivalents) and then by 6 times coupling with Fmoc-L-Arg(Pbf)-OH using the general method. Purification by HPLC afforded the title compound (17% yield).
MS (MALDI-TOF): m/z: 2634 g/mol; calculated: 2636 g/mol.
This product was prepared by deprotection of the compound described in example 1022.
MS (MALDI-TOF): m/z: 2213 g/mol; calculated: 2214 g/mol.
The fully protected peptidyl resin was synthesized according to the Fmoc strategy on an Applied Biosystems 431A peptide synthesizer in 0.25 mmol scale using the manufacturer supplied FastMoc UV protocols which employ 4 equivalents HBTU (2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium hexafluorophosphate) mediated couplings in DMF (N,N-dimethylformamide), and UV monitoring of the deprotection of the Fmoc protection group. The starting resin (0.25 mmol) used for the synthesis was Rink amide AM resin (Novabiochem) with a substitution capacity of 0.65 mmol/g.
The protected amino acid derivatives used were Fmoc-Arg(Pbf)-OH, Boc-Arg(Boc)2-OH, and Fmoc-Lys(Dde)-OH using 4 equivalents pr. coupling.
The acylation with 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid is done on the fully protected resin-bound peptide where only the ε-amino group to be acylated has been deprotected. The appropriately protected resin bound peptide was synthesized using Fmoc chemistry, eg.:
↓ 2% Hydrazine/DMF treatment to remove the Dde group.
↓ Acylation with 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid.
↓ TFA deprotection.
Analysis by LC-MS and analytical HPLC.
To the fully protected peptidyl resin was swelled in NMP (N-methylpyrrolidone) (20 mL) for 30 minutes and filtered, and a freshly prepared solution of hydrazine hydrate 2% in NMP (12 mL) was added to the resin and the mixture was shaken for 10 minutes and drained. More hydrazine hydrate 2% in NMP (20 mL) was added and the mixture was shaken for 20 minutes and drained. The resin was washed with NMP (6×20 mL).
To the Dde deprotected resin was added a solution of HOAt in NMP (1.4 g in 15 mL) followed by 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid (0.365 g, 4 eq), and DIC (diisopropylcarbodiimide) (0.15 mL). The reaction mixture was shaken for 16 hours at room temperature and drained. The resin was washed extensively with NMP (5×20 mL), dichloromethane 6×20 mL), 2-propanol and diethyl ether (2×20 mL).
Cleavage of the Acylated Peptide from the Resin:
The peptide was cleaved from the resin by stirring with a mixture of TFA (trifluoro acetic acid) (15 mL), triisopropylsilane (500 μL) for 16 hours at room temperature. The cleavage mixture was filtered the resin was washed with dichloromethane (8 mL) and drained. The combined filtrates were concentrated to approximately 1 mL by a stream of nitrogen. The crude peptide is precipitated with diethyl ether (50 mL), washed 3 times with diethyl ether (3 times 50 ml) and dried to afford 363 mg crude product.
The crude peptide was dissolved in water (3 mL) and acetic acid glacial (3 mL) (100 ml) adjusted to pH 7.5 with NH4OH and purified by semipreparative HPLC in 3 runs on a Jones Chromasil 15 mm×225 mm column packed with 5μ C-18 silica. The column was eluted with the following gradient: 0-10 minutes: 10% acetonitrile; 10-40 minutes: 10% to 50% acetonitrile, and 40-55 minutes: 50% to 90% acetonitrile against 0.1% TFA/water at 10 ml/min at a temperature of 40° C. The peptide containing fractions were collected and lyophilized. This afforded 34 mg of the title compound.
MALDI-TOF-MS: Found 2528 amu, calculated 2527 amu.
This compound was prepared similarly as described in example 1024 using the following amino acids: Fmoc-Arg(Pbf)-OH, Dde-Lys(Fmoc)-OH, and Boc-Arg(Boc)2-OH.
The resin-bound prepared fully protected peptide was:
Dde-Lys(Boc-Arg(Boc)2-Arg5-yl)-Arg7-NH-Resin; wherein Arg5 and Arg7 means 5, respectively 7 repeating units of Pbf-protected arginines.
↓ Dde-Lys(Boc-Arg(Boc)2-Arg5-yl)-Arg7-NH-Resin
↓ 2% Hydrazine/DMF treatment to remove the Dde group.
↓ Acylation with 4-[3-(1H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid.
↓ TFA deprotection.
Analysis by LC-MS and analytical HPLC.
Yield of crude product: 0.39 g
Yield of pure title compound: 36 mg.
MALDI-TOF-MS: Found 2526 amu; calculated 2527 amu.
This compound was prepared as described in example 1024 using the following modifications:
The resin used was only 0.125 mmol. First, Dde-Lys(Fmoc)-OH was attached, and after removal of the Fmoc-protection, 4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzoic acid was attached (HOAt/DIC, 4 eq.) overnight. After Dde removal (3% hydrazine in NMP, 12 mL, 15 minutes), the resin was washed with NMP (6×20 mL) and transferred to the Applied Biosystems 431A peptide synthesizer. Here, the following amino acids were attached to the resin: 2 cycles of Fmoc-Lys(Fmoc)-OH, and using double couplings 3 cycles of Fmoc-Arg(Pmc)-OH. The resin was treated with piperidine prior to cleavage to remove the terminal Fmoc-groups.
MALDI-TOF-MS: Found 2754 amu; calculated 2755 amu.
For pH-solubility profiles, 0.6 mM human insulin stock solutions containing 0.3 mM Zn2+, 30 mM phenol, 1.6% glycerol and 1.2 mM H-Arg6-Lys(5-[6-(5-Cyano-1H-[1,2,3]triazol-4-yl)naphthalen-2-yloxy]pentanoyl)-Arg6-NH2 (example 1016), 4-[3-(1H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Lys(Arg6-yl)-Arg7-NH2 (example 1025) or 4-[3-(1H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Glu(-Arg6-NH2)-Arg6-NH2 (example 1021) were prepared and the pH was adjusted to the desired value corresponding to the alkaline endpoint of the pH-solubility profile. From these stock solutions samples were withdrawn, the pH adjusted to the desired value in the pH 3-8 range, and 0.3 ml samples were incubated at 23° C. for at least 4 days. After centrifugation (20,000 g for 20 minutes at 23° C.) of each sample, pH was measured and the solubility was determined by quantification of insulin contents in the supernatant by SEC HPLC analysis.
In
The binding affinity of ligands to the metal site of insulin R6 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 10 mL quantum as described below. Buffer is always 50 mM tris buffer adjusted to pH=8.0 with NaOH/ClO4−.
1000 μL of 2.0 mM human insulin in buffer
66.7 μL of 10 mM Zn-acetate in buffer
800 μL of 500 mM phenol in H2O
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 1 below.
The UV/vis spectra resulting from a titration of the compound 3-hydroxy-2-naphthoic acid is shown in
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([ligand]free)=(absmax*[ligand]free)/(KD(obs)+[ligand]free)
The observed dissociation constant is recalculated to obtain the apparent dissociation constant
K
D(app)=KD(obs)/(1+[4H3N]/K4H3N)
The value of K4H3N=50 μM is taken from Huang et al., 1997, Biochemistry 36, 9878-9888.
The binding affinity of ligands to the metal site of insulin R6 hexamers are measured in a fluorescense 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 455 nm upon excitation at 410 nm.
Stock solution: 0.02 mM human insulin, 0.007 mM Zn-acetate, 40 mM phenol, 0.01 mM TZD in 50 mM tris buffer adjusted to pH=8.0 with NaOH/ClO4−.
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.
Fluorescence measurements were carried out on a Perkin Elmer Spectrofluorometer LS50B. 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.
The fluorescence spectra resulting from a titration of the compound 5-(4-dimethylaminobenzylidene)thiazolidine-2,4-dione (TZD) is shown in
This equation is fitted to the datapoints
ΔF(455 nm))=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 KTZD 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05100835.7 | Feb 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/050675 | 2/6/2006 | WO | 00 | 3/11/2008 |
