Novel ligands for the hisb10 zn2+ sites of the r-state insulin hexamer

FIELD OF THE INVENTION

[0001] The present invention discloses novel ligands for the His10 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. The novel preparations release insulin slowly following subcutaneous injection.

BACKGROUND OF THE INVENTION

[0002] Insulin Allostery. The insulin hexamer is an allosteric protein that exhibits both positive and negative cooperativity and half-of-the-sites reactivity in ligand binding. This allosteric behaviour consists of two interrelated allosteric transitions designated LA0 and LB0, three interconverting allosteric conformation states (eq. 1),

\begin{matrix} T_{6} \overset{L_{0}^{A}}{⟷} T_{3} R_{3} \overset{L_{0}^{B}}{⟷} R_{6} & (1) \end{matrix}

[0003] designated T6, T3R3, and R6 and two classes of allosteric ligand binding sites designated as the phenolic pockets and the HisB10 anion sites. These allosteric sites are associated only with insulin subunits in the R conformation.

[0004] Insulin Hexamer Structures and Ligand Binding. The T- to R-transition of the insulin hexamer involves transformation of the first nine residues of the B chain from an extended conformation in the T-state to an α-helical conformation in the R-state. This coil-to-helix transition causes the N-terminal residue, PheB1, to undergo an ˜30 Å change in position. This conformational change creates hydrophobic pockets (the phenolic pockets) at the sub-unit interfaces (three in T3R3, and six in R6), and the new B-chain helices form 3-helix bundles (one in T3R3 and two in R6) with the bundle axis aligned along the hexamer three-fold symmetry axis. The HisB10 Zn2+ in each R3 unit is forced to change coordination geometry from octahedral to either tetrahedral (monodentate ligands) or pentahedran (bidentate ligands). Formation of the helix bundle creates a narrow hydrophobic tunnel in each R3 unit that extends from the surface ˜12 Å down to the HisB10 metal ion. This tunnel and the HisB10 Zn2+ ion form the anion binding site.

[0005] Hexamer Ligand Binding and Stability of Insulin Formulations. The in vivo role of the T to R transition is unknown. However, the addition of allosteric ligands (e.g. phenol and chloride ion) to insulin preparations is widely used. Hexamerization is driven by coordination of Zn2+ at the HisB10 sites to give T6, and the subsequent ligand-mediated transition of T6 to T3R3 and to R6 is known to greatly enhance the physical and chemical stability of the resulting formulations.

[0006] Ligand Binding and Long Acting Insulin Formulations. Although the conversion of T6 to T3R3 and R6 improves the stability of the preparation, the rate of absorption following subcutaneous injection of a soluble hexameric preparation is not much affected by the addition of phenol and cloride.

[0007] Putative events following injection of a soluble hexameric preparation. The small molecule ligands initially diffuse away from the protein. The affinity of the ligands for insulin may help to slow this process. On the other hand, the affinity of Zn2+ for e.g. albumin and the large effective space available for diffusion of the lipophilic phenol will tend to speed up the separation. In about 10-15 minutes after injection, the distribution of insulin species in the subcutaneous tissue will roughly correspond to that of a zinc-free insulin preparation at the same dilution. Then, the equilibrium distribution of species at this point will determine the observed absorption rate. In this regimen, absorption rates vary between about 1 hour (for rapid-acting insulin analogues, such as AspB28 human insulin) and about 4 hours (Co3+-hexamer).

[0008] Current Approaches Toward Slow Acting Insulins. The inherent limitation of the absorption half-life to about 4 hours for a soluble human insulin hexamer necessitates further modifications to obtain the desired protraction. Traditionally, this has been achieved by the use of preparations wherein the constituent insulin is in the form of a crystalline and/or amorphous precipitate. In this type of formulation, the dissolution of the precipitate in the subcutaneous depot becomes rate-limiting for the absorption. NPH and Ultralente belong to this category of insulin preparations where crystallization/precipitation is effected by the addition of protamine and excessive zinc ion, respectively.

[0009] 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.

[0010] 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.

SUMMARY OF THE INVENTION

[0011] The present invention provides novel ligands for the HisB10 Zn2+ sites of the R-state insulin hexamer. The ligands stabilize the hexamers and modify solubility in the neutral range. The resulting preparations release insulin slowly following subcutaneous injection. In comparison with earlier slow release preparations, the present ligands work to modify the timing of both human insulin and insulin mutants/analogues. The ligands alone or in combination with new ligands for the phenol cavity also confer increased physical and chemical stability of the resulting preparations. Moreover, the preparations release active insulin more reproducibly that e.g. NPH preparations.

DEFINITIONS

[0012] The following is a detailed definition of the terms used to describe the invention:

[0013] “Halogen” designates an atom selected from the group consisting of F, Cl, Br and I.

[0014] 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.

[0015] 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.

[0016] 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-pentynyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3hexenyl, 2,4-hexadienyl, 5-hexenyl and the like.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] The term “aryloxy” as used herein denotes a group —O-aryl, wherein aryl is as defined above.

[0025] The term “aroyl” as used herein denotes a group —C(O)-aryl, wherein aryl is as defined above.

[0026] 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.

[0027] 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.

[0028] “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:

[0029] 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.

[0030] Furthermore, when polycyclic structures are substituted with one or more substituents, it is intended that substitutions at any available position in either of the rings that are part of the polycyclic structure are included.

[0031] 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.

[0032] 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.

[0033] The term “treatment” 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.

[0034] The term “fragment” as used herein is intended to mean a bivalent chemical group

[0035] 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.

[0036] The term “α amino acid” as used herein is intended to mean mean any natural (codable) and non-natural α-aminocarboxylic acid, including D-isomers of these.

[0037] The term “β amino acid” as used herein is intended to mean any β-aminocarboxylic acid, such as β-alanine, isoserine or the like.

[0038] 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.

[0039] 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 expression “insulin derivative” as used herein (and related expressions) refers to human insulin or an analogue thereof in which at least one organic substituent is bound to one or more of the amino acids.

[0040] By “analogue of human insulin” as used herein (and related expressions) is meant human insulin in which one or more amino acids have been deleted and/or replaced by other amino acids, including non-codeable amino acids, or human insulin comprising additional amino acids, i.e. more than 51 amino acids,such that the resulting analogue possesses insulin activity.

[0041] 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.

[0042] The term “physiologically relevant pH” as used herein is intended to mean a pH of about 7.1 to 7.9.

[0043] When calculating the ratio between precipitated and dissolved insulin in dual-acting insulin composition, i.e. a composition containing both rapid-acting insulin and insulin with a prolonged action, the term “precipitated insulin” as used herein is intended to mean insulin monomer which is part of a hexamer to which a ligand of the present invention is bound at physiologically relevant pH as defined above. Similarly the term “dissolved insulin” as used herein is intended to mean insulin which is not precipitated as defined above.

1Abbreviations:4H3N4-hydroxy-3-nitrobenzoic acidAbzAminobenzoic acidAcOHacetic acidBTBenzotriazol-5-oylDMFN,N-DimethylformamideDMSODimethylsulfoxideDICDiisopropylcarbodiimideEDAC1-ethyl-3-(3′-dimethylamino-propyl)carbodiimide, hydrochlorideFmoc9H-Fluorene-9-ylmethoxycarbonylG, GlyGlycineHOAt1-hydroxy-7-azabenzotriazoleHOBT1-HydroxybenzotriazoleL, LysLysineNMPN-methyl-2-pyrrolidonePbf2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonylPmc2,2,5,7,8-pentamethylchroman-6-sulfonylR, ArgArginineTFATrifluoroacetic acid

[0044] Abbreviations for non-natural amino acid residues:

BRIEF DESCRIPTION OF DRAWINGS

[0045]
FIG. 1: Effect of BTG2R5—NH2 on pH-solubility profile of an insulin preparation.

[0046]
FIG. 2: Effect of BTG2R4—NH2 on the pH-solubility profile of an insulin preparation.

[0047]
FIG. 3: Disappearance from the subcutaneous depot (pig model) of insulin preparations in the presence of BT—AbzG2R5—NH2 with phenol and 7-hydroxy indole (a-b); and BT—G2R5—NH2 and BT—G2R4 with phenol (c-d). The bottom panels (e-f) show slow- and dual release profiles, respectively, obtained from AspB28 human insulin formulated with variable concentration of TZD—Abz—G2R5

[0048]
FIG. 4: 4H3N-assay. UV/vis spectra resulting from a titration of hexameric insulin with the compound 3-hydroxy-2-naphthoic acid in the presence of 4-hydroxy-3-nitrobenzoic acid (4H3N). Inserted in the upper right corner is the absorbance at 444 nm vs. the concentration of ligand

[0049]
FIG. 5: TZD-assay. Fluorescence spectra resulting from a titration of hexameric insulin with 5-(3-methoxybenzylidene)thiazolidine-2,4-dione in the presence of 5-(4-dimethylamino-benzylidene)thiazolidine-2,4-dione (TZD). Inserted in the upper right corner is the fluorescence at 460 nm vs. the concentration of ligand

DESCRIPTION OF THE INVENTION

[0050] The present invention is based on the discovery that the two known 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, e.g. human Insulin or AspB28 human insulin.

[0051] The basic concept underlying the present invention involves reversible attachment of a ligand to the HisB10 Zn2+ site of the R-state hexamer. A suitable ligand binds to the hexamer metal site with one end while other moieties are covalently attachment 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.

[0052] 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.

[0053] Most ligand binding sites in proteins are highly asymmetric. Because the HisB10 Zn2+ 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.

[0054] The HisB10 Zn2+ 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 HisB10 Zn2+ 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.

[0055] The present invention originates from a search for compounds with suitable binding properties by using novel 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 from the initial search of compound databases.

[0056] These starter compounds provide the starting point for the task of constructing a chemical handle that allows for attachment of the positively charged fragment D (see below).

[0057] 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.

[0058] 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.

[0059] 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

[0060] The zinc-binding ligands of the present invention are characterised by the following formula (I):

A—B—C—D—X (I)

[0061] wherein:

[0062] A is a functionality capable of reversibly coordinating to a HisB10 Zn2+ site of an insulin hexamer;

[0063] B is a valence bond or a non-naturally occurring amino acid residue containing an aromatic ring;

[0064] C is a valence bond or a fragment consisting of 1 to 5 neutral α- or β-amino acids;

[0065] D is a fragment containing 1 to 20 positively charged groups independently selected from amino or guanidino groups, preferably a fragment consisting of 1 to 20 basic amino acids independently selected from the group consisting of Lys and Arg and D-isomers of these; and

[0066] X is OH, NH2 or a diamino group.

[0067] The length of the zinc-binding ligand should be such that it extends from the HisB10 Zn2+ site to beyond the hexamer surface.

[0068] A is preferably a chemical structure selected from the group consisting of carboxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles, benzimidazoles, benzotriazoles, purines, thiazolidinediones, naphthoic acids and salicylic acids.

[0069] More preferably, A comprises a benzotriazole, a 3-hydroxy 2-napthoic acid, a salicylic acid, a tetrazole or a thiazolidinedione structure.

[0070] A is is advantageously selected from one of the following chemical structures:

[0071] wherein

[0072] R1 is hydrogen, fluoro, chloro, bromo or iodo,

[0073] m is 0 or 1.

[0074] B is preferably a valence bond or one of the following amino acid residues:

[0075] C is preferably a valence bond or a fragment consisting of 1 to 5 amino acids independently selected from the group consisting of neutral amino acids, more preferably from the group of amino acids consisting of Gly, Ala, Thr, and Ser.

[0076] In a particular preferred embodiment, C consists of 1-5 Gly residues or 1-5 Ala residues. D preferably consists of 1-10 Arg residues.

[0077] X is preferably OH, NH2 or

[0078] The most preferred specific zinc-binding ligands of the present invention are:

[0079] Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-NH2

[0080] Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2

[0081] Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-NH2

[0082] Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-NH2

[0083] Benzotriazol-5-ylcarbonyl-Gly-Arg-Arg-Arg-Arg-Arg-NH2

[0084] Benzotriazol-5-ylcarbonyl-Gly-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2

[0085] Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-NH2

[0086] Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2

[0087] Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-NH2

[0088] Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-NH2

[0089] Benzotriazol-5-ylcarbonyl-4-Abz-Arg-Arg-Arg-Arg-Arg-NH2

[0090] Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2

[0091] Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-Arg-NH2

[0092] Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-NH2

[0093] Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-Arg-Arg-N H2

[0094] Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-Arg-NH2

[0095] Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-NH2

[0096] [4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2

[0097] [3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2

[0098] 4-(2H-Tetrazol-5-yl)benzoyl-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2

[0099] In another embodiment the invention provides a zinc-binding ligand of the following general formula (II)

A—B—C—D—X (II)

[0100] wherein:

[0101] A is a chemical group which reversibly binds to a HisB10 Zn2+ site of an insulin hexamer;

[0102] B is a linker selected from

[0103] A valence bond

[0104] A chemical group GB of the formula —B1—B2—C(O)—, —B1—B2—SO2—, —B1—B2—CH2—, or —B1—B2—NH—; wherein B1 is a valence bond, —O—, —S—, or —NR6—;

[0105] B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C-1-C18-alkyl-aryl-, —C2-C18-alkenyl-aryl-, —C2-C18-alkynyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O), —C(═O)—C1-C18-alkenyl-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)—, —C(═O)-aryl-C(═O)—, —C(═O)-heteroaryl-C(═O)—;

[0106] wherein the alkylene, alkenylene, and alkynyl enemoieties are optionally substituted by —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene and heteroarylene moieties are optionally substituted by halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl;

[0107] R6 and R7 are independently H, C1-C4-alkyl;

[0108] C is a fragment consisting of 1 to 5 neutral α- or β-amino acids

[0109] D is a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidine groups; and

[0110] X is —OH, —NH2 or a diamino group,

[0111] or a salt thereof with a pharmaceutically acceptable acid or base, or any optical isomer or mixture of optical isomers, including a racemic mixture, or any tautomeric forms.

[0112] In another embodiment A 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.

[0113] In another embodiment A is a chemical structure selected from the group consisting of benzotriazoles, 3-hydroxy 2-napthoic acids, salicylic acids, tetrazoles or thiazolidinediones

[0114] In another embodiment A is one of the following structures:

[0115] wherein

[0116] X is ═O, ═S or ═NH

[0117] Y is —S—, —O— or —NH—

[0118] R8 and R11 are independently hydrogen or C1-C6-alkyl,

[0119] R9 is hydrogen or C1-C6-alkyl or aryl, R8 and R9 may optionally be combined to form a double bond,

[0120] R10 and R12 are independently hydrogen, aryl, C1-C6-alkyl, or —C(O)NR16R17

[0121] E and G are independently 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 halogen, —CN, —CF3, —OCF3, aryl, —COOH and —NH2, and the arylene or heteroarylene is optionally substituted with up to three substituents R13, R14 and R15.

[0122] E and R10 may be connected through one or two valence bonds, G and R12 may be connected through one or two valence bonds;

[0123] R13, R14 and R15 are independently selected from

[0124] hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —OS(O)2CF3, —SCF3, —NO2, —OR6, —NR16R17, —SR16, —NR 16S(O)2R17, —S(O)2NR16R17, —S(O)NR16R17, —S(O)R16, —S(O)2R16, —OS(O)2R16, —C(O)NR16R17, —OC(O)NR16R17, —NR16C(O)R17, —CH2C(O)NR16R17, —OC1-C6-alkyl-C(O)NR16R17, —CH2OR16, —CH2OC(O)R16, —CH2NR16R17, —OC(O)R16, —OC1-C6-alkyl-C(O)OR16, —OC1-C6-alkyl-OR16, —SC1-C6-alkyl-C(O)OR16, —C2-C6-alkenyl-C(═O)OR16, —NR16-C(═O)—C1-C6-alkyl-C(═O)OR16, —NR16—C(═O)—C1-C16-alkenyl-C(═)OR16 , —C(═O)OR16, or —C2-C6-alkenyl-C(═O)R16,

[0125] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0126] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17

[0127] aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0128] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR16, —CH2C(O)OR16, —CH2OR16, —CN, —CF3, —OCF3, —NO2, —OR16, —NR16R17 and C1-C6-alkyl,

[0129] R16 and R17 independently are hydrogen, OH, C1-C6-alkyl, aryl-C1-C6-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OC1-C6-alkyl, —C(O)OC1-C6-alkyl, —COOH and —NH2, and the aryl groups may optionally be substituted by halogen, —C(O)OC1-C6-alkyl, —COOH, —CN, —CF3, —OCF3, —NO2, —OH, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl; R16 and R17 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

[0130] In another embodiment X is ═O or ═S

[0131] In another embodiment X is ═O

[0132] In another embodiment X is ═S

[0133] In another embodiment Y is —O— or —S—

[0134] In another embodiment Y is —O—

[0135] In another embodiment Y is —S—

[0136] In another embodiment E is arylene optionally substituted with up to three substituents R13, R14 and R15.

[0137] In another embodiment E is phenylene or naphtylene optionally substituted with up to three substituents R13, R14 and R15.

[0138] In another embodiment E is heteroarylene optionally substituted with up to three substituents R13, R14 and 15.

[0139] In another embodiment E is indolylene optionally substituted with up to three substituents R13, R14 and R15.

[0140] In another embodiment R8 is hydrogen.

[0141] In another embodiment R9 is hydrogen.

[0142] In another embodiment R8 and R9 are combined to form a double bond.

[0143] In another embodiment R10 is C1-C6-alkyl.

[0144] In another embodiment R10 is methyl.

[0145] In another embodiment G is phenylene optionally substituted with up to three substituents R13, R14 and R15.

[0146] In another embodiment R11 is hydrogen.

[0147] In another embodiment R12 is hydrogen.

[0148] In another embodiment R13, R14 and R15 are independently selected from

[0149] hydrogen, halogen, —NO2, —OR6, —NR16R17, —SR16, —NR16S(O)2R17, —S(O)2NR16R17, —S(O)NR16R17, —S(O)R16, —S(O)2R16, —OS(O)2R16, —NR16C(O)R17, —CH2OR16, —CH2OC(O)R16, —CH2NR16R17, —OC1-C6-alkyl-C(O)OR16, —OC1-C6-alkyl-C(O)NR16R17, —OC1-C6-alkyl-OR16, —SC1-C6-alkyl-C(O)OR16, —C2-C6-alkenyl-C(═O)OR16, or —C2-C6-alkenyl-C(═O)R16,

[0150] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0151] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17

[0152] aryl, aryloxy, aroyl, arylsulfanyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0153] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR16, —CH2C(O)OR16, —CH2OR16, —CN, —CF3, —OCF3, —NO2, —OR16, —NR16R17 and C1-C6-alkyl.

[0154] In another embodiment R13, R14and R15 are independently selected from

[0155] hydrogen, halogen, —NO2, —OR6, —NR16R17, —SR16, —S(O)2R16, —OS(O)2 R16, —CH2OC(O)R16, —OC(O)R16, —OC1-C6-alkyl-C(O)OR16, —OC1-C6-alkyl-OR16, —SC1-C6-alkyl-C(O)R16, —C(O)OR16, or —C2-C6-alkenyl-C(═O)R16,

[0156] C1-C6-alkyl or C1-C6-alkenyl which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17

[0157] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl,

[0158] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR16, —CH2C(O)OR16, —CH2OR16, —CN, —CF3, —OCF3, —NO2, —OR16, —NR16R17 and C1-C6-alkyl.

[0159] In another embodiment R13, R14 and R15 are independently selected from

[0160] hydrogen, halogen, —NO2, —OR6, —NR16R17, —SR16, —S(O)2R16, —OS(O)2R16, —CH2OC(O)R16, —OC(O)R16, —OC1-C6-alkyl-C(O)OR16, —OC1-C6-alkyl-OR16, —SC1-C6-alkyl-C(O)OR16, —C(O)OR16, or —C2-C6-alkenyl-C(═O)R16,

[0161] C1-C6-alkyl or C1-C6-alkenyl which may optionally be substituted with one or more substituents selected from halogen, —CF3, —OR16, and —NR16R17

[0162] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl,

[0163] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, C(O)OR16, —CN, —NO2, —OR16, —NR16R17 and C1-C6-alkyl.

[0164] In another embodiment R13, R14 and R15 are independently selected from

[0165] hydrogen, halogen, —OR16, —OC1-C6-alkyl-C(O)OR16, or —C(O)OR16,

[0166] C1-C6-alkyl which may optionally be substituted with one or more substituents selected from halogen, —OR16, and —NR16R17

[0167] aryl, aryloxy, aryl-C1-C6-alkoxy,

[0168] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, C(O)OR16, OR16, and C1-C6-alkyl.

[0169] In another embodiment R16 and R17 independently are hydrogen, C1-C6-alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, —CF3, —OCF3, —OC1-C6-alkyl, —COOH and —NH2, and the aryl groups may optionally be substituted by halogen, —COOH, —CN, —CF3, —OCF3, —NO2, —OH, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl; R16 and R17 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

[0170] In another embodiment R16 and R17 independently are hydrogen, C1-C6-alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, —CF3, —OC1-C6-alkyl, —COOH and —NH2, and the aryl groups may optionally be substituted by halogen, —COOH, —CN, —CF3, —OCF3, —OH, —NH2, or C1-C6-alkyl.

[0171] In another embodiment A is one of the following structures

[0172] wherein

[0173] R20 is hydrogen or C1-C6-alkyl,

[0174] R21 is hydrogen or C1-C6-alkyl,

[0175] U and V are a valence bond or C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl independently,

[0176] J is C1-C6-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R22, R23 and R24,

[0177] L is C1-C6-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R25, R26 and R27,

[0178] R18, R19, R22, R23, R24, R25, R26 and R27 are independently selected from

[0179] hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —SCF3, —NO2, —OR28, —NR28R29, —SR28, —NR28S(O)2R29, —S(O)2NR28R29, —S(O)NR28R29, —S(O)R28, —S(O)2R28, —C(O)NR28R29, —OC(O)NR28R29, —NR28C(O)R29, —NR28C(O)OR29, —CH2C(O)NR28R29, —OCH2C(O)NR28R29, —CH2OR28, —CH2NR28R29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —NR28-C(═O)—C1-C6-alkyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0180] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0181] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0182] aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0183] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl,

[0184] R28 and R29 independently are hydrogen, C1-C6-alkyl, aryl-C1-C6-alkyl or aryl, or R28 and R29 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds

[0185] In another embodiment U is a valence bond

[0186] In another embodiment U is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl

[0187] In another embodiment J is arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R22, R23 and R24

[0188] In another embodiment J is arylene optionally substituted with up to three substituents R22, R23 and R24

[0189] In another embodiment J is phenylene optionally substituted with up to three substituents R22, R23 and R24

[0190] In another embodiment R22, R23 and R24 are independently selected from

[0191] hydrogen, halogen, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —SCF3, —NO2, —OR28, —NR28R29, —SR28, —C(O)NR28R29, —OC(O)NR28R29, —NR28C(O)R29, —NR28C(O)OR29, —CH2C(O)NR28R29, —OCH2C(O)NR28R29, —CH2OR28, —CH2NR28R29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkenyl-C(═O)OR28—, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0192] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0193] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0194] aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0195] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0196] In another embodiment R22, R23 and R24 are independently selected from

[0197] hydrogen, halogen, —OCF3, —OR28, —NR28R29, —SR28, —NR28C(O)R29, —NR28C(O)OR29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0198] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0199] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0200] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0201] In another embodiment R22, R23 and R24 are independently selected from

[0202] hydrogen, halogen, —OCF3, —OR28, —NR28R29, —SR28, —NR28C(O)R29, —NR28C(O)OR29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0203] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, or —CF3

[0204] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0205] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OH, —CN, —CF3, —NO2, or C1-C6-alkyl

[0206] In another embodiment R20 is hydrogen or methyl

[0207] In another embodiment R20 is hydrogen

[0208] In another embodiment R28 is hydrogen, C1-C6-alkyl or aryl

[0209] In another embodiment R28 is hydrogen or C1-C6-alkyl

[0210] In another embodiment R29 is hydrogen or C1-C6-alkyl

[0211] In another embodiment V is a valence bond

[0212] In another embodiment V is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl

[0213] In another embodiment L is C1-C6-alkylene or arylene, wherein the arylene is optionally substituted with up to three substituents R25, R26 and R27

[0214] In another embodiment L is C1-C6-alkyl

[0215] In another embodiment L is phenylene optionally substituted with up to three substituents R25, R26 and R27

[0216] In another embodiment R25, R26 and R27 are independently selected from

[0217] hydrogen, halogen, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —SCF3, —NO2, —OR28, —NR28R29, —SR28, —C(O)NR28R29, —OC(O)NR28R29, —NR28C(O)R29, —NR28C(O)OR29, —CH2C(O)NR28R29, —OCH2C(O)NR28R29, —CH2OR28, —CH2NR28R29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkenyl-C(═O)OR28—, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0218] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0219] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0220] aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0221] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0222] In another embodiment R25, R26 and R27 are independently selected from

[0223] hydrogen, halogen, —OCF3, —OR28, —NR28R29, —SR28, —NR28C(O)R29, —NR28C(O)OR29, —OC(O)R28, —OC1-C6-alkyl-C(O)R28, —SC1-C6-alkenyl-C(O)OR28, C2-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0224] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0225] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0226] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0227] In anther embodiment R25, R26 and R27 are independently selected from

[0228] hydrogen, halogen, —OCF3, —OR28, —NR28R29, —SR28, —NR28C(O)R29, —NR28C(O)OR29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0229] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, or —CF3

[0230] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0231] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OH, —CN, —CF3, —NO2, or C1-C6-alkyl

[0232] In another embodiment R21 is hydrogen or methyl

[0233] In another embodiment R21 is hydrogen

[0234] In another embodiment R28 is Hydrogen, C1-C6-alkyl or aryl

[0235] In another embodiment R28 is Hydrogen or C1-C6-alkyl

[0236] In another embodiment R29 is Hydrogen or C1-C6-alkyl

[0237] In another embodiment R18 and R19 are independently selected from

[0238] hydrogen, halogen, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29, —SR28, —S(O)R28, —S(O)2R28, —C(O)NR28R29, —CH2OR28, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, or —C(O)OR28.

[0239] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0240] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0241] aryl, aryloxy, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0242] In another embodiment R18 and R19 are independently selected from

[0243] hydrogen, halogen, —CN, —CF3, —NO2, —OR28, —NR28R29, or —C(O)OR28

[0244] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0245] aryl, aryloxy, aryl-C1-C6-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0246] In another embodiment A is a compound of the form M—Q—T—

[0247] wherein M is one of the following structures

[0248] wherein W1, W2, and W3 are independently OH, SH or NH2 and the phenyl, naphthalene or benzocarbazole rings are optionally substituted by one or more R34 independently

[0249] Q is selected from the following:

[0250] a valence bond

[0251] —CH2N(R30 )— or —SO2N(R31)—

[0252] A compound of the formula

[0253] wherein Z1 is S(O)2 or CH2, Z2 is N, —O— or —S—, and n is 1 or 2;

[0254] T is

[0255] A valence bond

[0256] C1-C6-alkylene, C2-C6-alkenylene or C2-C6-alkynylene, which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR32, and —NR32R33

[0257] Arylene, -aryloxy-, -aryloxycarbonyl-, -aroyl-, -aryl-C1-C6-alkoxy-, -aryl-C1-C6-alkyl-, -aryl-C2-C6-alkenyl-, -aryl-C2-C6-alkynyl-, heteroarylene, -heteroaryl-C1-C6-alkyl-, -heteroaryl-C2-C6-alkenyl- or -heteroaryl-C2-C6-alkynyl-, wherein the cyclic moieties are optionally substituted by one or more substituents selected from halogen, —C(O)OR32, —C(O)H, —CN, —CF3, —OCF3, —NO2, —OR32R33, C1-C6-alkyl or C1-C6-alkanoyl,

[0258] R32 and R33 independently are hydrogen, C1-C6-alkyl, aryl-C1-C6-alkyl or aryl, or R32 and R33 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds, R30 and R31 are independently hydrogen, C1-C6-alkyl or C1-C6-alkanoyl.

[0259] R34 is hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —SCF3, —NO2, —OR32, —C(O)R32, —NR32R33, —SR32, —NR32S(O)2R33, —S(O)2NR32R33, —S(O)NR32R33 —S(O)R32, —S(O)2R32, —C(O)NR32R33, —OC(O)NR32R33, —NR32C(O)R33, —CH2C(O)NR32R33, —OCH2C(O)NR32R33, —CH2OR32, —CH2NR32R33, —OC(O)R32, —OC1-C6-alkyl-C(O)OR32, —SC1-C6-alkyl-C(O)OR32—C2-C6-alkenyl-C(═O)OR32, —NR32—C(═O)—C1-C6-alkyl-C(═O)OR32, —NR32-C(═O)-C1-C6-alkenyl-C(═O)OR32—, C1-C6-alkyl, C1-C6-alkanoyl or —C(O)OR32,

[0260] In another embodiment M is one of the following structures

[0261] In another embodiment M is

[0262] In another embodiment M is

[0263] In another embodiment the salicylic acid moiety is of the formula

[0264] In another embodiment the napthoic acid moiety is of the formula

[0265] In another embodiment Q is a valence bond, —CH2N(R30)—, or —SO2N(R31)—

[0266] In another embodiment Q is a valence bond

[0267] In another embodiment T is

[0268] A valence bond

[0269] C1-C6-alkylene, C2-C6-alkenylene or C2-C6-alkynylene,

[0270] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR32, and —NR32R33

[0271] Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70

[0272] In another embodiment T is

[0273] A valence bond

[0274] Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70

[0275] In another embodiment T is phenylene or naphthalene

[0276] In another embodiment the cyclic moiety in T is optionally substituted by halogen, —C(O)OR32, —CN, —CF3, —OR32, —NR32R33, C1-C6-alkyl or C1-C6-alkanoyl

[0277] In another embodiment the cyclic moiety in T is optionally substituted by halogen, —C(O)OR32, —OR32, —NR32R33, C1-C6-alkyl or C1-C6-alkanoyl

[0278] In another embodiment the cyclic moiety in T is optionally substituted by halogen, —C(O)OR32 or —OR32

[0279] In another embodiment T is a valence bond

[0280] In another embodiment R30 and R31 are independently hydrogen or C1-C6-alkyl

[0281] In another embodiment R34 is hydrogen, halogen, —CN, —CF3, —OCF3, —SCF3, —NO2, —OR32, —C(O)R32, —NR32R33, —SR32, —C(O)NR32R33, —OC(O)NR32R33, —NR32C(O)R33, —OC(O)R32, —OC1-C6-alkyl-C(O)OR32, —SC1-C6-alkyl-C(O)OR32 or —C(O)OR32

[0282] In another embodiment R34 is hydrogen, halogen, —CF3, —NO2, —OR32, —NR32R33, —SR32, —NR32C(O)R33, or —C(O)OR32

[0283] In another embodiment R34 is hydrogen, halogen, —CF3, —NO2, —OR32, —NR32R33, or —NR32C(O)R33

[0284] In another embodiment R34 is hydrogen, halogen, or —OR32

[0285] In another embodiment R32 and R33 independently are hydrogen, C1-C6-alkyl, or aryl

[0286] In another embodiment R32 and R33 independently are hydrogen or C1-C6-alkyl

[0287] In another embodiment C consists of 1-5 neutral amino acids independently selected from the group consisting of Gly, Ala, Thr, and Ser

[0288] In another embodiment C consists of 1-5 Gly

[0289] In another embodiment GB is of the formula —B1—B2—C(O)—, —B1—B2—SO2— or —B1—B2—CH2—, wherein B1 and B2 are as defined above

[0290] In another embodiment GB is of the formula —B1—B2—C(O)—, —B1—B2—SO2— or —B1—B2—NH—, wherein B1 and B2 are as defined above

[0291] In another embodiment GB is of the formula —B1—B2—C(O)—, —B1—B2—CH2— or —B1—B2—NH—, wherein B1 and B2 are as defined above

[0292] In another embodiment GB is of the formula —B1—B2—CH2—, —B1—B2—SO2— or —B1—B2—NH—, wherein B1 and B2 are as defined above

[0293] In another embodiment GB is of the formula —B1—B2—C(O)— or —B1—B2—SO2—, wherein B1 and B2 are as defined above

[0294] In another embodiment GB is of the formula —B1—B2—C(O)— or —B1—B2—CH2—, wherein B1 and B2 are as defined above

[0295] In another embodiment GB is of the formula —B1—B2—C(O)— or —B1—B2—NH—, wherein B1 and B2 are as defined above

[0296] In another embodiment GB is of the formula —B1—B2—CH2— or —B1—B2—SO2—, wherein B1and B2 are as defined above

[0297] In another embodiment GB is of the formula —B1—B2—NH— or —B1—B2—SO2—, wherein B1 and B2 are as defined above

[0298] In another embodiment GB is of the formula —B1—B2—CH2— or —B1—B2—NH—, wherein B1 and B2 are as defined above

[0299] In another embodiment GB is of the formula —B1—B2—C(O)—

[0300] In another embodiment GB is of the formula —B1—B2—CH2—

[0301] In another embodiment GB is of the formula —B1—B2—SO2—

[0302] In another embodiment GB is of the formula —B1—B2—NH—

[0303] In another embodiment B1 is a valence bond, —O—, or —S—

[0304] In another embodiment B1 is a valence bond, —O—, or —N(R6)—

[0305] In another embodiment B1 is a valence bond, —S—, or —N(R6)—

[0306] In another embodiment B1 is —O—, —S— or —N(R6)—

[0307] In another embodiment B1 is a valence bond or —O—

[0308] In another embodiment B1 is a valence bond or —S—

[0309] In another embodiment B1 is a valence bond or —N(R6)—

[0310] In another embodiment B1 is —O— or —S—

[0311] In another embodiment B1 is —O— or —N(R6)—

[0312] In another embodiment B1 is —S— or —N(R6)—

[0313] In another embodiment B1 is a valence bond

[0314] In another embodiment B1 is —O—

[0315] In another embodiment B1 is —S—

[0316] In another embodiment B1 is —N(R6)—

[0317] 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-C18alkyl-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 above

[0318] 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 alkyl and aryl moieties are optionally substituted as defined above

[0319] 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 above

[0320] 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 above

[0321] 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 above

[0322] 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 above

[0323] In another embodiment B2 is a valence bond or C1-C18-alkylene, and the alkylene moiety is optionally substituted as defined above

[0324] In another embodiment D comprises 1 to 16 positively charged groups

[0325] In another embodiment D comprises 1 to 12 positively charged groups

[0326] In another embodiment D comprises 1 to 10 positively charged groups

[0327] In another embodiment D is a fragment containing basic amino acids independently selected from the group consisting of Lys and Arg and D-isomers of these.

[0328] In another embodiment the basic amino acid is Arg

[0329] In another embodiment X is —OH or —NH2

[0330] In another embodiment X is —NH2

[0331] Also provided by the present invention is an R-state insulin hexamer comprising: 6 molecules of insulin, at least 2 zinc ions, and a zinc-binding ligand according to any one of the preceding claims.

[0332] In one embodiment the insulin forming the R-state insulin hexamer is selected from the group consisting of human insulin, an analogue thereof, a derivative thereof, and combinations of any of these

[0333] In another embodiment the insulin is an analogue of human insulin selected from the group consisting of

[0334] i.An analogue wherein position B28 is Asp, Lys, Leu, Val, or Ala and position B29 is Lys or Pro; and

[0335] ii.des(B28-B30), des(B27) or des(B30) human insulin.

[0336] 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.

[0337] In another embodiment the insulin is des(B30) human insulin.

[0338] In another embodiment the insulin is a derivative of human insulin having one or more lipophilic substituents.

[0339] 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.

[0340] In another embodiment the insulin derivative is B29-Nε-myristoyl-des(B30) human insulin.

[0341] In another embodiment the insulin hexamer of the invention further comprises at least 3 phenolic molecules.

[0342] In another embodiment the invention provides an insulin preparation comprising R-state insulin hexamers as defined above

[0343] In another embodiment the invention provides a method of prolonging the action of an insulin preparation which comprises adding a zinc-binding ligand as defined above to the insulin preparation.

[0344] In another embodiment the invention provides an aqueous insulin preparation as defined above wherein the ratio between precipitated insulin and dissolved insulin is in the range from 99:1 to 1:99.

[0345] In another embodiment the ratio between precipitated insulin and dissolved insulin is in the range from 95:5 to 5:95

[0346] In another embodiment the ratio between precipitated insulin and dissolved insulin is in the range from 80:20 to 20:80

[0347] In another embodiment the ratio between precipitated insulin and dissolved insulin is in the range from 70:30 to 30:70.

[0348] In another embodiment the invention provides a zinc-binding ligand of the following general formula (III)

A—B—C—D—X (III)

[0349] wherein:

[0350] A is a chemical group which reversibly binds to a HisB10 Zn2+ site of an insulin hexamer;

[0351] B is a linker selected from

[0352] A valence bond

[0353] A chemical group GB of the formula —B1—B2—C(O)—, —B1—B2—SO2—, —B1—B2—CH2—, or —B1—B2—NH—; wherein B1 is a valence bond, —O—, —S—, or —NR6—,

[0354] B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C2-C18-alkenyl-aryl-, —C2-C18-alkynyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkenyl-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), —C(═O)aryl-C(═O)—, —C(═O)-heteroaryl-C(═O)—;

[0355] wherein the alkylene, alkenylene, and alkynylene moieties are optionally substituted by —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene and heteroarylene moieties are optionally substituted by halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl;

[0356] R6 and R7 are independently H, C1-C4-alkyl;

[0357] C is a fragment consisting of 0 to 5 neutral amino acids, wherein the individual neutral amino acids are the same or different

[0358] D is a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidino groups, wherein the individual positively charged groups are the same or different; and

[0359] X is —OH, —NH2 or a diamino group,

[0360] or a salt thereof with a pharmaceutically acceptable acid or base, or any optical isomer or mixture of optical isomers, including a racemic mixture, or any tautomeric forms.

[0361] In another embodiment of the invention A 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.

[0362] In another embodiment of the invention A is a chemical structure selected from the group consisting of benzotriazoles, 3-hydroxy 2-napthoic acids, salicylic acids, tetrazoles, thiazolidinediones, 5-mercaptotetrazoles, or 4-cyano-1,2,3-triazoles.

[0363] In another embodiment of the invention A is

[0364] wherein

[0365] X is ═O, ═S or ═NH

[0366] Y is —S—, —O— or —NH—

[0367] R8 and R11 are independently hydrogen or C1-C6-alkyl,

[0368] R9 is hydrogen or C1-C6-alkyl or aryl, R8 and R9 may optionally be combined to form a double bond,

[0369] R10 and R12 are independently hydrogen, aryl, C1-C6-alkyl, or —C(O)NR16R17

[0370] E and G are independently 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 halogen, —CN, —CF3, —OCF3, aryl, —COOH and —NH2, and the arylene or heteroarylene is optionally substituted with up to four substituents R13, R14, R15, and R15A

[0371] E and R10 may be connected through one or two valence bonds, G and R12 may be connected through one or two valence bonds;

[0372] R13, R14, R15 and R15A are independently selected from

[0373] hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —OS(O)2CF3, —SCF3, —NO2, —OR16, —NR16R17, —SR16, —NR16S(O)2R17, —S(O)2NR1617, —S(O)NR16R17, —S(O)R16, —S(O)2R16, —OS(O)2R16, —C(O)NR16R17, —OC(O)NR16R17, —NR16C(O)R17, —CH2C(O)NR16R17, —OC1-C6-alkyl-C(O)NR16R17, —CH2OR16, —CH2OC(O)R16, —CH2NR16R17, —OC(O)R16, —OC1-C6-alkyl-C(O)OR16, —OC1-C6-alkyl-OR16, —SC1-C6-alkyl-C(O)OR16, —C2-C6-alkenyl-C(═O)OR16, —NR16—C(═O)—C1-C6-alkyl-C(═O)OR16, —NR16—C(═O)—C1-C6-alkenyl-C(═O)OR16, —C(O)OR16, or —C2-C6-alkenyl-C(═O)R16, ═O, or —C2-C6-alkenyl-C(═O)NR16R17,

[0374] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0375] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17

[0376] aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0377] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR16, —CH2C(O)O16, —CH2OR16, —CN, —CF3, —OCF3, —NO2, —OR16, —NR16R17, S(O)2R16, aryl and C1-C6-alkyl,

[0378] R16 and R17 independently are hydrogen, OH, C1-C20-alkyl, aryl-C1-C6-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OC1-C6-alkyl, —C(O)OC1-C6-alkyl, —COOH and —NH2, and the aryl groups may optionally be substituted by halogen, —C(O)OC1-C6-alkyl, —COOH, —CN, —CF3, —OCF3, —NO2, —OH, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl; R16 and R17 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

[0379] In another embodiment of the invention X is ═O or ═S

[0380] In another embodiment of the invention X is ═O

[0381] In another embodiment of the invention X is ═S

[0382] In another embodiment of the invention Y is —O— or —S—

[0383] In another embodiment of the invention Y is —O—

[0384] In another embodiment of the invention Y is —S—

[0385] In another embodiment of the invention E is arylene optionally substituted with up to four substituents, R13, R14, R15, and R15A.

[0386] In another embodiment of the invention E is phenylene or naphtylene optionally substituted with up to four substituents, R13, R14, R15, and R15A.

[0387] In another embodiment of the invention E is

[0388] In another embodiment of the invention E is

[0389] In another embodiment of the invention E is phenylene

[0390] In another embodiment of the invention E is heteroarylene optionally substituted with up to four substituents, R13, R14, R15, and R15A.

[0391] In another embodiment of the invention E is benzofuranylidene optionally substituted with up to four substituents R13, R14, R15, and R15A.

[0392] In another embodiment of the invention E is

[0393] In another embodiment of the invention E is carbazolylidene optionally substituted with up to four substituents R13, R14, R15, and R15A.

[0394] In another embodiment of the invention E is

[0395] In another embodiment of the invention E is quinolylidene optionally substituted with up to four substituents R13, R14, R15, and R15A.

[0396] In another embodiment of the invention E is

[0397] In another embodiment of the invention E is indolylene optionally substituted with up to four substituents R13, R14R , R15, and R15A.

[0398] In another embodiment of the invention E is

[0399] In another embodiment of the invention R8 is Hydrogen.

[0400] In another embodiment of the invention R9 is Hydrogen.

[0401] In another embodiment of the invention R8 and R9 are combined to form a double bond.

[0402] In another embodiment of the invention R10 is C1-C6-alkyl.

[0403] In another embodiment of the invention R10 is methyl.

[0404] In another embodiment of the invention G is phenylene optionally substituted with up to four substituents, R13, R14, R15, and R15A.

[0405] In another embodiment of the invention R11 is Hydrogen.

[0406] In another embodiment of the invention R12 is Hydrogen.

[0407] In another embodiment of the invention R13, R14, R15 and R15A are independently selected from

[0408] hydrogen, halogen —NO2, —OR6, —NR16R17, —SR16, —NR16S(O)2R17, —S(O)2NR16R17, —S(O)NR16R17, —S(O)R16, —S(O)2R16, —OS(O)2R16, —NR16C(O)R17, —CH2OR16, —CH2OC(O)R16, —CH2NR16R17, —OC(O)R16, —OC1-C6-alkyl-C(O)OR16, —OC1-C6-alkyl-C(O)NR16R17, —OC1-C6-alkyl-OR16, —SC1-C6-alkyl-C(O)OR16, —C2-C6-alkenyl-C(═O)OR16, —C(O)O16, or —C2-C6-alkenyl-C(═O)R16,

[0409] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0410] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17

[0411] aryl, aryloxy, aroyl, arylsulfanyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0412] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR16, —CH2C(O)OR16, —CH2OR16, —CN, —CF3, —OCF3, —NO2, —OR16, —NR16R17 and C1-C6-alkyl.

[0413] In another embodiment of the invention R13, R14, R15 and R15A are independently selected from

[0414] hydrogen, halogen, —NO2, —OR6, —NR16R17, —S16, —S(O)2R16, —OS(O)2R16, —CH2OC(O)R16, —OC(O)16, —OC1-C6-alkyl-C(O)OR16, —OC1-C6-alkyl-OR16, —SC1-C6-alkyl-C(O)OR16, —C(O)OR16, or —C2-C6-alkenyl-C(═O)R16,

[0415] C1-C6-alkyl or C1-C6-alkenyl which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17

[0416] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl,

[0417] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR16, —CH2C(O)OR16, —CH2OR16, —CN, —CF3, —OCF3, —NO2, —OR16, —NR16R17 and C1-C6-alkyl.

[0418] In another embodiment of the invention R13, R14, R15 and R15A are independently selected from

[0419] hydrogen, halogen, —NO2, —OR6, —NR16R17, —SR16, —S(O)2R16, —OS(O)2R16, —CH2OC(O)R16, —OC(O)R16, —OC1-C6-alkyl-C(O)OR16, —OC1-C6-alkyl-OR16, —SC1-C6-alkyl-C(O)OR16, —C(O)OR16, or —C2-C6-alkenyl-C(═O)R18,

[0420] C1-C6-alkyl or C1-C6-alkenyl which may optionally be substituted with one or more substituents selected from halogen, —CF3, —OR16, and —NR16R17

[0421] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl

[0422] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, C(O)OR16, —CN, —NO2, —OR16, —NR16R17 and C1-C6-alkyl.

[0423] In another embodiment of the invention R13, R14, R15 and R15A are independently selected from

[0424] hydrogen, halogen, —OR16, —OC1-C6-alkyl-C(O)OR16, or —C(O)OR16,

[0425] C1-C6-alkyl which may optionally be substituted with one or more substituents selected from halogen, —OR16, and —NR16R17

[0426] aryl, aryloxy, aryl-C1-C6-alkoxy,

[0427] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, C(O)OR16, OR16, and C1-C6-alkyl.

[0428] In another embodiment of the invention R16 and R17 independently are hydrogen, C1-C20-alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, —CF3, —OCF3, —OC1-C6-alkyl, —COOH and —NH2, and the aryl groups may optionally be substituted by halogen, —COOH, —CN, —CF3, —OCF3, —NO2, —OH, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl; R16 and R17 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

[0429] In another embodiment of the invention R16 and R17 independently are hydrogen, C1-C20-alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, —CF3, —OC1-C6-alkyl, —COOH and —NH2, and the aryl groups may optionally be substituted by halogen, —COOH, —CN, —CF3, —OCF3, —OH, —NH2, or C1-C6-alkyl.

[0430] In another embodiment of the invention A is

[0431] wherein

[0432] R20 is hydrogen or C1-C6-alkyl,

[0433] R21 is hydrogen or C1-C6-alkyl,

[0434] U and V are a valence bond or C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl independently,

[0435] J is C1-C6-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R22, R23 and R24,

[0436] L is C1-C6-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R25, R26 and R27,

[0437] R18, R19, R22, R23R24, R25, R26 and R27 are independently selected from

[0438] hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —SCF3, —NO2, —OR28, —NR28R29, —SR28, —NR28S(O)2R29, —S(O)2NR28R29, —S(O)NR28R29, —S(O)R28, —S(O)2R28, —C(O)NR28R29, —OC(O)NR28R29, —NR28C(O)R29, —NR28C(O)R29, —CH2C(O)NR28R29, —OCH2C(O)NR28R29, —CH2OR28, —CH2NR28R29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0439] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0440] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0441] aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl,- heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0442] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl,

[0443] R28 and R29 independently are hydrogen, C1-C6-alkyl, aryl-C1-C6-alkyl or aryl, or R28 and R29 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds

[0444] In another embodiment of the invention U is a valence bond

[0445] In another embodiment of the invention U is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl

[0446] In another embodiment of the invention J is arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R22, R23 and R24

[0447] In another embodiment of the invention J is arylene optionally substituted with up to three substituents R22, R23 and R24

[0448] In another embodiment of the invention J is phenylene optionally substituted with up to three substituents R22, R23 and R24

[0449] In another embodiment of the invention J is

[0450] In another embodiment of the invention R22, R23 and R24 are independently selected from

[0451] hydrogen, halogen, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —SCF3, —NO2, —OR28, —NR28R29, —SR28—C(O)NR28R29, —OC(O)NR28R29, —NR28C(O)R29, —NR28C(O)OR29, —CH2C(O)NR28R29, —OCH2C(O)NR28R29, —CH2OR28, —CH2NR28R29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkenyl-C(═O)OR28—, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0452] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0453] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0454] aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0455] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0456] In another embodiment of the invention R22, R23 and R24 are independently selected from

[0457] hydrogen, halogen, —OCF3, —OR28, —NR28R29, —SR28, —NR28C(O)R29, —NR28C(O)OR29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0458] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0459] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0460] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0461] In another embodiment of the invention R22, R23 and R24 are independently selected from

[0462] hydrogen, halogen, —OCF3, —OR28, —NR28R29, —SR28, —NR28C(O)R29, —NR28C(O)OR29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0463] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, or —CF3

[0464] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0465] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OH, —CN, —CF3, —NO2, or C1-C6-alkyl

[0466] In another embodiment of the invention R20 is hydrogen or methyl

[0467] In another embodiment of the invention R20 is hydrogen

[0468] In another embodiment of the invention R28 is Hydrogen, C1-C6-alkyl or aryl

[0469] In another embodiment of the invention R28 is Hydrogen or C1-C6-alkyl

[0470] In another embodiment of the invention R29 is Hydrogen or C1-C6-alkyl

[0471] In another embodiment of the invention V is a valence bond

[0472] In another embodiment of the invention V is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl

[0473] In another embodiment of the invention L is C1-C6-alkylene or arylene, wherein the arylene is optionally substituted with up to three substituents R25, R26 and R27

[0474] In another embodiment of the invention L is C1-C6-alkylene

[0475] In another embodiment of the invention L is phenylene optionally substituted with up to three substituents R25, R26 and R27

[0476] In another embodiment of the invention R25, R26 and R27 are independently selected from

[0477] hydrogen, halogen, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —SCF3, —NO2, —OR28, —NR28R29, —SR28, —C(O)NR28R29, —OC(O)NR28R29, —NR28C(O)R29, —NR28C(O)OR29, —CH2C(O)NR28R29, —OCH2C(O)NR28R29, —CH2OR28, —CH2NR28R29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —NR28—C(═O)—C1-C6-alkyl-C(═O)OR28, —NR28—C(═)—C1-C6-alkenyl-C(═O)OR28—, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0478] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0479] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0480] aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0481] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0482] In another embodiment of the invention R25, R26 and R27 are independently selected from

[0483] hydrogen, halogen, —OCF3, —OR28, —NR28R29, —SR28, —NR28C(O)R29, —NR28C(O)OR29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0484] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0485] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6- alkyl,

[0486] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0487] In another embodiment of the invention R25, R26 and R27 are independently selected from

[0488] hydrogen, halogen, —OCF3, —OR28, —NR28R29, —SR28, —NR28C(O)R29, —NR28C(O)OR29, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, —C2-C6-alkenyl-C(═O)OR28, —C(═O)NR28—C1-C6-alkyl-C(═O)OR28, —C1-C6-alkyl-C(═O)OR28, or —C(O)OR28,

[0489] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, or —CF3

[0490] aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl,

[0491] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OH, —CN, —CF3, —NO2, or C1-C6-alkyl

[0492] In another embodiment of the invention R21 is hydrogen or methyl

[0493] In another embodiment of the invention R21 is hydrogen

[0494] In another embodiment of the invention R28 is Hydrogen, C1-C6-alkyl or aryl

[0495] In another embodiment of the invention R28 is Hydrogen or C1-C6-alkyl

[0496] In another embodiment of the invention R29 is Hydrogen or C1-C6-alkyl

[0497] In another embodiment of the invention R18 and R19 are independently selected from

[0498] hydrogen, halogen, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29, —SR28, —S(O)R28, —S(O)2R28, —C(O)NR28R29, —CH2OR28, —OC(O)R28, —OC1-C6-alkyl-C(O)OR28, —SC1-C6-alkyl-C(O)OR28, or —C(O)OR28,

[0499] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0500] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0501] aryl, aryloxy, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl, heteroaryl-C1-C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0502] In another embodiment of the invention R18 and R19 are independently selected from

[0503] hydrogen, halogen, —CN, —CF3, —NO2, —OR28, —NR28R29, or —C(O)OR28,

[0504] C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29

[0505] aryl, aryloxy, aryl-C1-C6-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR28, —CN, —CF3, —OCF3, —NO2, —OR28, —NR28R29 and C1-C6-alkyl

[0506] In another embodiment of the invention A is

[0507] In another embodiment of the invention A is of the form M—Q—T—

[0508] wherein M is

[0509] wherein W1, W2, and W3 are independently OH, SH or NH2 and the phenyl, naphthalene or benzocarbazole rings are optionally substituted by one or more R34 independently

[0510] Q is selected from the following:

[0511] a valence bond

[0512] —CH2N(R30)— or —SO2N(R31)—

[0513] A compound of the formula

[0514] wherein Z1 is S(O)2 or CH2, Z2 is N, —O— or —S—, and n is 1 or 2;

[0515] T is

[0516] C1-C6-alkylene, C2-C6-alkenylene or C2-C6-alkynylene, which may optionally be substituted with; one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR32, and —NR32R33

[0517] Arylene, arylene-oxy, -aryl-oxycarbonyl-, -aroyl-, -aryl-C1-C6-alkoxy-, -aryl-C1-C6-alkyl-, -aryl-C2-C6-alkenyl-, -aryl-C2-C6-alkynyl-, heteroarylene, -heteroaryl-C1-C6-alkyl-, -heteroaryl-C2-C6-alkenyl- or -heteroaryl-C2-C6-alkynyl-, wherein the cyclic moieties are optionally substituted by one or more substituents selected from halogen, —C(O)OR32, —C(O)H, —CN, —CF3, —OCF3, —NO2, —OR32, —NR32R33, C1-C6-alkyl or C1-C6-alkanoyl,

[0518] A valence bond

[0519] R32 and R33 independently are hydrogen, C1-C6-alkyl, aryl-C1-C6-alkyl or aryl, or R32 and R33 when attached to the same nitrogen atom together with the said nitrogen atom may form a 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,

[0520] R30 and R31 are independently hydrogen, C1-C6-alkyl or C1-C6-alkanoyl.

[0521] R34 is hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —SCF3, —NO2, —OR32, —C(O)R32, —NR2R33, —SR32, —NR32S(O)2R33, —S(O)2NR32R33, —S(O)NR32R33 —S(O)R32, —S(O)2R32, —C(O)NR32R33, —OC(O)NR32R33, —NR32C(O)R33, —CH2C(O)NR32R33, —OCH2C(O)NR32R33, —CH2OR32, —CH2NR32R33, —OC(O)R32, —OC1-C6-alkyl-C(O)OR32, —SC1-C6-alkyl-C(O)OR32—C2-C6-alkenyl-C(═O)OR32, —NR32—C(═O)—C1-C6-alkyl-C(═O)OR32, —NR32—C(═O)—C1-C6-alkenyl-C(═O)OR32—, C1-C6-alkyl, C1-C6-alkanoyl or —C(O)OR32,

[0522] In another embodiment of the invention M is

[0523] In another embodiment of the invention M is

[0524] In another embodiment of the invention M is

[0525] In another embodiment of the invention M is

[0526] In another embodiment of the invention M is

[0527] In another embodiment of the invention Q is a valence bond, —CH2N(R30)—, or —SO2N(R31)—

[0528] In another embodiment of the invention Q is a valence bond

[0529] In another embodiment of the invention T is

[0530] A valence bond

[0531] C1-C6-alkylene, C2-C6-alkenylene or C2-C6-alkynylene, which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR32, and —NR32R33

[0532] Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70

[0533] In another embodiment of the invention T is

[0534] A valence bond

[0535] Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70

[0536] In another embodiment of the invention T is phenylene or naphthalene

[0537] In another embodiment of the invention the cyclic moiety in T is optionally substituted by halogen, —C(O)OR32, —CN, —CF3, —OR32, —NR32R33, C1-C6-alkyl or C1-C6-alkanoyl

[0538] In another embodiment of the invention the cyclic moiety in T is optionally substituted by halogen, —C(O)OR32, —OR32, —NR32R33, C1-C6-alkyl or C1-C6-alkanoyl

[0539] In another embodiment of the invention the cyclic moiety in T is optionally substituted by halogen, —C(O)OR32 or —OR32

[0540] In another embodiment of the invention T is a valence bond

[0541] In another embodiment of the invention R30 and R31 are independently hydrogen or C1-C6-alkyl

[0542] In another embodiment of the invention R34 is hydrogen, halogen, —CN, —CF3, —OCF3, —SCF3, —NO2, —OR32, —C(O)R32, —NR32R33, —SR32, —C(O)NR32R33, —OC(O)NR32R33, —NR32C(O)R33, —OC(O)R32, —OC1-C6-alkyl-C(O)OR32, —SC1-C6-alkyl-C(O)OR32 or —C(O)OR32

[0543] In another embodiment of the invention R34 is hydrogen, halogen, —CF3, —NO2, —OR32, —NR32R33, —SR32, —NR32C(O)R33, or —C(O)OR32

[0544] In another embodiment of the invention R34 is hydrogen, halogen, —CF3, —NO2, —OR32, —NR32R33, or —NR32C(O)R33

[0545] In another embodiment of the invention R34 is hydrogen, halogen, or —OR32

[0546] In another embodiment of the invention R32 and R33 independently are hydrogen, C1-C6-alkyl, or aryl

[0547] In another embodiment of the invention R32 and R33 independently are hydrogen or C1-C6-alkyl

[0548] In another embodiment of the invention A is

[0549] wherein A1 is a valence bond, C1-C6-alkylene, —NH—C(═O)—A2—, —C1-C6-alkyl-S—, —C1-C6-alkyl-O—, —C(═O)—, or —C(═O)—NH—, wherein any C1-C6-alkyl moiety is optionally substituted by R1A;

[0550] A2 is a valence bond, C1-C6-alkylene, C1-C6-alkenylene, or —C1-C6-alkyl-O—;

[0551] R1 A is C1-C6-alkyl, aryl, wherein the alkyl or aryl moieties are optionally substituted by one or more halogen, cyano, nitro, amino;

[0552] AR1 is a valence bond, arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted by one or more R1B independently

[0553] R1B is selected from

[0554] hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —OS(O)2CF3, —SCF3, —NO2, —OR1C, —NR1CR1D, —SR1C, —NR1CS(O)2R1D, —S(O)2NR1CR1D, —S(O)NR1CR1D, —S(O)R1C, —S(O)2R1C, —OS(O)2R1C, —C(O)NR1CR1D, —OC(O)NR1CR1D, —NR1CC(O)R1D, —CH2C(O)NR1CR1D, —OC1-C6-alkyl-C(O)NR1CR1D, —CH2OR1C, —CH2OC(O)R1C, —CH2NR1CR1D, —OC(O)R1C, —OC1-C6-alkyl-C(O)OR1C, —OC1-C6-alkyl-OR1C, —S—C1-C6-alkyl-C(O)OR1C, —C2-C6-alkenyl-C(═O)OR1C, —NR1C—C(═O)—C1-C6-alkyl-C(═O)OR1C, —NR1C—C(═O)—C1-C6-alkenyl-C(═O)OR1C, —C2-C6-alkenyl-C(═O)R1C, ═O, —NH—C(═O)—O—C1-C6-alkyl, or —NH—C(═O)—C(═O)—O—C1-C6-alkyl

[0555] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0556] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR1C, and —NR1CR1D

[0557] aryl, aryloxy, aryloxycarbonyl , arylsulfanyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0558] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR1C, —CH2C(O)OR1C, —CH2OR1C, —CN, —CF3, —OCF3, —NO2, —OR1C, —NR1CR1D and C1-C6-alkyl,

[0559] R1C and R1D independently are hydrogen, —OH, C1-C6-alkyl, C1-C6-alkenyl, aryl-C1-C6-alkyl or aryl, wherein the alkyl moieties may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —O—C1-C6-alkyl, —C(O)—O—C1-C6-alkyl, —COOH and —NH2, and the aryl moieties may optionally be substituted by halogen, —C(O)OC1-C6-alkyl, —COOH, —CN, —CF3, —OCF3, —NO2, —OH, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl; R1C and R1D 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,

[0560] C1 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(R1E) wherein the alkyl moieties are optionally substituted by one or more R1F independently

[0561] R1E and R1F are independently selected from C1-C6-alkyl, aryl optionally substituted by one or more halogen, —COOH;

[0562] AR2 is

[0563] a valence bond

[0564] C1-C6-alkylene, C2-C6-alkenylene, C2-C6-alkynylene wherein the alkyl, alkenyl and alkynyl moieties are optionally substituted by one or more R2A independently;

[0565] arylene, -aryloxy-, -aryloxy-carbonyl-, aryl-C1-C6-alkyl, -aroyl-, aryl-C1-C6-alkoxy-, aryl-C2-C6-alkenyl-, aryl-C2-C6-alkynyl-, heteroarylene, -heteroaryl-C1-C6-alkyl-, -heteroaryl-C2-C6-alkenyl-, -heteroaryl-C2-C6-alkynyl- wherein the aryl and heteroaryl moieties are optionally substituted by one or more R2A independently;

[0566] R2A is C1-C6-alkyl, C1-C6-alkoxy, aryl, aryloxy, aryl-C1-C6-alkoxy, —C(═O)—NH—C1-C6-alkyl-aryl, heteroaryl, heteroaryl-C1-C6-alkoxy, —C1-C6-alkyl-COOH, —O—C1-C6-alkyl-COOH, —S(O)2R2B, —C2-C6-alkenyl-COOH, —OR2B, —NO2, halogen, —COOH, —CF3, —CN, —N(R2BR2C), wherein the aryl or heteroaryl moieties are optionally substituted by one or more C1-C6-alkyl, C1-C6-alkoxy, -C1-C6-alkyl-COOH, —C2-C6-alkenyl-COOH, —OR2B, —NO2, halogen, —COOH, —CF3, —CN, or —N(R2BR2C)

[0567] R2B and R2C are independently selected from hydrogen and C1-C6-alkyl

[0568] In another embodiment of the invention A1 is a valence bond, C1-C6-alkylene, —NH—C(═O)—A2—, —C1-C6-alkyl-S—, —C1-C6-alkyl-O—, or —C(═O)—, wherein any C1-C6-alkyl moiety is optionally substituted by R1A

[0569] In another embodiment of the invention A1 is a valence bond, C1-C6-alkylene, —NH—C(═O)—A2—, —C1-C6-alkyl-S—, or —C1-C6-alkyl-O, wherein any C1-C6-alkyl moiety is optionally substituted by R1A

[0570] In another embodiment of the invention A1 is a valence bond, C1-C6-alkylene, or —NH—C(═O)—A2, wherein any C1-C6-alkyl moiety is optionally substituted by R1A

[0571] In another embodiment of the invention A1 is a valence bond or C1-C6-alkylene, wherein any C1-C6-alkyl moiety is optionally substituted by R1A

[0572] In another embodiment of the invention A1 is a valence bond

[0573] In another embodiment of the invention A2 is a valence bond or —C1-C6-alkyl-O—

[0574] In another embodiment of the invention A2 is a valence bond

[0575] In another embodiment of the invention AR1 is arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted by one or more R1B independently

[0576] In another embodiment of the invention AR1 is selected from the group of compounds consisting of phenylene, biphenylylene, naphthylene, anthracenylene, phenanthrenylene, fluorenylene, indenylene, azulenylene, 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, or acridinylene, optionally substituted by one or more R1B independently

[0577] In another embodiment of the invention AR1 is selected from phenylene, biphenylylene, naphthylene, pyridinylene, fyrylene, indolylene, or carbazolylene, optionally substituted by one or more R1B independently

[0578] In another embodiment of the invention AR1 is selected from the group of compounds consisting of phenylene, indolylene, or carbazolylene, optionally substituted by one or more R1B independently

[0579] In another embodiment of the invention AR1 is phenylene optionally substituted by one or more R1B independently

[0580] In another embodiment of the invention AR1 is indolylene

[0581] In another embodiment of the invention AR1 is carbazolylene

[0582] In another embodiment of the invention AR1 is

[0583] In another embodiment of the invention AR1 is

[0584] In another embodiment of the invention R1B is selected from

[0585] hydrogen, halogen, —CN, —CF3, —OCF3, —NO2, —OR1C, —NR1CR1D, —SR1C, —S(O)2R1C, —NR1CC(O)R1D, —OC1-C6-alkyl-C(O)NR1CR1D, —C2-C6-alkenyl-C(═O)OR1C, —C(O)OR1C, ═O, —NH—C(═O)—O—C1-C6-alkyl, or —NH—C(═O)—C(═O)—O—C1-C6-alkyl

[0586] C1-C6-alkyl or C2-C6-alkenyl

[0587] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR1C, and —NR1CR1D

[0588] aryl, aryloxy, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, heteroaryl, heteroaryl-C1-C6-alkyl, or heteroaryl-C2-C6-alkenyl

[0589] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR1C, —CN, —CF3, —OCF3, —NO2, —OR1C, —NR1CR1D and C1-C6-alkyl

[0590] In another embodiment of the invention R1B is selected from

[0591] hydrogen, halogen, —CF3, —NO2, —OR1C, —NR1CR1D, —C(O)OR1C, ═O, —NH—C(═O)—O—C1-C6-alkyl, or —NH—C(═O)—C(═O)—O—C1-C6-alkyl

[0592] C1-C6-alkyl

[0593] In another embodiment of the invention R1C and R1D independently are hydrogen, C1-C6-alkyl, or aryl, wherein the aryl moieties may optionally be substituted by halogen or —COOH

[0594] In another embodiment of the invention R1C and R1D independently are hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties may optionally be substituted by halogen or —COOH

[0595] In another embodiment of the invention C1 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(R1E)— wherein the alkyl moieties are optionally substituted by one or more R1F independently

[0596] In another embodiment of the invention C1 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)—

[0597] In another embodiment of the invention R1E and R1F are independently selected from C1-C6-alkyl

[0598] In another embodiment of the invention AR2 is

[0599] a valence bond

[0600] C1-C6-alkylene, wherein the alkyl is optionally substituted by one or more R2A independently

[0601] arylene, aryl-C1-C6-alkyl, heteroarylene, wherein the aryl and heteroaryl moieties are optionally substituted by one or more R2A independently

[0602] In another embodiment of the invention AR2 is

[0603] a valence bond

[0604] C1-C6-alkylene, wherein the alkyl is optionally substituted by one or more R2A independently

[0605] phenyl, phenyl-C1-C6-alkyl, wherein the phenyl moieties are optionally substituted by one or more R2A independently

[0606] In another embodiment of the invention R2A is C1-C6-alkyl, C1-C6-alkoxy, aryl, aryloxy, heteroaryl, —C1-C6-alkyl-COOH, —O—C1-C6-alkyl-COOH, —S(O)2R2B, —C2-C6-alkenyl-COOH, —OR2B, —NO2, halogen, —COOH, —CF3, —CN, —N(R2BR2C), wherein the aryl or heteroaryl moieties are optionally substituted by one or more C1-C6-alkyl, C1-C6-alkoxy, —C1-C6-alkyl-COOH, —C2-C6-alkenyl-COOH, —OR2B, —NO2, halogen, —COOH, —CF3, —CN, or —N(R2BR2C)

[0607] In another embodiment of the invention R2A is C1-C6-alkyl, C1-C6-alkoxy, aryl, —OR2B, —NO2, halogen, —COOH, —CF3, —CN, —N(R2BR2C), wherein the aryl is optionally substituted by one or more C1-C6-alkyl, C1-C6-alkoxy, —OR2B, —NO2, halogen, —COOH, —CF3, —CN, or —N(R2BR2C)

[0608] In another embodiment of the invention R2A is C1-C6-alkyl, C1-C6-alkoxy, aryl, halogen, —CF3, wherein the aryl is optionally substituted by one or more C1-C6-alkyl, halogen, —COOH, —CF3, or —CN

[0609] In another embodiment of the invention R2A is C1-C6-alkyl, C1-C6-alkoxy, phenyl, halogen, —COOH, —CF3, or —CN

[0610] In another embodiment of the invention A is

[0611] wherein AR3 is C1-C6-alkylene, arylene, heteroarylene, -aryl-C1-6-alkyl- or -aryl-C2-6-alkenyl-, wherein the alkylene or alkenylene is optionally substituted with one or more substituents independently selected from halogen, —CN, —CF3, —OCF3, aryl, —COOH and —NH2, and the arylene or heteroarylene is optionally substituted with one or more R3A independently

[0612] R3A is independently selected from

[0613] hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —OS(O)2CF3, —SCF3, —NO2, —OR3B, —NR3BR3C, —SR3B, —NR3BS(O)2R3C, —S(O)2NR3BR4C, —S(O)NR3BR3C, —S(O)R3B, —S(O)2R3B, —OS(O)2R3B, —C(O)NR3BR3C, —OC(O)NR3BR3C, —NR3BC(O)R3C, —CH2C(O)NR3CR3B, —OC1-C6-alkyl-C(O)NR3BR3C, —CH2OR3B, —CH2OC(O)R3B, —CH2NR3BR3C, —OC(O)R3B, —OC1-C6-alkyl-C(O)OR3B, —OC1-C6-alkyl-OR3B, —SC1-C6-alkyl-C(O)OR3B, —C2-C6-alkenyl-C(═O)OR3B, —NR3B—C(═O)—C1-C6-alkyl-C(═O)OR3B, —NR3B—C(═O)—C1-C6-alkenyl-C(═O)OR3B, —C(O)OR3B, or —C2-C6-alkenyl-C(═O)R3B,

[0614] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0615] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR3B, and —NR3BR3C

[0616] aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0617] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR3B, —CH2C(O)OR3B, —CH2OR3B, —CN, —CF3, —OCF3, —NO2, —OR3B, —NR3BR3C and C1-C6-alkyl,

[0618] R3B and R3C are independently hydrogen, OH, CF3, C1-C2-alkyl, aryl-C1-C6-alkyl, —C(═O)—C1-C6-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OC1-C6-alkyl, —C(O)OC1-C6-alkyl, —C(═O)—R3D, —COOH and —NH2, and the aryl groups may optionally be substituted by halogen, —C(O)OC1-C6-alkyl, —COOH, —CN, —CF3, —OCF3, —NO2, —OH, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl; R3B and R3C 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

[0619] R3D is C1-C6-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl optionally substituted with one or more C1-C6-alkyl.

[0620] In another embodiment of the invention AR3 is arylene, heteroarylene, or aryl-C1-6-alkyl, wherein the alkyl is optionally substituted with one or more substituents independently selected from halogen, —CN, —CF3, —OCF3, aryl, —COOH and —NH2, and the arylene or heteroarylene is optionally substituted with one or more R3A independently

[0621] In another embodiment of the invention AR3 is arylene optionally substituted with one or more R3A independently

[0622] In another embodiment of the invention AR3 is phenylene, naphthalene or anthranylene optionally substituted with one or more R3A independently

[0623] In another embodiment of the invention AR3 is phenylene optionally substituted with one or more R3A independently

[0624] In another embodiment of the invention R3A is independently selected from

[0625] halogen, —CN, —CF3, —NO2, —OR3B, —NR3BR3C, —SR3B, —OC1-C6-alkyl-C(O)OR3B or —C(O)OR3B

[0626] C1-C6-alkyl optionally substituted with one or more substituents selected from halo gen, —CN, —CF3, —OCF3, —OR3B, and —NR3BR3C

[0627] aryl, aryl-C1-C6-alkyl, heteroaryl, or heteroaryl-C1-C6-alkyl of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR3B, —CN, —CF3, —OCF3, —NO2, —OR3B, —NR3BR3C and C1-C6-alkyl

[0628] In another embodiment of the invention R3A is independently selected from halogen, —OR3B, —NR3BR3C, —C(O)OR3B, —OC1-C6-alkyl-C(O)OR3B, or C1-C6-alkyl

[0629] In another embodiment of the invention R3B and R3C are independently hydrogen, CF3, C1-C12-alkyl, or —C(═O)—C1-C6-alkyl; R3B and R3C when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom

[0630] In another embodiment of the invention A is

[0631] wherein AR4 is C1-C6-alkylene, arylene, heteroarylene, -aryl-C1-6-alkyl- or -aryl-C2-6-alkenyl, wherein the alkylene or alkenylene is optionally substituted with one or more substituents independently selected from halogen, —CN, —CF3, —OCF3, aryl, —COOH and —NH2, and the arylene or heteroarylene is optionally substituted with one or more R4A independently

[0632] R4A is independently selected from

[0633] hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —OS(O)2CF3, —SCF3, —NO2, —OR4B, —NR4BR4C, —SR4B, —NR4BS(O)2R4C, —S(O)2NR4BR4C, —S(O)NR4BR4C, —S(O)R4B, —S(O)R4B, —OS(O)2R4B, —C(O)NR4BR4C, —OC(O)NR4BR4C, —NR4BC(O)R4C, —CH2C(O)NR4BR4C, —OC1-C6-alkyl-C(O)NR4BR4C, —CH2OR4B, —CH2OC(O)R4B, —CH2NR4BR4C, —OC(O)R4B, —OC1-C6-alkyl-C(O)O4B, —OC1-C6-alkyl-OR4B, —SC1-C6-alkyl-C(O)OR4B, —C2-C6-alkenyl-C(═O)OR4B, —NR4B—C(═O)—C1-C6-alkyl-C(═O)OR4B, —NR4B—C(═O)—C1-C6-alkenyl-C(═O)OR4B, —C(O)OR4B, or —C2-C6-alkenyl-C(═O)R4B,

[0634] C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,

[0635] which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR4B, and —NR4BR4C

[0636] aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, aroyl-C2-C6-alkenyl, aryl-C2-C6-alkynyl, heteroaryl, heteroaryl-C1-C6-alkyl, heteroaryl-C2-C6-alkenyl or heteroaryl-C2-C6-alkynyl,

[0637] of which the cyclic moieties optionally may be substituted with one or more substituents selected from halogen, —C(O)OR4B, —CH2C(O)OR4B, —CH2OR4B, —CN, —CF3, —OCF3, —NO2, —OR4B, —NR4BR4C and C1-C6-alkyl,

[0638] R4B and R4C are independently hydrogen, OH, CF3, C1-C12-alkyl, aryl-C1-C6-alkyl, —C(═O)—R4D, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OC1-C6-alkyl, —C(O)OC1-C6-alkyl, —COOH and —NH2, and the aryl groups may optionally be substituted by halogen, —C(O)OC1-C6-alkyl, —COOH, —CN, —CF3, —OCF3, —NO2, —OH, —OC1-C6-alkyl, —NH2, C(═O) or C1-C6-alkyl; R4Band R4C 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

[0639] R4D is C1-C6-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl optionally substituted with one or more C1-C6-alkyl.

[0640] In another embodiment of the invention AR4 is arylene, heteroarylene or aryl-C1-6-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently selected from halogen, —CN, —CF3, —OCF3, aryl, —COOH and —NH2, and the arylene or heteroaryl is optionally substituted with one or more R4A independently

[0641] In another embodiment of the invention AR4 is arylene or heteroarylene optionally substituted with one or more R4A independently

[0642] In another embodiment of the invention AR4 is phenylene, naphtylene, anthrylene, thienylene, pyridylene, or benzodioxylene optionally substituted with one or more R4A independently

[0643] In another embodiment of the invention AR4 is phenylene optionally substituted with one or more R4A independently

[0644] In another embodiment of the invention R4A is independently selected from hydrogen, halogen, —CF3, —OR4B, —NR4BR4C, C1-C6-alkyl, aryl-C2-C6-alkenyl or aryl optionally substituted with one or more substituents selected from halogen, —CF3, or —OR4B

[0645] In another embodiment of the invention R4B and R4C are independently hydrogen, CF3, C1-C12-alkyl, —C(═O)—R4D, or aryl

[0646] In another embodiment of the invention R4D is C1-C6-alkyl, phenyl optionally substituted with one or more halogen, or a heteroaryl selected from isoxazole and thiadiazole optionally substituted with one or more C1-C6-alkyl

[0647] In another embodiment of the invention C consists of 0 to 5 neutral amino acids independently selected from the group consisting of Abz, Gly, Ala, Thr, and Ser

[0648] In another embodiment of the invention C consists of 0 to 5 Gly

[0649] In another embodiment of the invention C consists of 0 Gly

[0650] In another embodiment of the invention C consists of 1 Gly

[0651] In another embodiment of the invention C consists of 2 Gly

[0652] In another embodiment of the invention C consists of 3 Gly

[0653] In another embodiment of the invention C consists of 4 Gly

[0654] In another embodiment of the invention C consists of 5 Gly

[0655] In another embodiment of the invention GB is of the formula —B1—B2—C(O)—, —B1—B2—SO2— or —B1—B2—CH2—, wherein B1 and B2 are as defined in claim 1

[0656] In another embodiment of the invention GB is of the formula —B1—B2—C(O)—, —B1—B2—SO2— or —B1—B2—NH—, wherein B1 and B2 are as defined in claim 1

[0657] In another embodiment of the invention GB is of the formula —B1—B2—C(O)—, —B1—B2—CH2, or —B1—B2—NH—, wherein B1 and B2 are as defined in claim 1

[0658] In another embodiment of the invention GB is of the formula —B1—B2—CH2—, —B1—B2—SO2— or —B1—B2—NH—, wherein B1 and B2 are as defined in claim 1

[0659] In another embodiment of the invention GB is of the formula —B1—B2—C(O)— or —B1—B2—SO2—, wherein B1 and B2 are as defined in claim 1

[0660] In another embodiment of the invention GB is of the formula —B1—B2—C(O)— or —B1—B2—CH2—, wherein B1 and B2 are as defined in claim 1

[0661] In another embodiment of the invention GB is of the formula —B1—B2—C(O)— or —B1—B2—NH—, wherein B1 and B2 are as defined in claim 1

[0662] In another embodiment of the invention GB is of the formula —B1—B2—CH2— or —B1—B2—SO2—, wherein B1 and B2 are as defined in claim 1

[0663] In another embodiment of the invention GB is of the formula —B1—B2—NH— or —B1—B2—SO2—, wherein B1 and B2 are as defined in claim 1

[0664] In another embodiment of the invention GB is of the formula —B1—B2—CH2— or —B1—B2—NH—, wherein B1 and B2 are as defined in claim 1

[0665] In another embodiment of the invention GB is of the formula —B1—B2—C(O)—

[0666] In another embodiment of the invention GB is of the formula —B1—B2—CH2—

[0667] In another embodiment of the invention GB is of the formula —B1—B2—SO2—

[0668] In another embodiment of the invention GB is of the formula —B1—B2—NH—

[0669] In another embodiment of the invention B1 is a valence bond, —O—, or —S—

[0670] In another embodiment of the invention B1 is a valence bond, —O—, or —N(R6)—

[0671] In another embodiment of the invention B1 is a valence bond, —S—, or —N(R6)—

[0672] In another embodiment of the invention B1 is —O—, —S— or —N(R6)—

[0673] In another embodiment of the invention B1 is a valence bond —O—

[0674] In another embodiment of the invention B1 is a valence bond or —S—

[0675] In another embodiment of the invention B1 is a valence bond or —N(R6)—

[0676] In another embodiment of the invention B1 is —O— or —S—

[0677] In another embodiment of the invention B1 is —O— or —N(R6)—

[0678] In another embodiment of the invention B1 is —S— or —N(R6)—

[0679] In another embodiment of the invention B1 is a valence bond

[0680] In another embodiment of the invention B1 is —O—

[0681] In another embodiment of the invention B1 is —S—

[0682] In another embodiment of the invention B1 is —N(R6)—

[0683] In another embodiment of the invention 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-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

[0684] In another embodiment of the invention 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

[0685] In another embodiment of the invention 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

[0686] In another embodiment of the invention 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

[0687] In another embodiment of the invention 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

[0688] In another embodiment of the invention 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

[0689] In another embodiment of the invention B2 is a valence bond or —C1-C18-alkylene, and the alkylene moieties are optionally substituted as defined in claim 1

[0690] In another embodiment of the invention D comprises 1 to 16 positively charged groups

[0691] In another embodiment of the invention D comprises 1 to 12 positively charged groups

[0692] In another embodiment of the invention D comprises 1 to 10 positively charged groups

[0693] In another embodiment of the invention D is a fragment containing basic amino acids independently selected from the group consisting of Lys and Arg and D-isomers of these.

[0694] In another embodiment of the invention the basic amino acid is Arg

[0695] In another embodiment of the invention X is —OH or —NH2

[0696] In another embodiment of the invention X is —NH2

[0697] The invention furthermore provides an R-state insulin hexamer comprising:

[0698] 6 molecules of insulin, at least 2 zinc ions, and a zinc-binding ligand as defined above

[0699] In another embodiment of the invention the insulin is selected from the group consisting of human insulin, an analogue thereof, a derivative thereof, and combinations of any of these

[0700] In another embodiment of the invention the insulin is an analogue of human insulin selected from the group consisting of

[0701] iii.An analogue wherein position B28 is Asp, Lys, Leu, Val, or Ala and position B29 is Lys or Pro; and

[0702] iv.des(B28-B30), des(B27) or des(B30) human insulin.

[0703] In another embodiment of the invention the insulin is an analogue of human insulin wherein position B28 is Asp or Lys, and position B29 is Lys or Pro.

[0704] In another embodiment of the invention the insulin is des(B30) human insulin.

[0705] In another embodiment of the invention the insulin is a derivative of human insulin having one or more lipophilic substituents.

[0706] In another embodiment of the invention 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.

[0707] In another embodiment of the invention the insulin derivative is B29-Nεmyristoyl-des(B30) human insulin.

[0708] In another embodiment of the invention the insulin hexamer as defined above further comprises at least 3 phenolic molecules.

[0709] The invention furthermore provides an aqueous insulin preparation comprising R-state insulin hexamers as defined above

[0710] The invention furthermore provides a method of prolonging the action of an insulin preparation which comprises adding a zinc-binding ligand as defined above to the insulin preparation.

[0711] In another embodiment of the invention the ratio between precipitated insulin and dissolved insulin is in the range from 99:1 to 1:99.

[0712] In another embodiment of the invention the ratio between precipitated insulin and dissolved insulin is in the range from 95:5 to 5:95

[0713] In another embodiment of the invention the ratio between precipitated insulin and dissolved insulin is in the range from 80:20 to 20:80

[0714] In another embodiment of the invention the ratio between precipitated insulin and dissolved insulin is in the range from 70:30to 30:70

[0715] The invention furthermore provides a method of preparing a zinc-binding ligand as defined above comprising the steps of

[0716] Identifying starter compounds that are able to displace a ligand from the R-state HisB10-Zn2+ site

[0717] optionally attaching a fragment consisting of 0 to 5 neutral α- or β-amino acids

[0718] attaching a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidino groups

[0719] The compounds of the present invention may be chiral, and it is intended that any enantiomers, as separated, pure or partially purified enantiomers or racemic mixtures thereof are included within the scope of the invention.

[0720] Furthermore, when a double bond or a fully or partially saturated ring system or more than one centre of asymmetry or a bond with restricted rotatability is present in the molecule diastereomers may be formed. It is intended that any diastereomers, as separated, pure or partially purified diastereomers or mixtures thereof are included within the scope of the invention.

[0721] Furthermore, some of the compounds of the present invention may exist in different tautomeric forms and it is intended that any tautomeric forms, which the compounds are able to form, are included within the scope of the present invention.

[0722] 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.

[0723] Also intended as pharmaceutically acceptable acid addition salts are the hydrates, which the present compounds, are able to form.

[0724] Furthermore, the pharmaceutically acceptable salts comprise basic amino acid salts such as lysine, arginine and ornithine.

[0725] 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.

[0726] 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.

Pharmaceutical Compositions

[0727] The present invention also relates to a pharmaceutical composition 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.

[0728] In one embodiment of the invention the insulin preparation comprises 60 to 3000 nmol/ml of insulin.

[0729] In another embodiment of the invention the insulin preparation comprises 240 to 1200 nmol/ml of insulin.

[0730] In another embodiment of the invention the insulin preparation comprises about 600 nmol/ml of insulin.

[0731] 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.

[0732] 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.

[0733] In one embodiment of the invention 0.5 to 4.0 mg/ml of phenolic compound may be employed.

[0734] In another embodiment of the invention 0.6 to 4.0 mg/ml of m-cresol may be employed.

[0735] In another embodiment of the invention 0.5 to 4.0 mg/ml of phenol may be employed.

[0736] In another embodiment of the invention 1.4 to 4.0 mg/ml of phenol may be employed.

[0737] In another embodiment of the invention 0.5 to 4.0 mg/ml of a mixture of m-cresol or phenol may be employed.

[0738] In another embodiment of the invention 1.4 to 4.0 mg/ml of a mixture of m-cresol or phenol may be employed.

[0739] The pharmaceutical preparation may further comprises a buffer substance, such as a TRIS, phosphate, glycine or glycylglycine (or another zwitterionic substance) buffer, an isotonicity agent, such as NaCl, glycerol, mannitol and/or lactose. Chloride would be used at moderate concentrations (e.g. up to 50 mM) to avoid competition with the zinc-site ligands of the present invention.

[0740] 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.

[0741] In a particular embodiment the insulin preparation of the invention comprises between 0.001% by weight and 1% by weight of anon-ionic surfactant, for example tween 20 or Polox 188.

[0742] A nonionic detergent can be added to stabilise insulin against fibrillation during storage and handling.

[0743] The insulin preparation of the present invention may have a pH value in the range of 3.5 to 8.5, more preferably 7.4 to 7.9.

EXAMPLES

[0744] 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.

[0745] HPLC-MS (Method A)

[0746] The following instrumentation was used:

[0747] Hewlett Packard series 1100 G1312A Bin Pump

[0748] Hewlett Packard series 1100 Column compartment

[0749] Hewlett Packard series 1100 G13 15A DAD diode array detector

[0750] Hewlett Packard series 1100 MSD

[0751] The instrument was controlled by HP Chemstation software.

[0752] The HPLC pump was connected to two eluent reservoirs containing:

[0753] A: 0.01% in water

[0754] B: 0.01% TFA in acetonitrile

[0755] 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.

[0756] The HPLC conditions, detector settings and mass spectrometer settings used are given in the following table.

2ColumnWaters Xterra MS C-18 × 3 mm idGradient10%-100% acetonitrile lineary during 7.5 min at 1.0 mL/minDetectionUV: 210 nm (analog output from DAD)MSIonisation mode: API-ESScan 100-1000 amu step 0.1 amu

[0757] HPLC-MS (Method B)

[0758] The following instrumentation was used:

[0759] Sciex API 100 Single quadropole mass spectrometer

[0760] Perkin Elmer Series 200 Quard pump

[0761] Perkin Elmer Series 200 autosampler

[0762] Applied Biosystems 785A UV detector

[0763] Sedex 55 evaporative light scattering detector

[0764] A Valco column switch with a Valco actuator controlled by timed events from the pump.

[0765] The Sciex Sample control software running on a Macintosh PowerPC 7200 Computer was used for the instrument control and data acquisition.

[0766] The HPLC pump was connected to four eluent reservoirs containing:

[0767] A: acetonitrile

[0768] B: water

[0769] C: 0.5% TFA in water

[0770] D: 0.02 M ammonium acetate

[0771] 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.)

[0772] 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.

[0773] 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 100 spectrometer.

[0774] The remaining 1.48 mL/min was passed through the UV detector and to the ELS detector.

[0775] During the LC-analysis the detection data were acquired concurrently from the mass spectrometer, the UV detector and the ELS detector.

[0776] The LC conditions, detector settings and mass spectrometer settings used for the different methods are given in the following table.

3ColumnYMC ODS-A 120 Å s - 5μ 3 mm × 50 mm idGradient5%-90% acetonitrile in 0.05% TFA linearly during 7.5 min at1.5 mL/minDetectionUV: 214 nmELS: 40° C.MSExperiment:Start: 100 amuStop: 800 amuStep:0.2 amuDwell:0.571 msecMethod:Scan 284 times =9.5 min

[0777] HPLC-MS (Method C)

[0778] The Following Instrumentation is Used:

[0779] Hewlett Packard series 1100 G1312A Bin Pump

[0780] Hewlett Packard series 1100 Column compartment

[0781] Hewlett Packard series 1100 G1315A DAD diode array detector

[0782] Hewlett Packard series 1100 MSD

[0783] Sedere 75 Evaporative Light Scattering detector

[0784] The instrument is controlled by HP Chemstation software.

[0785] The HPLC pump is connected to two eluent reservoirs containing:

4A0.01% TFA in waterB0.01% TFA in acetonitrile

[0786] 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.

[0787] The HPLC conditions, detector settings and mass spectrometer settings used are given in the following table.

5ColumnWaters Xterra MS C-18 × 3 mm id 5 μmGradient50%-100% acetonitrile linear during 7.5 min at 1.5 ml/minDetection210 nm (analogue output from DAD)ELS (analogue output from ELS)MSionisation mode API-ESScan 100-1000 amu step 0.1 amu

[0788] After the DAD the flow is divided yielding approximately 1 ml/min to the ELS and 0.5 ml/min to the MS.

[0789] HPLC-MS (Method D)

[0790] The following instrumentation was used:

[0791] Sciex API 150 Single Quadropole mass spectrometer

[0792] Hewlett Packard Series 1100 G1312A Bin pump

[0793] Gilson 215 micro injector

[0794] Hewlett Packard Series 1100 G1315A DAD diode array detector

[0795] 15 Sedex 55 evaporative light scattering detector

[0796] A Valco column switch with a Valco actuator controlled by timed events from the pump.

[0797] The Sciex Sample control software running on a Macintosh Power G3 Computer was used for the instrument control and data acquisition.

[0798] The HPLC pump was connected to two eluent reservoirs containing:

[0799] A: Acetonitrile containing 0.05% TFA

[0800] B: Water containing 0.05% TFA

[0801] 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.)

[0802] 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.

[0803] 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.

[0804] The LC conditions, detector settings and mass spectrometer settings used for the different methods are given in the following table.

6ColumnWaters X-terra C18 5μ 3 mm × 50 mm idGradient5%-90% acetonitrile in 0.05% TFA linearly during 7.5 min at1.5 ml/minDetectionUV: 214 nmELS: 40° C.MSExperiment:Start: 100 amuStop: 800 amuStep:0.2 amuDwell:0.571 msecMethod:Scan 284 times =9.5 min

EXAMPLES

Example 1

[0805] 1H-Benzotriazole

Example 2

[0806] 5,6-Dimethyl-1H-benzotriazole

Example 3

[0807] 1H-Benzotriazole-5-carboxylic acid

Example 4

[0808] 4-Nitro-1H-benzotriazole

Example 5

[0809] 5-Amino-1H-benzotriazole

Example 6

[0810] 5-Chloro-1H-benzotriazole

Example 7

[0811] 5-Nitro-1H-benzotriazole

Example 8

[0812] 4-[(1H-Benzotriazole-5-carbonyl)amino]benzoic acid

[0813] 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).

[0814]

1
H-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.

[0815] General procedure (A) for preparation of compounds of general formula I1:

[0816] wherein U, J and R20 are as defined above, and J is optionally containing up to three substituents, R22, R23 and R24 as defined above.

[0817] 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 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.

[0818] The general procedure (A) is further illustrated in the following example:

Example 9 (General Procedure (A))

[0819] 1H-Benzotriazole-5-carboxylic acid phenylamide

[0820] 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 aliqot 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.

[0821] HPLC-MS (Method B): m/z: 239 (M+1); Rt=3.93 min.

[0822] The compounds in the following examples were similarly made. Optionally, the compounds may be isolated by filtration or by chromatography.

Example 10 (General Procedure (A))

[0823] 1H-Benzotriazole-5-carboxylic acid (4-methoxyphenyl)amide

[0824] HPLC-MS (Method A): m/z: 269 (M+1) & 291 (M+23); Rt=2.41 min HPLC-MS (Method B): m/z: 239 (M+1); Rt=3.93 min.

Example 11 (General Procedure (A))

[0825] {4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}carbamic acid tert-butyl ester

[0826] HPLC-MS (Method B): m/z: 354 (M+1); Rt=4.58 min.

Example 12 (General Procedure (A))

[0827] 1H-Benzotriazole-5-carboxylic acid (4-acetylaminophenyl)amide

[0828] HPLC-MS (Method B): m/z: 296 (M+1); Rt=3.32 min.

Example 13 (General Procedure (A))

[0829] 1H-Benzotriazole-5-carboxylic acid (3-fluorophenyl)amide

[0830] HPLC-MS (Method B): m/z: 257 (M+1); Rt=4.33 min.

Example 14 (General Procedure (A))

[0831] 1H-Benzotriazole-5-carboxylic acid (2-chlorophenyl)amide

[0832] HPLC-MS (Method B): m/z: 273 (M+1); Rt=4.18 min.

Example 15 (General Procedure (A))

[0833] 4-[(1H-Benzotriazole-5-carbonyl)amino]benzoic acid methyl ester

[0834] HPLC-MS (Method A): m/z: 297 (M+1); Rt: 2,60 min. HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.30 min.

Example 16 (General Procedure (A))

[0835] 1H-Benzotriazole-5-carboxylic acid (4-butylphenyl )amide

[0836] HPLC-MS (Method B): m/z: 295 (M+1); Rt=5.80 min.

Example 17 (General Procedure (A))

[0837] 1H-Benzotriazole-5-carboxylic acid (1-phenylethyl)amide

[0838] HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.08 min.

Example 18 (General Procedure (A))

[0839] 1H-Benzotriazole-5-carboxylic acid benzylamide

[0840] HPLC-MS (Method B): m/z: 253 (M+1); Rt=3.88 min.

Example 19 (General Procedure (A))

[0841] 1H-Benzotriazole-5-carboxylic acid 4-chlorobenzylamide

[0842] HPLC-MS (Method B): m/z: 287(M+1); Rt=4.40 min.

Example 20 (General Procedure (A))

[0843] 1H-Benzotriazole-5-carboxylic acid 2-chlorobenzylamide

[0844] HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.25 min.

Example 21 (General Procedure (A))

[0845] 1H-Benzotriazole-5-carboxylic acid 4-methoxybenzylamide

[0846] HPLC-MS (Method B): m/z: 283 (M+1); Rt=3.93 min.

Example 22 (General Procedure (A))

[0847] 1H-Benzotriazole-5-carboxylic acid 3-methoxybenzylamide

[0848] HPLC-MS (Method B): m/z: 283 (M+1); Rt=3.97 min.

Example 23 (General Procedure (A))

[0849] 1H-Benzotriazole-5-carboxylic acid (1,2-diphenylethyl)amide

[0850] HPLC-MS (Method B): m/z: 343 (M+1); Rt=5.05 min.

Example 24 (General Procedure (A))

[0851] 1H-Benzotriazole-5-carboxylic acid 3-bromobenzylamide

[0852] HPLC-MS (Method B): m/z: 331 (M+1); Rt=4.45 min.

Example 25 (General Procedure (A))

[0853] 4-{[(1H-Benzotriazole-5-carbonyl)amino]methyl}benzoic acid

[0854] HPLC-MS (Method B): m/z: 297 (M+1); Rt=3.35 min.

Example 26 (General Procedure (A))

[0855] 1H-Benzotriazole-5-carboxylic acid phenethylamide

[0856] HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.08 min.

Example 27 (General Procedure (A))

[0857] 1H-Benzotriazole-5-carboxylic acid [2-(4-chlorophenyl)ethyl]amide

[0858] HPLC-MS (Method B): m/z: 301 (M+1); Rt=4.50 min.

Example 28 (General Procedure (A))

[0859] 1H-Benzotriazole-5-carboxylic acid [2-(4-methoxyphenyl)ethyl]amide

[0860] HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.15 min.

Example 29 (General Procedure (A))

[0861] 1H-Benzotriazole-5-carboxylic acid [2-(3-methoxyphenyl)ethyl]amide

[0862] HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.13 min.

Example 30 (General Procedure (A))

[0863] 1H-Benzotriazole-5-carboxylic acid [2-(3-chlorophenyl)ethyl]amide

[0864] HPLC-MS (Method B): m/z: 301 (M+1); Rt=4.55 min.

Example 31 (General Procedure (A))

[0865] 1H-Benzotriazole-5-carboxylic acid (2,2-diphenylethyl)amide

[0866] HPLC-MS (Method B): m/z: 343 (M+1); Rt=5.00 min.

Example 32 (General Procedure (A))

[0867] 1H-Benzotriazole-5-carboxylic acid (3,4-dichlorophenyl)methylamide

[0868] HPLC-MS (Method B): m/z: 321 (M+1); Rt=4.67 min.

Example 33 (General Procedure (A))

[0869] 1H-Benzotriazole-5-carboxylic acid methylphenylamide

[0870] HPLC-MS (Method B): m/z: 253 (M+1); Rt=3.82 min.

Example 34 (General Procedure (A))

[0871] 1H-Benzotriazole-5-carboxylic acid benzylmethylamide

[0872] HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.05 min.

Example 35 (General Procedure (A))

[0873] 1H-Benzotriazole-5-carboxylic acid [2-(3-chloro-4-methoxyphenyl)ethyl]methyl-amide

[0874] HPLC-MS (Method B): m/z: 345 (M+1); Rt=4.37 min.

Example 36 (General Procedure (A))

[0875] 1H-Benzotriazole-5-carboxylic acid methylphenethylamide

[0876] HPLC-MS (Method B): m/z-281(M+1); Rt 4.15 min.

Example 37 (General Procedure (A))

[0877] 1H-Benzotriazole-5-carboxylic acid [2-(3,4-dimethoxyphenyl)ethyl]methylamide

[0878] HPLC-MS (Method B): m/z: 341 (M+1); Rt=3.78 min;

Example 38 (General Procedure (A))

[0879] 1H-Benzotriazole-5-carboxylic acid (2-hydroxy-2-phenylethyl)methylamide

[0880] HPLC-MS (Method B): m/z: 297 (M+1); Rt=3.48 min.

Example 39 (General Procedure (A))

[0881] 1H-Benzotriazole-5-carboxylic acid (3-bromophenyl)amide

[0882] HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.19 min.

Example 40 (General Procedure (A))

[0883] 1H-Benzotriazole-5-carboxylic acid (4-bromophenyl)amide

[0884] HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.18 min.

Example 41 (General Procedure (A))

[0885] {4-[(1H-Benzotriazole-5-carbonyl)amino]benzoylamino}acetic acid

[0886] HPLC-MS (Method A): m/z: 340 (M+1); Rt=1.71 min.

Example 42 (General Procedure (A))

[0887] {4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acetic acid

[0888] HPLC-MS (Method A): m/z: 297 (M+1); Rt=2.02 min.

Example 43 (General Procedure (A))

[0889] 3-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acrylic acid

[0890] HPLC-MS (Method A): m/z: 309 (M+1); Rt=3.19 min.

Example 44 (General Procedure (A))

[0891] {3-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acetic acid

[0892] HPLC-MS (Method A): m/z: 297 (M+1); Rt=2.10 min.

Example 45 (General Procedure (A))

[0893] 2-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenoxy}-2-methylpropionic acid

[0894] HPLC-MS (Method A): m/z: 341 (M+1); Rt=2.42 min.

Example 46 (General Procedure (A))

[0895] 3-{4-[(1H-Benzotriazole-5-carbonyl)amino]benzoylamino}propionic acid

[0896] HPLC-MS (Method A): m/z: 354 (M+1); Rt=1.78 min.

Example 47 (General Procedure (A))

[0897] 3-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}propionic acid

[0898] HPLC-MS (Method A): m/z: 311 (M+1); Rt 2.20 min.

Example 48 (General Procedure (A))

[0899] 1H-Benzotriazole-5-carboxylic acid (4-benzyloxyphenyl)amide

[0900] HPLC-MS (Method A): m/z: 345 (M+1); Rt=3.60 min.

Example 49 (General Procedure (A))

[0901] 1H-Benzotriazole-5-carboxylic acid (3-chloro4-methoxyphenyl)amide

[0902] HPLC-MS (Method A): m/z: 303 (M+1); Rt=2.88 min.

Example 50 (General Procedure (A))

[0903] 1H-Benzotriazole-5-carboxylic acid (4-phenoxyphenyl)amide

[0904] HPLC-MS (Method A): m/z: 331 (M+1); Rt=3.62 min.

Example 51 (General Procedure (A))

[0905] 1H-Benzotriazole-5-carboxylic acid (4-butoxyphenyl)amide

[0906] HPLC-MS (Method A): m/z: 311 (M+1); Rt=3.59 min.

Example 52 (General Procedure (A))

[0907] 1H-Benzotriazole-5-carboxylic acid (3-bromo-4-trifluoromethoxyphenyl)amide

[0908] HPLC-MS (Method A): m/z: 402 (M+1); Rt=3.93 min.

Example 53 (General Procedure (A))

[0909] 1H-Benzotriazole-5-carboxylic acid (3,5-dichloro-4-hydroxyphenyl)amide

[0910] HPLC-MS (Method A): m/z: 323 (M+1); Rt=2.57 min.

Example 54 (General Procedure (A))

[0911] 4-{[(1H-Benzotriazole-5-carbonyl)amino]methyl}benzoic acid

[0912] HPLC-MS (Method A): m/z: 297 (M+1); Rt=1.86 min.

Example 55 (General Procedure (A))

[0913] {4-[(1H-Benzotriazole-5-carbonyl)amino]phenylsulfanyl}acetic acid

[0914] HPLC-MS (Method A): m/z: 329 (M+1); Rt=2.34 min.

Example 56

[0915] N-(1H-Benzotriazol-5-yl)acetamide

[0916] HPLC-MS (Method A): m/z: 177 (M+1); Rt=0.84 min.

Example 57 (General Procedure (A))

[0917] 1H-Benzotriazole-5-carboxylic acid 4-nitrobenzylamide

[0918] General procedure (B) for preparation of compounds of general formula I2:

[0919] wherein X, Y, E and R10 are as defined above and E is optionally containing up to four optional substituents, R13, R14, R15, and R15A as defined above.

[0920] 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), rhodanine (X=Y=S) and hydantoin (X=O; Y=NH) in the presence of a base, such as sodium acetate, potassium acetate, ammonium acetate, piperidinium benzoate or an amine (eg piperidine, triethylamine and the like) in a solvent (eg acetic acid, ethanol, methanol, DMSO, DMF, NMP, toluene, benzene) or in a mixture of two or more of these solvents. The reaction is performed at room temperature or at elevated temperature, most often at or near the boiling point of the mixture. Optionally, azeotropic removal of the formed water can be done.

[0921] This general procedure (B) is further illustrated in the following example:

Example 58(General Procedure (B))

[0922] 5-(3-Phenoxybenzylidene)thiazolidine-2,4-dione

[0923] 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.

[0924] HPLC-MS (Method A): m/z: 298 (M+1); Rt=4.54 min.

[0925] The compounds in the following examples were similarly prepared. Optionally, the compounds can be further purified by filtration and washing with water, ethanol and/or heptane instead of concentration in vacuo. Also optionally the compounds can be purified by washing with ethanol, water and/or heptane, or by chromatography, such as preparative HPLC.

Example 59 (General Procedure (B))

[0926] 5-(4-Dimethylaminobenzylidene)thiazolidine-2,4-dione

[0927] HPLC-MS (Method C): m/z: 249 (M+1); Rt=4.90 min

Example 60 (General Procedure (B))

[0928] 5-Naphthalen-1-ylmethylenethiazolidine-2,4-dione

[0929] HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.16 min.

Example 61 (General Procedure (B))

[0930] 5-Benzylidene-thiazolidine-2,4-dione

100

[0931] HPLC-MS (Method A): m/z: 206 (M+1); Rt=4.87 min.

Example 62 (General Procedure (B))

[0932] 5-(4-Methoxy-benzylidene)-thiazolidine-2,4-dione

101

[0933] HPLC-MS (Method A): m/z: 263 (M+1); Rt=4.90 min.

Example 63 (General Procedure (B))

[0934] 5-(4-Chloro-benzylidene)-thiazolidine-2,4-dione

102

[0935] HPLC-MS (Method A): m/z: 240 (M+1); Rt=5.53 min.

Example 64 (General Procedure (B))

[0936] 5-(4-Nitro-benzylidene)-thiazolidine-2,4-dione

103

[0937] HPLC-MS (Method A): m/z: 251 (M+1); Rt=4.87 min.

Example 65 (General Procedure (B))

[0938] 5-(4-Hydroxy-3-methoxy-benzylidene)thiazolidine-2,4-dione

104

[0939] HPLC-MS (Method A): m/z: 252 (M+1); Rt=4.07 min.

Example 66 (General Procedure (B))

[0940] 5-(4-Methylsulfanyl-benzylidene)-thiazolidine-2,4-dione

105

[0941] HPLC-MS (Method A): m/z: 252 (M+1); Rt=5.43 min.

Example 67 (General Procedure (B))

[0942] 5-(3-Fluoro4-methoxy-benzylidene)-thiazolidine-2,4-dione

106

[0943] HPLC-MS (Method A): m/z: 354 (M+1); Rt=4.97 min.

Example 68 (General Procedure (B))

[0944] 5-(4-tert-Butylbenzylidene)thiazolidine-2,4-dione

107

[0945] HPLC-MS (Method A): m/z: 262 (M+1); Rt=6.70 min.

Example 69 (General Procedure (B))

[0946] N-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acetamide

108

[0947] HPLC-MS (Method A): m/z: 263 (M+1); Rt=3.90 min.

Example 70 (General Procedure (B))

[0948] 5-Biphenyl4-ylmethylene-thiazolidine-2,4-dione

109

[0949] HPLC-MS (Method A): m/z: 282 (M+1); Rt=4.52 min.

Example 71 (General Procedure (B))

[0950] 5-(4-Phenoxy-benzylidene)-thiazolidine-2,4-dione

110

[0951] HPLC-MS (Method A): m/z: 298 (M+1); Rt=6.50 min.

Example 72 (General Procedure (B))

[0952] 5-(3-Benzyloxybenzylidene)thiazolidine-2,4-dione

111

[0953] HPLC-MS (Method A): m/z: 312 (M+1); Rt=6.37 min.

Example 73 (General Procedure (B))

[0954] 5-(3-p-Tolyloxybenzylidene)thiazolidine-2,4-dione

112

[0955] HPLC-MS (Method A): m/z: 312 (M+1); Rt=6.87 min.

Example 74 (General Procedure (B))

[0956] 5-Napthalen-2-ylmethylene-thiazolidine-2,4-dione

113

[0957] HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.15 min.

Example 75 (General Procedure (B))

[0958] 5-Benzo[1,3]dioxol-5-ylmethylenethiazolidine-2,4-dione

114

[0959] HPLC-MS (Method A): m/z: 250 (M+1), Rt=3.18 min.

Example 76 (General Procedure (B))

[0960] 5-(4-Chlorobenzylidene)-2-thioxothiazolidin-4-one

115

[0961] HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.51 min.

Example 77 (General Procedure (B))

[0962] 5-(4-Dimethylaminobenzylidene)-2-thioxothiazolidin-4-one

116

[0963] HPLC-MS (Method A): m/z: 265 (M+1); Rt=5.66 min.

Example 78 (General Procedure (B))

[0964] 5-(4-Nitrobenzylidene)-2-thioxothiazolidin-4-one

117

[0965] HPLC-MS (Method A): m/z: 267 (M+1); Rt=3.94 min.

Example 79 (General Procedure (B))

[0966] 5-(4-Methylsulfanylbenzylidene)-2-thioxothiazolidin4-one

118

[0967] HPLC-MS (Method A): m/z: 268 (M+l); Rt=6.39 min.

Example 80 (General Procedure (B))

[0968] 5-(3-Fluoro4-methoxybenzylidene)-2-thioxothiazolidin4-one

119

[0969] HPLC-MS (Method A): m/z: 270 (M+1); Rt=5.52 min.

Example 81 (General Procedure (B))

[0970] 5-Naphthalen-2-ylmethylene-2-thioxothiazolidin-4-one

120

[0971] HPLC-MS (Method A): m/z: 272 (M+1); Rt=6.75 min.

Example 82 (General Procedure (B))

[0972] 5-(4-Diethylaminobenzylidene)-2-thioxothiazolidin4-one

121

[0973] HPLC-MS (Method A): m/z: 293 (M+1); Rt=5.99 min.

Example 83 (General Procedure (B))

[0974] 5-Biphenyl-4-ylmethylene-2-thioxothiazolidin-4-one

122

[0975] HPLC-MS (Method A): m/z: 298 (M+1); Rt=7.03 min.

Example 84 (General Procedure (B))

[0976] 5-(3-Phenoxybenzylidene)-2-thioxothiazolidin-4-one

123

[0977] HPLC-MS (Method A): m/z: 314 (M+1); Rt=6.89 min.

Example 85 (General Procedure (B))

[0978] 5-(3-Benzyloxybenzylidene)-2-thioxothiazolidin-4-one

124

[0979] HPLC-MS (Method A): m/z: 328 (M+1); Rt=6.95 min.

Example 86 (General Procedure (B))

[0980] 5-(4-Benzyloxybenzylidene)-2-thioxothiazolidin-4-one

125

[0981] HPLC-MS (Method A): m/z: 328 (M+1); RT=6.89 min.

Example 87 (General Procedure (B))

[0982] 5-Naphthalen-1-ylmethylene-2-thioxothiazolidin4-one

126

[0983] HPLC-MS (Method A): m/z: 272 (M+1); Rt=6.43 min.

Example 88 (General Procedure (B))

[0984] 5-(3-Methoxybenzyl)thiazolidine-2,4-dione

127

[0985] HPLC-MS (Method A): m/z: 236 (M+1 ); Rt=3.05 min.

Example 89 (General Procedure (D))

[0986] 4-[2-Chloro4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid ethyl ester

128

[0987] HPLC-MS (Method A): m/z: 392 (M+23), Rt=4.32 min.

Example 90 (General Procedure (D))

[0988] 4-[2-Bromo-4-(2,4dioxothiazolidin-5-ylidenemethyl)-phenoxy]-butyric acid

129

[0989] HPLC-MS (Method A): m/z: 410 (M+23); Rt=3.35 min.

Example 91 (General Procedure (B))

[0990] 5-(3-Bromobenzylidene)thiazolidine-2,4-dione

130

[0991] HPLC-MS (Method A): m/z: 285 (M+1); Rt=4.01 min.

Example 92 (General Procedure (B))

[0992] 5-(4-Bromobenzylidene)thiazolidine-2,4-dione

131

[0993] HPLC-MS (Method A): m/z: 285 (M+1); Rt=4.05 min.

Example 93 (General Procedure (B))

[0994] 5-(3-Chlorobenzylidene)thiazolidine-2,4-dione

132

[0995] HPLC-MS (Method A): m/z: 240 (M+1); Rt=3.91 min.

Example 94 (General Procedure (B))

[0996] 5-Thiophen-2-ylmethylenethiazolidine-2,4-dione

133

[0997] HPLC-MS (Method A): m/z: 212 (M+1); Rt=3.09 min.

Example 95 (General Procedure (B))

[0998] 5-(4-Bromothiophen-2-ylmethylene)thiazolidine-2,4-dione

134

[0999] HPLC-MS (Method A): m/z: 291 (M+1); Rt=3.85 min.

Example 96 (General Procedure (B))

[1000] 5-(3,5-Dichlorobenzylidene)thiazolidine-2,4-dione

135

[1001] HPLC-MS (Method A): m/z: 274 (M+1); Rt=4.52 min.

Example 97 (General Procedure (B))

[1002] 5-(1-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

136

[1003] HPLC-MS (Method A): m/z: 259 (M+1); Rt=3.55 min.

Example 98 (General Procedure (B))

[1004] 5-(1H-Indol-3-ylmethylene)thiazolidine-2,4-dione

137

[1005] HPLC-MS (Method A): m/z: 245 (M+1); Rt=2.73 min.

Example 99 (General Procedure (B))

[1006] 5-Fluoren-9-ylidenethiazolidine-2,4-dione

138

[1007] HPLC-MS (Method A): m/z: 280 (M+1); Rt=4.34 min.

Example 100 (General Procedure (B))

[1008] 5-(1-Phenylethylidene)thiazolidine-2,4-dione

139

[1009] HPLC-MS (Method A): m/z: 220 (M+1); Rt=3.38 min.

Example 101 (General Procedure (B))

[1010] 5-[1-(4-Methoxyphenyl)-ethylidene]-thiazolidine-2,4-dione

140

[1011] HPLC-MS (Method A): m/z: 250 (M+1); Rt=3.55 min.

Example 102 (General Procedure (B))

[1012] 5-(1-Naphthalen-2-yl-ethylidene)-thiazolidine-2,4-dione

141

[1013] HPLC-MS (Method A): m/z: 270 (M+1); Rt=4.30 min.

Example 103 (General Procedure (B))

[1014] 5-[1-(4-Bromophenyl)-ethylidene]-thiazolidine-2,4-dione

142

[1015] HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.18 min.

Example 104 (General Procedure (B))

[1016] 5-(2,2-Diphenylethylidene)-thiazolidine-2,4-dione

143

[1017] HPLC-MS (Method A): m/z: 296 (M+1); Rt=4.49 min.

Example 105 (General Procedure (B))

[1018] 5-[1-(3-Methoxyphenyl)-ethylidene]-thiazolidine-2,4-dione

144

[1019] HPLC-MS (Method A): m/z: 250 (M+1); Rt=3.60 min.

Example 106 (General Procedure (B))

[1020] 5-[1-(6-Methoxynaphthalen-2-yl)-ethylidene]-thiazolidine-2,4-dione

145

[1021] HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.26 min.

Example 107 (General Procedure (B))

[1022] 5-[1-(4-Phenoxyphenyl)-ethylidene]-thiazolidine-2,4-dione

146

[1023] HPLC-MS (Method A): m/z: 312 (M+1); Rt=4.68 min.

Example 108 (General Procedure (B))

[1024] 5-[1-(3-Fluoro-4-methoxyphenyl)ethylidene]thiazolidine-2,4-dione

147

[1025] HPLC-MS (Method A): m/z: 268 (M+1); Rt=3.58 min.

Example 109 (General Procedure (B))

[1026] 5-[1-(3-Bromophenyl)-ethylidene]-thiazolidine-2,4-dione

148

[1027] HPLC-MS (Method A): m/z: 300 (M+1); Rt=4,13 min.

Example 110 (General Procedure (B))

[1028] 5-Anthracen-9-ylmethylenethiazolidine-2,4-dione

149

[1029] HPLC-MS (Method A): m/z: 306 (M+1); Rt=4.64 min.

Example 111 (General Procedure (B))

[1030] 5-(2-Methoxynaphthalen-1-ylmethylene)-thiazolidine-2,4-dione

150

[1031] HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.02 min.

Example 112 (General Procedure (B))

[1032] 5-(4-Methoxynaphthalen-1-ylmethylene)-thiazolidine-2,4-dione

151

[1033] HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.31 min.

Example 113 (General Procedure (B))

[1034] 5-(4-Dimethylaminonaphthalen-1-ylmethylene)-thiazolidine-2,4-dione

152

[1035] HPLC-MS (Method A): m/z: 299 (M+1); Rt=4.22 min.

Example 114 (General Procedure (B))

[1036] 5-(4-Methylnaphthalen-1-ylmethylene)-thiazolidine-2,4-dione

153

[1037] HPLC-MS (Method A): m/z: 270 (M+1); Rt=4.47 min.

Example 115 (General Procedure (B))

[1038] 5-Pyridin-2-ylmethylene-thiazolidine-2,4-dione

154

Example 116

[1039] 5-Pyridin-2-ylmethyl-thiazolidine-2,4-dione

155

[1040] 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.

[1041] TLC: Rf=0.30 (SiO2; EtOAc:heptane 1:1)

Example 117 (General Procedure (B))

[1042] 5-(1H-Imidazol-4-ylmethylene)-thiazolidine-2,4-dione

156

Example 118 (General Procedure (B))

[1043] 5-(4-Benzyloxy-benzylidene)-thiazolidine-2,4-dione

157

[1044] HPLC-MS (Method A): m/z: 6.43 min; 99% (2A)

Example 119 (General Procedure (B))

[1045] 5-[4-(4-Fluorobenzyloxy)benzylidene]-2-thioxothiazolidin-4-one

158

Example 120 (General Procedure (B))

[1046] 5-(4-Butoxybenzylidene)-2-thioxothiazolidin-4-one

159

Example 121 (General Procedure (B))

[1047] 5-(3-Methoxybenzylidene)thiazolidine-2,4-dione

160

[1048] HPLC-MS (Method A): m/z: 236 (M+1); Rt=4.97 min

Example 122 (General Procedure (B))

[1049] 5-(3-Methoxybenzylidene)imidazolidine-2,4-dione

161

[1050] HPLC-MS (Method A): m/z: 219 (M+1); Rt=2.43 min.

Example 123 (General Procedure (B))

[1051] 5-(4-Methoxybenzylidene)imidazolidine-2,4-dione

162

[1052] HPLC-MS (Method A): m/z: 219 (M+1); Rt=2.38 min.

Example 124 (General Procedure (B))

[1053] 5-(2,3-Dichlorobenzylidene)thiazolidine-2,4-dione

163

Example 125 (General Procedure (B))

[1054] 5-Benzofuran-7-ylmethylenethiazolidine-2,4-dione

164

[1055] HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.57 min.

Example 126 (General Procedure (B))

[1056] 5-Benzo[1,3]dioxol-4-ylmethylenethiazolidine-2,4-dione

165

[1057] HPLC-MS (Method C): m/z: 250 (M+1); Rt=4.00 min.

Example 127 (General Procedure (B))

[1058] 5-(4-Methoxy-2,3-dimethylbenzylidene)thiazolidine-2,4-dione

166

[1059] HPLC-MS (Method C): m/z: 264 (M+1); Rt=5,05 min.

Example 128 (General Procedure (B))

[1060] 5-(2-Benzyloxy-3-methoxybenzylidene)thiazolidine-2,4-dione

167

[1061] HPLC-MS (Method C): m/z: 342 (M+1); Rt=5,14 min.

Example 129 (General Procedure (B))

[1062] 5-(2-Hydroxybenzylidene)thiazolidine-2,4-dione

168

[1063] HPLC-MS (Method C): m/z: 222 (M+1); Rt=3,67 min.

Example 130 (General Procedure (B))

[1064] 5-(2,4-Dichlorobenzylidene)thiazolidine-2,4-dione

169

[1065]

1
H-NMR (DMSO-d6): 7.60 (2H, “s”), 7.78 (1H, s), 7.82 (1H, s).

Example 131 (General Procedure (B))

[1066] 5-(2-Chlorobenzylidene)thiazolidine-2,4-dione

170

[1067]

1
H-NMR (DMSO-d6): 7.40 (1H, t), 7.46 (1H, t), 7.57 (1H, d), 7.62 (1H, d), 7.74 (1H, s).

Example 132 (General Procedure (B))

[1068] 5-(2-Bromobenzylidene)thiazolidine-2,4-dione

171

[1069]

1
H-NMR (DMSO-d6): 7.33 (1H, t), 7.52 (1H, t), 7.60 (1H, d), 7.71 (1H, s), 7.77 (1H, d).

Example 133 (General Procedure (B))

[1070] 5-(2,4-Dimethoxybenzylidene)thiazolidine-2,4-dione

172

[1071] HPLC-MS (Method C): m/z: 266 (M+1) Rt=4.40 min.

Example 134 (General Procedure (B))

[1072] 5-(2-Methoxybenzylidene)thiazolidine-2,4-dione

173

[1073] HPLC-MS (Method C): m/z: 236 (M+1); Rt=4.17 min.

Example 135 (General Procedure (B))

[1074] 5-(2,6-Difluorobenzylidene)thiazolidine-2,4-dione

174

[1075] HPLC-MS (Method C): m/z: 242 (M+1); Rt=4.30 min.

Example 136 (General Procedure (B))

[1076] 5-(2,4-Dimethylbenzylidene)thiazolidine-2,4-dione

175

[1077] HPLC-MS (Method C): m/z: 234 (M+1); Rt=5,00 min.

Example 137 (General Procedure (B))

[1078] 5-(2,4,6-Trimethoxybenzylidene)thiazolidine-2,4-dione

176

[1079] HPLC-MS (Method C): m/z: 296 (M+1); Rt=4.27 min.

Example 138 (General Procedure (B))

[1080] 5-(4-Hydroxy-2-methoxybenzylidene)thiazolidine-2,4-dione

177

[1081] HPLC-MS (Method C): m/z: 252 (M+1); Rt=3,64 min.

Example 139 (General Procedure (B))

[1082] 5-(4-Hydroxynaphthalen-1-ylmethylene)thiazolidine-2,4-dione

178

[1083]

1
H-NMR (DMSO-d6): δ=7.04 (1H, d), 7.57 (2H, m), 7.67 (1H, t), 8.11 (1H, d), 8.25 (1H, d), 8.39 (1H, s) 11.1 (1H, s), 12.5 (1H, bs). HPLC-MS (Method C): m/z: 272 (M+1); Rt=3.44 min.

Example 140 (General Procedure (B))

[1084] 5-(2-Trifluoromethoxybenzylidene)thiazolidine-2,4-dione

179

[1085] HPLC-MS (Method C): m/z: 290 (M+1); Rt=4.94 min.

Example 141 (General Procedure (B))

[1086] 5-Biphenyl-2-ylmethylenethiazolidine-2,4-dione

180

[1087] HPLC-MS (Method C): m/z: 282 (M+1); Rt=5,17 min.

Example 142 (General Procedure (B))

[1088] 5-(2-Benzyloxybenzylidene)thiazolidine-2,4-dione

181

[1089] HPLC-MS (Method C): m/z: 312 (M+1); Rt=5,40 min.

Example 143 (General Procedure (B))

[1090] 5-Adamantan-2-ylidenethiazolidine-2,4-dione

182

[1091] HPLC-MS (Method A): m/z: 250 (M+1); Rt=4.30 min.

[1092] General procedure (C) for preparation of compounds of general formula I2:

183

[1093] wherein X, Y, E, and R10 are as defined above and E is optionally containing up to four optional substituents, R13, R14, R15, and R15A as defined above.

[1094] This general procedure (C) is quite similar to general procedure (B) and is further illustrated in the following example:

Example 144 (General Procedure (C))

[1095] 5-(3,4-Dibromobenzylidene)thiazolidine-2,4-dione

184

[1096] 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.

[1097]

1
H 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.

[1098] The compounds in the following examples were similarly prepared. Optionally, the compounds can be further purified by filtration and washing with water instead of concentration in vacuo. Also optionally the compounds can be purified by washing with ethanol, water and/or heptane, or by preparative HPLC.

Example 145 (General Procedure (C))

[1099] 5-(4-Hydroxy-3-iodo-5-methoxybenzylidene)thiazolidine-2,4-dione

185

[1100] 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) δ=168.0, 167.7, 149.0, 147.4, 133.0, 131.2, 126.7, 121.2, 113.5, 85.5, 56.5; HPLC-MS (Method A): m/z: 378 (M+1); Rt=3.21 min.

Example 146 (General Procedure (C))

[1101] 5-(4-Hydroxy-2,6-dimethylbenzylidene)thiazolidine-2,4-dione

186

[1102] HPLC-MS (Method C): m/z 250 (M+1); Rt.=2.45 min.

Example 147 (General Procedure (C))

[1103] 4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-2-yloxymethyl]-benzoic acid

187

[1104] HPLC-MS (Method C): m/z: 506 (M+23); Rt.=4.27 min.

Example 148 (General Procedure (C))

[1105] 5-(4-Bromo-2,6-dichlorobenzylidene)thiazolidine-2,4-dione

188

[1106] HPLC-MS (Method C): m/z: 354 (M+1); Rt.=4.36 min.

Example 149 (General Procedure (C))

[1107] 5-(6-Hydroxy-2-naphthylmethylene)thiazolidine-2,4-dione

189

[1108] 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.

[1109] Preparation of the starting material, 6-hydroxy-2-naphtalenecarbaldehyde:

[1110] 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.

[1111] 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).

[1112] Alternative preparation of 6-hydroxy-2-naphtalenecarbaldehyde:

[1113] 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 RT. 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.

Example 150 (General Procedure (C))

[1114] 5-(3-Iodo-4-methoxybenzylidene)thiazolidiene-2,4-dione

190

[1115]

1
H NMR (DMSO-d6): δH 12.55 (s,broad,1H), 8.02 (d,1H), 7.72 (s,1H) 7.61 (d,1H),7.18(d,1H), 3.88 (s,3H); 3C NMR (DMSO-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.

[1116] Preparation of the starting material, 3-iodo-4-methoxybenzaldehyde:

[1117] 4-Methoxybenzaldehyde (0.5 g, 3.67 mmol) and silver trifluoroacetate (0.92 g, 4.19 mmol) were mixed in dichloromethane (25 mL). Iodine (1.19 g, 4.7 mmol) was added in small portions and the mixture was stirred overnight at room temperature under nitrogen. The mixture was subsequently filtered and the residue washed with DCM. The combined filtrates were treated with an acqueous sodium thiosulfate solution (1 M) until the colour disappeared. Subsequent extraction with dichloromethane (3×20 mL) followed by drying with MgSO4 and evaporation in vacuo afforded 0.94 g of 3-iodo-4-methoxybenzaldehyde.

[1118] Mp 104-107° C.; HPLC-MS (Method A): m/z: 263 (M+1); Rt=3.56 min.; 1H NMR (CDCl3): δH=8.80 (s,1H), 8.31 (d,1H), 7.85 (dd,1H) 6.92 (d,1H), 3.99 (s, 3H).

Example 151 (General Procedure (C))

[1119] 5-(1-Bromonaphthalen-2-ylmethylene)thiazolidine-2,4-dione

191

[1120] HPLC-MS (Method A): m/z:=336 (M+1); Rt=4.46 min.

Example 152 (General Procedure (C))

[1121] 1-[5-(2,4-Dioxothiazolidin-5-ylidenemethyl)thiazol-2-yl]piperidine-4-carboxylic acid ethyl ester

192

[1122]

1
H 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.

Example 153 (General Procedure (C))

[1123] 5-(2-Phenyl-[1,2,3]triazol-4-ylmethylene)thiazolidine-2,4-dione

193

[1124]

1
H 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.

Example 154 (General Procedure (C))

[1125] 5-(Quinolin-4-ylmethylene)thiazolidine-2,4-dione

194

[1126] HPLC-MS (Method A): m/z: 257 (M+1); Rt=2.40 min.

Example 155 (General Procedure (C))

[1127] 5-(6-Methylpyridin-2-ylmethylene)thiazolidine-2,4-dione

195

[1128]

1
H NMR (DMSO-d6): δH=12.35 (s,broad,1H), 7.82 (t,1H), 778 (s,1H), 7.65 (d,1H), 7.18 (d,1H), 2.52 (s,3 H); HPLC-MS (Method A): m/z: 221 (M+1); Rt=3.03 min.

Example 156 (General Procedure (C))

[1129] 5-(2,4-dioxothiazolidin-5-ylidenemethyl)-furan-2-ylmethylacetate

196

[1130]

1
H 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.

Example 157 (General Procedure (C))

[1131] 5-(2,4-Dioxothiazolidin-5-ylidenemethyl)furan-2-sulfonic acid

197

[1132] HPLC-MS (Method A): m/z: 276 (M+1); Rt=0.98 min.

Example 158 (General Procedure (C))

[1133] 5-(5-Benzyloxy-1H-pyrrolo[2,3-c]pyridin-3-ylmethylene)-thiazolidine-2,4-dione

198

[1134] HPLC-MS (Method A): m/z: 352 (M+1); Rt=3.01 min.

Example 159 (General Procedure (C))

[1135] 5-(Quinolin-2-ylmethylene)thiazolidine-2,4-dione

199

[1136] HPLC-MS (Method A): m/z: 257 (M+1); Rt=3.40 min.

Example 160 (General Procedure (C))

[1137] 5-(2,4-Dioxothiazolidin-5-ylidenemethyl)thiophene-2-carboxylic acid

200

[1138] HPLC-MS (Method A): m/z: 256 (M+1); Rt=1.96 min.

Example 161 (General Procedure (C))

[1139] 5-(2-Phenyl-1H-imidazol-4-ylmethylene)thiazolidine-2,4-dione

201

[1140] HPLC-MS (Method A): m/z: 272 (M+1); Rt=2.89 min.

Example 162 (General Procedure (C))

[1141] 5-(4-Imidazol-1-yl-benzylidene)thiazolidine-2,4-dione

202

[1142] HPLC-MS (Method A): m/z: 272 (M+1); Rt=1.38 min.

Example 163 (General Procedure (C))

[1143] 5-(9-Ethyl-9H-carbazol-3ylmethylene)thiazolidine-2,4-dione

203

[1144] HPLC-MS (Method A): m/z: 323 (M+1); Rt=4.52 min.

Example 164 (General Procedure (C))

[1145] 5-(1,4-Dimethyl-9H-carbazol-3-ylmethylene)thiazolidine-2,4-dione

204

[1146] HPLC-MS (Method A): m/z: 323 (M+1); Rt=4.35 min.

Example 165 (General Procedure (C))

[1147] 5-(2-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

205

[1148] HPLC-MS (Method A): m/z: 259 (M+1); Rt=3.24 min.

Example 166 (General Procedure (C))

[1149] 5-(2-Ethylindol-3-ylmethylene)thiazolidine-2,4-dione

206

[1150] 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 residur was purified by column chromatography on silica gel using heptane/ether (4/1) as eluent. This afforded 720 mg (69%) of 2-ethylindole.

[1151]

1
H 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).

[1152] 2-Ethylindole (0.5 g, 3.4 mmol) dissolved in DMF (2 mL) was added to a cold (020 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 4020 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).

[1153] HPLC-MS (Method C): m/z: 174 (M+1); Rt.=2.47 min.

[1154] 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).

[1155] HPLC-MS (Method C): m/z: 273 (M+1); Rt.=3.26 min.

Example 167 (General Procedure (C))

[1156] 5-[2-(4-Bromophenylsulfanyl)-1-methyl-1H-indol-3-ylmethylene]thiazolidine-2,4-dione

207

[1157] HPLC-MS (Method A): m/z: 447 (M+1); Rt=5.25 min.

Example 168 (General Procedure (C))

[1158] 5-[2-(2,4-Dichlorobenzyloxy)-naphthalen-1-ylmethylene]thiazolidine-2,4-dione

208

[1159] HPLC-MS (Method A): (anyone 1) m/z: 430 (M+1); Rt=5.47 min.

Example 169 (General Procedure (C))

[1160] 5-{4-[3-(4-Bromophenyl)-3-oxopropenyl]-benzylidene}thiazolidine-2,4-dione

209

[1161] HPLC-MS (Method A): m/z: 416 (M+1); Rt=5.02 min.

Example 170 (General Procedure (C))

[1162] 5-(4-Pyridin-2-ylbenzylidene)thiazolidine-2,4-dione

210

[1163] HPLC-MS (Method A): m/z: 283 (M+1), Rt=2.97 min.

Example 171 (General Procedure (C))

[1164] 5-(3,4-Bisbenzyloxybenzylidene)thiazolidine-2,4-dione

211

[1165] HPLC-MS (Method A): m/z: 418 (M+1); Rt=5.13 min.

Example 172 (General Procedure (C))

[1166] 5-[4-(4-Nitrobenzyloxy)-benzylidene]thiazolidine-2,4-dione

212

[1167] HPLC-MS (Method A): m/z: 357 (M+1); Rt=4.45 min.

Example 173 (General Procedure (C))

[1168] 5-(2-Phenyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

213

[1169] HPLC-MS (Method A): m/z: 321 (M+1); Rt=3.93 min.

Example 174 (General-Procedure-(C))

[1170] 5-(5-Benzyloxy-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

214

[1171] HPLC-MS (Method A): m/z: 351 (M+1); Rt=4.18 min.

Example 175 (General Procedure (C))

[1172] 5-(4-Hydroxybenzylidene)thiazolidine-2,4-dione

215

[1173] HPLC-MS (Method A); m/z: 222 (M+1); Rt=2.42 min.

Example 176 (General Procedure (C))

[1174] 5-(1-Methyl-1H-indol-2-ylmethylene)thiazolidine-2,4-dione

216

[1175]

1
H 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.

Example 177 (General Procedure (C))

[1176] 5-(5-Nitro-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

217

[1177] .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.

Example 178 (General Procedure (C))

[1178] 5-(6-Methoxynaphthalen-2-ylmethylene)thiazolidine-2,4-dione

218

[1179] HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.27 min.

Example 179 (General Procedure (C))

[1180] 5-(3-Bromo-4-methoxybenzylidene)thiazolidine-2,4-dione

219

[1181] HPLC-MS (Method A): m/z: 314 (M+1), Rt=3.96 min.

Example 180 (General Procedure (C))

[1182] 3-{(2-Cyanoethyl)-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]amino}propionitrile

220

[1183] HPLC-MS (Method A): m/z: 327 (M+1); Rt=2.90 min.

Example 181 (General Procedure (C))

[1184] 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methyl ester

221

[1185] HPLC-MS (Method A): m/z: 303 (M+1); Rt=3.22-3-90 min.

Example 182

[1186] 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid pentyl ester

222

[1187] (3-(2,4-Dioxohiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methyl ester (example 181, 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).

[1188] HPLC-MS (Method C): m/z: 359 (M+1); Rt.=4.25 min.

Example 183 (General Procedure (C))

[1189] 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carboxylic acid

223

[1190] HPLC-MS (Method A): m/z: 289 (M+1); Rt=2.67 min.

Example 184 (General Procedure (C))

[1191] 5-(1-Benzylindol-3-ylmethylene)thiazolidine-2,4-dione

224

[1192] HPLC-MS (Method A): m/z: 335 (M+1); Rt=4.55 min.

Example 185 (General Procedure (C))

[1193] 5-(1-Benzenesulfonylindol-3-ylmethylene)thiazolidine-2,4-dione

225

[1194] HPLC-MS (Method A): m/z:=385 (M+1); Rt=4.59 min.

Example 186 (General Procedure (C))

[1195] 5-(4-[1,2,3]Thiadiazol-4-ylbenzylidene)thiazolidine-2,4-dione

226

[1196] HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.45 min.

Example 187 (General Procedure (C))

[1197] 5-[4-(4-Nitrobenzyloxy)-benzylidene]thiazolidine-2,4-dione

227

[1198] HPLC-MS (Method A): m/z: 357 (M+1); Rt=4.42 min.

Example 188 (General Procedure (C))

[1199] 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-1-carboxylic acid ethyl ester

228

[1200] HPLC-MS (Method A): m/z: 317 (M+1); Rt=4.35 min.

Example 189 (General Procedure (C))

[1201] 5-[2-(4-Pentylbenzoyl)-benzofuran-5-ylmethylene]thiazolidine-2,4-dione

229

[1202] HPLC-MS (Method A): m/z: 420 (M+1); Rt=5.92 min.

Example 190 (General Procedure (C))

[1203] 5-[1-(2-Fluorobenzyl)-4-nitroindol-3-ylmethylene]thiazolidine-2,4-dione

230

[1204] HPLC-MS (Method A): (Anyone 1) m/z: 398 (M+1); Rt=4.42 min.

Example 191 (General Procedure (C))

[1205] 5-(4-Benzyloxyindol-3-ylmethylene)thiazolidine-2,4-dione

231

[1206] HPLC-MS (Method A): m/z: 351 (M+1); Rt=3.95 min.

Example 192 (General Procedure (C))

[1207] 5-(4-Isobutylbenzylidene)-thiazolidine-2,4-dione

232

[1208] HPLC-MS (Method A): m/z: 262 (M+1); Rt=4.97 min.

Example 193 (General Procedure (C))

[1209] Trifluoromethanesulfonic acid 4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yl ester

233

[1210] HPLC-MS (Method A): m/z: 404 (M+1); Rt=4.96 min.

[1211] Preparation of Starting Material:

[1212] 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.

Example 194 (General Procedure (C))

[1213] 5-(4-Nitroindol-3-ylmethylene)thiazolidine-2,4-dione

234

[1214] HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.14 min.

Example 195 (General Procedure (C))

[1215] 5-(3,5-Dibromo-4-hydroxy-benzylidene)thiazolidine-2,4-dione

235

[1216]

1
H 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.

Example 196 (General Procedure (C))

[1217]

236

[1218] HPLC-MS (Method A): m/z: 385 (M+1); Rt=5.08 min.

[1219] General procedure for preparation of starting materials for examples 196-199: 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.

[1220] 20 mL portions of this solution were mixed with equimolar amounts of arylmethylbromides in acetone (10 mL). The mixtures were stirred at RT for 4 days and subsequently evaporated to dryness and checked by HPLC-MS. The crude products, 1-benzylated indole-3-carbaldehydes, were used for the reaction with thiazolidine-2,4-dione using the general procedure C.

Example 197 (General Procedure (C))

[1221] 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-ylmethyl]benzoic acid methyl ester

237

[1222] HPLC-MS (Method A): m/z: 393 (M+1); Rt=4.60 min.

Example 198 (General Procedure (C))

[1223] 5-[1-(9,10-Dioxo-9,10-dihydroanthracen-2-ylmethyl)-1H-indol-3-ylmethylene]thiazolidine-2,4-dione

238

[1224] HPLC-MS (Method A): m/z: 465 (M+1); Rt=5.02 min.

Example 199 (General Procedure (C))

[1225] 4′-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-ylmethyl]biphenyl-2-carbonitrile

239

[1226] HPLC-MS (Method A): m/z: 458 (M+23); Rt=4.81 min.

Example 200 (General Procedure (C))

[1227] 3-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-methylindol-1-ylmethyl]benzonitrile.

240

[1228] 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).

[1229] HPLC-MS (Method C): m/z: 374 (M+1); Rt.=3.95 min.

Example 201 (General Procedure (C))

[1230] 5-(1-Benzyl-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione

241

[1231] This compound was prepared in analogy with the compound described in example 200 from benzyl bromide and 2-methylindole-3-carbaldehyde, followed by reaction with thiazolidine-2,4-dione resulting in 50 mg of the title compound.

[1232] HPLC-MS (Method C): m/z: 349 (M+1); Rt.=4.19 min.

Example 202

[1233] 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-methylindol-1-ylmethyl]benzoic acid methyl ester

242

[1234] This compound was prepared in analogy with the compound described in example 200 from 4-(bromomethyl)benzoic acid methyl ester and 2-methylindole-3-carbaldehyde, followed by reaction with thiazolidine-2,4-dione.

[1235] HPLC-MS (Method C): m/z: 407 (M+1); Rt.=4.19 min.

Example 203 (General Procedure (C))

[1236] 5-(2-Chloro-1-methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

243

[1237] HPLC-MS (Method A): m/z: 293 (M+1); Rt=4.10 min.

Example 204 (General Procedure (C))

[1238] 5-(4-Hydroxy-3,5-diiodo-benzylidene)-thiazolidine-2,4-dione

244

[1239] HPLC-MS (Method A): m/z: 474 (M+1); Rt=6.61 min.

Example 205 (General Procedure (C))

[1240] 5-(4-Hydroxy-3-iodobenzylidene)thiazolidine-2,4-dione

245

[1241] 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).

Example 206 (General Procedure (C))

[1242] 5-(2,3,6-Trichlorobenzylidene)thiazolidine-2,4-dione

246

[1243] H PLC-MS (Method C): m/z: 309 (M+1); Rt.=4.07 min

Example 207 (General Procedure (C))

[1244] 5-(2,6-Dichlorobenzylidene)thiazolidine-2,4-dione

247

[1245] 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).

Example 208 (General Procedure (C))

[1246] 5-[1-(2,6-Dichloro-4-trifluoromethylphenyl)-2,5-dimethyl-1H-pyrrol-3-ylmethylene]thiazolidine-2,4-dione

248

[1247] HPLC-MS (Method C): m/z: 436 (M+1); Rt.=4.81 min

Example 209 (General Procedure (C))

[1248] 5-[1-(3,5-Dichlorophenyl)-5-(4-methanesulfonylphenyl)-2-methyl-1H-pyrrol-3-ylmethylene]-thiazolidine-2,4-dione

249

[1249] HPLC-MS (Method C): m/z: 508 (M+1); Rt.=4.31 min

Example 210 (General Procedure (C))

[1250] 5-[1-(2,5-Dimethoxyphenyl)-5-(4-methanesulfonylphenyl)-2-methyl-1H-pyrrol-3-ylmethylene]-thiazolidine-2,4-dione

250

[1251] HPLC-MS (Method C): m/z: 499 (M+1); Rt.=3.70 min

Example 211 (General Procedure (C))

[1252] 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2,5-dimethylpyrrol-1-yl]benzoic acid

251

[1253] HPLC-MS (Method C): m/z: 342 (M+1); Rt.=3.19 min

Example 212 (General Procedure (C))

[1254] 5-(4-Hydroxy-2,6-dimethoxybenzylidene)thiazolidine-2,4-dione

252

[1255] HPLC-MS (Method C): m/z: 282(M+1); Rt.=2.56, mp=331-333° C.

Example 213 (General Procedure (C))

[1256] 5-(2,6-Dimethylbenzylidene)thiazolidine-2,4-dione

253

[1257] M.p: 104-105° C. HPLC-MS (Method C): m/z: 234 (M+1); Rt.=3.58 min,

Example 214 (General Procedure (C))

[1258] 5-(2,6-Dimethoxybenzylidene)thiazolidine-2,4-dione

254

[1259] Mp: 241-242° C. HPLC-MS (Method C): m/z: 266 (M+1); Rt.=3.25 min;

Example 215 (General Procedure (C))

[1260] 5-[4-(2-Fluoro-6-nitrobenzyloxy)-2,6-dimethoxybenzylidene]thiazolidine-2,4-dione

255

[1261] Mp: 255-256° C. HPLC-MS (Method C): m/z: 435 (M+1), Rt=4.13 min,

Example 216 (General Procedure (C))

[1262] 5-Benzofuran-2-ylmethylenethiazolidine-2,4-dione

256

[1263] HPLC-MS (Method C): m/z: 246 (M+1); Rt.=3.65 min, mp=265-266° C.

Example 217 (General Procedure (C)

[1264] 5-[3-(4-Dimethylaminophenyl)allylidene]thiazolidine-2,4-dione

257

[1265] HPLC-MS (Method C): m/z: 276(M+1); Rt.=3.63, mp=259-263° C.

[1266]

1
H-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).

Example 218 (General Procedure (C))

[1267] 5-(2-Methyl-3-phenylallylidene)thiazolidine-2,4-dione

258

[1268] Mp: 203-210° C. HPLC-MS (Method C): m/z: 246 (M+1); Rt=3.79 min.

Example 219 (General Procedure (C))

[1269] 5-(2-Chloro-3-phenylallylidene)thiazolidine-2,4-dione

259

[1270] Mp: 251-254° C. HPLC-MS (Method C): m/z: 266 (M+1; Rt=3.90 min

Example 220 (General Procedure (C))

[1271] 5-(2-Oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione

260

[1272] Mp: 338-347° C. HPLC-MS (Method C): m/z: 273 (M+1); Rt.=2.59 min.

Example 221 (General Procedure (C))

[1273] 5-(2,4,6-Tribromo-3-hydroxybenzylidene)thiazolidine-2,4-dione

261

[1274] HPLC-MS (Method C): m/z: 459 (M+1); Rt.=3.65 min.

Example 222 (General Procedure (C))

[1275] 5-(5-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione

262

[1276] HPLC-MS (Method C): m/z: 339 (M+1); Rt=3.37 min.

Example 223 (General Procedure (C))

[1277] 5-(7-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione

263

[1278] HPLC-MS (Method C): m/z: 319 (M+1); Rt=3.48 min.

Example 224 (General Procedure (C))

[1279] 5-(6-Bromoindol-3-ylmethylene)thiazolidine-2,4-dione

264

[1280] HPLC-MS (Method C): m/z: 325 (M+1); Rt=3.54 min.

Example 225 (General Procedure (C))

[1281] 5-(8-Methyl-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione

265

[1282] HPLC-MS (Method C): m/z: 287 (M+1); Rt=2.86 min.

Example 226 (General Procedure (C))

[1283] 5-(6-Methoxy-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione

266

[1284] HPLC-MS (Method C): m/z: 303 (M+1); Rt=2.65 min.

Example 227 (General Procedure (C))

[1285] 5-Quinolin-3-ylmethylenethiazolidine-2,4-dione

267

[1286] HPLC-MS (Method C): m/z: 257 (M+1); Rt=2.77 min.

Example 228 (General Procedure (C))

[1287] 5-(8-Hydroxyquinolin-2-ylmethylene)thiazolidine-2,4-dione

268

[1288] HPLC-MS (Method C): m/z: 273 (M+1); Rt=3.44 min.

Example 229 (General Procedure (C))

[1289] 5-Quinolin-8-ylmethylenethiazolidine-2,4-dione

269

[1290] HPLC-MS (Method C): m/z: 257 (M+1); Rt=3.15 min.

Example 230 (General Procedure (C))

[1291] 5-(1-Bromo-6-methoxynaphthalen-2-ylmethylene)thiazolidine-2,4-dione

270

[1292] HPLC-MS (Method C): m/z: 366 (M+1); Rt=4.44 min.

Example 231 (General Procedure (C))

[1293] 5-(6-Methyl-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione

271

[1294] HPLC-MS (Method C): m/z: 287 (M+1); Rt.=2.89 min.

Example 232 (General Procedure (D))

[1295] 5-(2,6-Dichloro-4-dibenzylaminobenzylidene)thiazolidine-2,4-dione

272

[1296] HPLC-MS (Method C): m/z: 469 (M+1); Rt=5.35 min.

[1297] Other preferred compounds include

[1298] 3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-carboxylic acid:

273

[1299] The following compounds are commercially available and may be prepared using general procedures (B) and/or (C).

Example 233

[1300] 5-(5-Bromo-1H-indol-3-ylmethylene)thiazolidine-2,4-dione

274

Example 234

[1301] 5-Pyridin-4-ylmethylenethiazolidine-2,4-dione

275

Example 235

[1302] 5-(3-Bromo-4-methoxybenzylidene)thiazolidine-2,4-dione

276

[1303] HPLC-MS (Method A):

Example 236

[1304] 5-(3-Nitrobenzylidene)thiazolidine-2,4-dione

277

[1305] HPLC-MS (Method A):

Example 237

[1306] 5-Cyclohexylidene-1,3-thiazolidine-2,4-dione

278

[1307] HPLC-MS (Method A):

Example 238

[1308] 5-(3,4-Dihydroxybenzylidene)thiazolidine-2,4-dione

279

Example 239

[1309] 5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione

280

Example 240

[1310] 5-(4-Hydroxy-3-methoxy-5-nitrobenzylidene)thiazolidine-2,4-dione

281

Example 241

[1311] 5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione

282

Example 242

[1312] 5-(4-Hydroxy-3,5-dimethoxybenzylidene)thiazolidine-2,4-dione

283

Example 243

[1313] 5-(3-Bromo-5-ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione

284

Example 244

[1314] 5-(3-Ethoxy-4-hydroxy-5-nitrobenzylidene)thiazolidine-2,4-dione

285

Example 245

[1315]

286

Example 246

[1316]

287

Example 247

[1317]

288

Example 248

[1318]

289

Example 249

[1319]

290

Example 250

[1320]

291

Example 251

[1321]

292

Example 252

[1322]

293

Example 253

[1323]

294

Example 254

[1324]

295

Example 255

[1325]

296

Example 256

[1326]

297

Example 257

[1327] 5-(3-Hydroxy-5-methyl-phenylamino)-thiazolidine-2,4-dione

298

Example 258

[1328]

299

Example 259

[1329]

300

Example 260

[1330]

301

Example 261

[1331]

302

Example 262

[1332]

303

Example 263

[1333]

304

Example 264

[1334]

305

Example 265

[1335]

306

Example 266

[1336]

307

Example 267

[1337]

308

Example 268

[1338]

309

Example 269

[1339]

310

Example 270

[1340]

311

Example 271

[1341]

312

Example 272

[1342]

313

Example 273

[1343]

314

Example 274

[1344]

315

Example 275

[1345]

316

Example 276

[1346]

317

Example 277

[1347]

318

Example 278

[1348]

319

Example 279

[1349]

320

Example 280

[1350]

321

Example 281

[1351]

322

[1352] General procedure (D) for preparation of compounds of general formula I3:

323

[1353] wherein X, Y, R10 are as defined above,

[1354] n is 1 or 3-20,

[1355] E is arylene or heterarylene (including up to four optional substituents, R13, R14, R15, and R15A as defined above),

[1356] 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.

[1357] 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.

[1358] Step 2 is a hydrolysis of the product from step 1.

[1359] Step 3 is similar to general procedure (B) and (C).

[1360] This general procedure (D) is further illustrated in the following examples:

Example 282 (General Procedure (D))

[1361] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid

324

[1362] Step 1:

[1363] 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.

[1364] Step 2:

[1365] 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.

[1366] Step 3:

[1367] 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.

[1368]

1
H-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.

Example 283 (General Procedure (D))

[1369] [3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid

325

[1370] Step 3:

[1371] 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.

[1372]

1
H-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.

[1373] The compounds in the following examples were similarly prepared.

Example 284 (General Procedure (D))

[1374] 3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acrylic acid

326

[1375]

1
H-NMR (DMSO-d6): δ 6.63 (1H, d), 7.59-7.64 (3H, m), 7.77 (1H, s), 7.83 (2H, m).

Example 285 (General Procedure (D))

[1376] [4-(-2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid

327

[1377] Triethylamine salt: 1H-NMR (DMSO-d6): δ 4.27 (2H, s), 6.90 (2H, d), 7.26 (1H, s), 7.40 (2H, d).

Example 286 (General Procedure (D))

[1378] 4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid

328

Example 287 (General Procedure (D))

[1379] 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid

329

[1380]

1
H-NMR (DMSO-d6): δ 7.57 (1H, s), 7.60 (1H, t), 7.79 (1H, dt), 7.92 (1H, dt), 8.14 (1H, t).

Example 288 (General Procedure (D))

[1381] 4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid

330

[1382]

1
H-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.

Example 289 (General Procedure (D))

[1383] 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric Acid

331

[1384]

1
H-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.

Example 290 (General Procedure (D))

[1385] 4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyric Acid

332

[1386]

1
H-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

Example 291 (General Procedure (D))

[1387] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid

333

[1388]

1
H-NMR (DMSO-d6): δ 2.12 (2H, p), 2.5 (below DMSO), 4.28 (2H, t), 7.12 (1H, d), 7.6-7.7 (3H, m), 8.12 (1H, d), 8.31 (1H, d), 8.39 (1H, s), 12.2 (1H, bs, 12.6 (1H, bs). HPLC-MS (method A): m/z: 380 (M+23); Rt=3.76 min.

Example 292 (General Procedure (D))

[1389] 5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoic acid

334

[1390] 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).

Example 293

[1391] 5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoic acid

335

[1392] 5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]pentanoic acid (example 292, 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.

[1393] HPLC-MS (Method C): m/z: 473 (M+23), Rt.=3.77 min

Example 294

[1394] 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid

336

[1395] Starting with 4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyric acid (example 291, 0.5 mmol) using the same method as in example 293 afforded 66 mg of the title compound.

[1396] HPLC-MS (Method C): m/z: 459 (M+23); Rt.=3.59 min.

Example 295 (General Procedure (D))

[1397] [2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid

337

[1398]

1
H-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.

Example 296 (General Procedure (D))

[1399] 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid

338

[1400]

1
H-NMR (DMSO-d6): δ 1.98 (2H, p), 2.42 (2H, t), 4.04 (2H, t), 7.05 (1H, dd), 7.15 (2H, m), 7.45 (1H, t), 7.77 (1H, s), 12.1 (1H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 330 (M+23); Rt=3.05 min.

Example 297 (General Procedure (D))

[1401] [4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-3-methoxyphenoxy]acetic Acid

339

[1402] HPLC-MS (Method B): m/z: 310 (M+1); Rt=3,43 min.

Example 298 (General Procedure (D))

[1403] [4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetic acid

340

[1404] HPLC-MS (Method A): m/z: 330 (M+1); Rt=3.25 min.

Example 299 (General Procedure (D))

[1405] 8-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalene-1-carboxylic acid

341

[1406] HPLC-MS (Method A): m/z: 299 (M+1); Rt=2,49 min.

Example 300 (General Procedure (D))

[1407] [3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]acetic acid

342

[1408] HPLC-MS (Method A): m/z: 303 (M+1); Rt=2.90 min.

[1409] Preparation of starting material:

[1410] 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.

[1411]

1
H-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).

[1412] (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.

[1413]

1
H-NMR (DMSO-d6): δH=5.20 (2H, s), 7.3 (2H, m), 7.55 (1H, d), 8.12 (1H, d), 8.30 (1H, s), 9.95 (1H, s), 13.3 (1H, bs).

Example 301 (General Procedure (D))

[1414] 3-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]propionic acid

343

[1415] HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.08 min.

[1416] Preparation of starting material:

[1417] A mixture of 3-formylindol (10 g, 69 mmol), ethyl 3-bromopropionate (10.5 mL, 83 mmol) and potassium carbonate (28.5 g, 207 mmol) and acetonitrile (100 mL) was stirred vigorously at refux temperature for 2 days. After cooling, the mixture was filtered and the filtrate was concentrated in vacuo to afford 17.5 g (quant.) of 3-(3-formylindol-1-yl)propionic acid ethyl ester as a solid.

[1418]

1
H-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).

[1419] 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.

[1420]

1
H-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).

Example 302 (General Procedure (D))

[1421] {5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetic acid

344

[1422] HPLC-MS (Method A): m/z: 429 (M+23); Rt=3.89 min.

Example 303 (General Procedure (D))

[1423] 6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxyoctanoic acid

345

[1424] HPLC-MS (Method C): m/z: 436 (M+23); Rt.=4.36 min

[1425] 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.

[1426] HPLC-MS (Method C): m/z 315 (M+1); Rt.=4.24 min.

Example 304 (General Procedure (D))

[1427] 12-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]dodecanoic acid

346

[1428] HPLC-MS (Method C): m/z: 492 (M+23); Rt.=5.3 min.

[1429] The intermediate aldehyde was prepared similarly as described in example 303.

Example 305 (General Procedure (D))

[1430] 11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoic Acid

347

[1431] HPLC-MS (Method C): m/z: 478 (M+23); Rt.=5.17 min.

[1432] The intermediate aldehyde was prepared similarly as described in example 303.

Example 306 (General Procedure (D))

[1433] 15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoic acid

348

[1434] HPLC-MS (Method C): m/z: 534 (M+23); Rt.=6.07 min.

[1435] The intermediate aldehyde was prepared similarly as described in example 303.

Example 307 (General procedure (D))

[1436] 6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoic acid

349

[1437] HPLC-MS (Method C): m/z: 408 (M+23); Rt.=3.71 min.

Example 308 (General Procedure (D))

[1438] 4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyric Acid

350

[1439] HPLC-MS (Method C): m/z: 380 (M+23); Rt.=3.23 min.

Example 309 (General Procedure (D))

[1440] 6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoic acid ethyl ester

351

[1441] HPLC-MS (Method C): m/z: 436 (M+23); Rt.=4.64 min.

Example 310 (General Procedure (D))

[1442] 4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyric acid ethyl ester

352

[1443] HPLC-MS (Method C): m/z: 408 (M+23); Rt.=4.28 min.

Example 311

[1444] N-(3-Aminopropyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyramide

353

[1445] To a mixture of 4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid (example 291, 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.

[1446] HPLC-MS (Method C): m/z: 515 (M+1); Rt=3.79 min.

[1447] (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.

[1448] HPLC-MS (Method C): m/z: 414 (M+1); Rt=2,27 min.

[1449] Preferred compounds of the invention includes:

354

[1450] The following compounds are commercially available and may be prepared according to general procedure (D):

Example 312

[1451]

355

Example 313

[1452]

356

Example 314

[1453]

357

Example 315

[1454]

358

Example 316

[1455]

359

Example 317

[1456]

360

Example 318

[1457]

361

[1458] The following salicylic acid derivatives do all bind to the His B10 Zn2+ site of the insulin hexamer:

Example 319

[1459] Salicylic Acid

362

Example 320

[1460] Thiosalicylic Acid (or: 2-Mercaptobenzoic acid)

363

Example 321

[1461] 2-Hydroxy-5-nitrobenzoic acid

364

Example 322

[1462] 3-Nitrosalicyclic acid

365

Example 323

[1463] 5,5′-Methylenedisalicylic acid

366

Example 324

[1464] 2-Amino-5-trifluoromethylbenzoesyre

367

Example 325

[1465] 2-Amino-4-chlorobenzoic acid

368

Example 326

[1466] 2-Amino-5-methoxybenzoesyre

369

Example 327

[1467]

370

Example 328

[1468]

371

Example 329

[1469]

372

Example 330

[1470]

373

Example 331

[1471]

374

Example 332

[1472]

375

Example 333

[1473] 5-Iodosalicylic acid

376

Example 334

[1474] 5-Chlorosalicylic acid

377

Example 335

[1475] 1-Hydroxy-2-naphthoic acid

378

Example 336

[1476] 3,5-Dihydroxy-2-naphthoic acid

379

Example 337

[1477] 3-Hydroxy-2-naphthoic acid

380

Example 338

[1478] 3,7-Dihydroxy-2-naphthoic acid

381

Example 339

[1479] 2-Hydroxybenzo[a]carbazole-3-carboxylic acid

382

Example 340

[1480] 7-Bromo-3-hydroxy-2-naphthoic Acid

383

[1481] This compound was prepared according to Murphy et al., J. Med. Chem. 1990, 33, 171-8. HPLC-MS (Method A): m/z: 267 (M+1), Rt:=3.78 min.

Example 341

[1482] 1,6-Dibromo-2-hydroxynaphthalene-3-carboxylic Acid

384

[1483] This compound was prepared according to Murphy et al., J. Med. Chem. 1990, 33, 171-8. HPLC-MS (Method A): m/z: 346 (M+1); Rt:=4,19 min.

Example 342

[1484] 7-Formyl-3-hydroxynaphthalene-2-carboxylic Acid

385

[1485] A solution of 7-bromo-3-hydroxynaphthalene-2-carboxylic acid (15.0 g, 56.2 mmol) (example 340) 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 5020 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.

[1486]

1
H-NMR (DMSO-d6): δ 11.95 (1H, bs), 10.02 (1H, s), 8.61 (1H, s), 8.54 (1H, s), 7.80 (2H, bs), 7.24 (1H, s); HPLC-MS (Method (A)): m/z: 217 (M+1); Rt=2.49 min.

Example 343

[1487] 3-Hydroxy-7-methoxy-2-naphthoic Acid

386

Example 344

[1488] 4-Amino-2-hydroxybenzoic Acid

387

Example 345

[1489] 5-Acetylamino-2-hydroxybenzoic Acid

388

Example 346

[1490] 2-Hydroxy-5-methoxybenzoic Acid

389

[1491] The following compounds were prepared as described below:

Example 347

[1492] 4-Bromo-3-hydroxynaphthalene-2-carboxylic Acid

390

[1493] 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.

[1494]

1
H-NMR (DMSO-d6): δ 7.49 (1H, t), 7.75 (1H, t), 8.07 (2H, “t”), 8.64 (1H, s). The substitution pattern was confirmed by a COSY experiment, showing connectivities between the 3 (4 hydrogen) “triplets”. HPLC-MS (Method A): m/z: 267 (M+1); Rt=3.73 min.

Example 348

[1495] 3-Hydroxy-4-iodonaphthalene-2-carboxylic Acid

391

[1496] 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.

[1497]

1
H-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.

Example 349

[1498] 2-Hydroxy-5-[(4-methoxyphenylamino)methyl]benzoic Acid

392

[1499] p-Anisidine (1.3 g, 10.6 mmol) was dissolved in methanol (20 mL) and 5-formylsalicylic acid (1.75 g, 10.6 mmol)was added and the resulting mixture was stirred at room temperature for 16 hours. The solid formed was isolated by filtration, re-dissolved in N-methyl pyrrolidone (20 mL) and methanol (2 mL). To the mixture was added sodium cyanoborohydride (1.2 g) and the mixture was heated to 7020 C. for 3 hours. To the cooled mixture was added ethyl acetate (100 mL) and the mixture was extracted with water (100 mL) and saturated aqueous ammonium chloride (100 mL). The combined aqueous phases were concentrated in vacuo and a 2 g aliquot was purified by SepPac chromatography eluting with mixtures of aetonitrile and water containing 0.1% trifluoroacetic acid to afford the title compound.

[1500] 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).

Example 350

[1501] 2-Hydroxy5-(47methoxyphenylsulfamoyl)benzoic Acid

393

[1502] A solution of 5chlrosulfonylsalicylic 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.

[1503] 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).

[1504] General procedure (E) for preparation of compounds of general formula I4:

394

[1505] 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 T is as defined above.

[1506] 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.

[1507] The general procedure (E) is further illustrated in the following example:

Example 351 (General Procedure (E))

[1508] 7-(4-Acetylphenyl)-3-hydroxynaphthalene-2-carboxylic Acid

395

[1509] To 7-bromo-3-hydroxynaphthalene-2-carboxylic acid (100 mg, 0.37 mmol) (example 340) 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): δ 12.60 (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.

[1510] The compounds in the following examples were prepared in a similar fashion. Optionally, the compounds can be further purified by recrystallization from e.g. ethanol or by chromatography.

Example 352 (General Procedure (E))

[1511] 3-Hydroxy-7-(3-methoxyphenyl)naphthalene-2-carboxylic Acid

396

[1512] HPLC-MS (Method (A)): m/z: 295 (M+1); Rt=4.60 min.

Example 353 (General Procedure (E))

[1513] 3-Hydroxy-7-phenylnaphthalene-2-carboxylic Acid

397

[1514] HPLC-MS (Method (A)): m/z: 265 (M+1); Rt=4.6 min.

Example 354 (General Procedure (E))

[1515] 3-Hydroxy-7-p-tolylnaphthalene-2-carboxylic Acid

398

[1516] HPLC-MS (Method (A)): m/z: 279 (M+1); Rt=4.95 min.

Example 355 (General Procedure (E))

[1517] 7-(4-Formylphenyl)-3-hydroxynaphthalene-2-carboxylic Acid

399

[1518] HPLC-MS (Method (A)): m/z: 293 (M+1); Rt=4.4 min.

Example 356 (General Procedure (E))

[1519] 6-Hydroxy-[1,2]binaphthalenyl-7-carboxylic Acid

400

[1520] HPLC-MS (Method (A)): m/z: 315 (M+1); Rt=5.17 min.

Example 357 (General Procedure (E))

[1521] 7-(4-Carboxy-phenyl)-3-hydroxynaphthalene-2-carboxylic Acid

401

[1522] HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=3.60 min.

Example 358 (General Procedure (E))

[1523] 7-Benzofuran-2-yl-3-hydroxynaphthalene-2-carboxylic Acid

402

[1524] HPLC-MS (Method (A)): m/z: 305 (M+1); Rt=4.97 min.

Example 359 (General Procedure (E))

[1525] 3-Hydroxy-7-(4-methoxyphenyl)-naphthalene-2-carboxylic Acid

403

[1526] HPLC-MS (Method (A)): m/z: 295 (M+1); Rt=4.68 min.

Example 360 (General Procedure (E))

[1527] 7-(3-Ethoxyphenyl)-3-hydroxynaphthalene-2-carboxylic Acid

404

[1528] HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=4.89 min.

Example 361 (General Procedure (E))

[1529] 7-Benzo[1,3]dioxol-5-yl-3-hydroxynaphthalene-2-carboxylic Acid

405

[1530] HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=5.61 min.

Example 362 (General Procedure (E))

[1531] 7-Biphenyl-3-yl-3-hydroxynaphthalene-2-carboxylic Acid

406

[1532] HPLC-MS (Method (A)): m/z: 341 (M+1); Rt=5.45 min.

[1533] General procedure (F) for preparation of compounds of general formula I5:

407

[1534] wherein R30 is hydrogen or C1-C6-alkyl and T is as defined above

[1535] This general procedure (F) is further illustrated in the following example:

Example 363 (General Procedure (F))

[1536] 3-Hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylic Acid

408

[1537] 7-Formyl-3-hydroxynaphthalene-2-carboxylic acid (40 mg, 0.19 mmol) (example 342) 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.

[1538]

1
H-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.

[1539] The compounds in the following examples were made using this general procedure (F).

Example 364 (General Procedure (F))

[1540] 7-{[(4-Bromophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid

409

[1541] HPLC-MS (Method C): m/z: 372 (M+1); Rt=4.31min.

Example 365 (General Procedure (F))

[1542] 7-{[(3,5-Dichlorophenyl)amino]methyl)-3-hydroxynaphthalene-2-carboxylic Acid

410

[1543] HPLC-MS (Method C): m/z: 362 (M+1); Rt=4.75 min.

Example 366 (General Procedure (F))

[1544] 7-{[(Benzothiazol-6-yl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid

411

[1545] HPLC-MS (Method C): m/z 351 (M+1); Rt=3.43 min.

Example 367 (General Procedure (F))

[1546] 3-Hydroxy-7-{[(quinolin-6-yl)amino]methyl}naphthalene-2-carboxylic Acid

412

[1547] HPLC-MS (Method C): m/z: 345 (M+1); Rt=2.26 min.

Example 368 (General Procedure (F))

[1548] 3-Hydroxy-7-{[(4-methoxyphenyl)amino]methyl}naphthalene-2-carboxylic Acid

413

[1549] HPLC-MS (Method C): m/z: 324 (M+1); Rt=2.57min.

Example 369 (General Procedure (F))

[1550] 7-{[(2,3-Dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid

414

[1551] HPLC-MS (Method C): m/z: 350 (M+1); Rt=2.22 min.

Example 370 (General Procedure (F))

[1552] 7-([(4-Chlorobenzyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid

415

[1553] HPLC-MS (Method C): m/z: 342 (M+1); Rt=2.45 min.

Example 371 (General Procedure (F))

[1554] 3-Hydroxy-7-{[(naphthalen-1-ylmethyl)amino]methyl}naphthalene-2-carboxylic Acid

416

[1555] HPLC-MS (Method C): m/z: 357 (M+1); Rt=2.63 min.

Example 372 (General Procedure (F))

[1556] 7-{[(Biphenyl-2-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-arboxylic Acid

417

[1557] HPLC-MS (Method C): m/z: 384 (M+1); Rt=2.90 min.

Example 373 (General Procedure (F))

[1558] 3-Hydroxy-7-{[(4-phenoxybenzyl)amino]methyl}naphthalene-2-carboxylic Acid

418

[1559] HPLC-MS (Method C): m/z: 400 (M+1); Rt=3.15 min.

Example 374 (General Procedure (F))

[1560] 3-Hydroxy-7-{[(4-methoxybenzyl)amino]methyl}naphthalene-2-carboxylic Acid

419

[1561] HPLC-MS (Method C): m/z: 338 (M+1); Rt-32 2.32 min.

[1562] General procedure (G) for preparation of compounds of general formula I6:

420

[1563] wherein T is as defined above and the moiety (C1-C6-alkanoyl)2O is an anhydride.

[1564] The general procedure (G) is illustrated by the following example:

Example 375 (General Procedure (G))

[1565] N-Acetyl-3-hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylic Acid

421

[1566] 3-Hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylic acid (25 mg, 0.07 mmol) (example 363) 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.

[1567]

1
H-NMR (DMSO-d6): δ 10.96 (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.

[1568] The compounds in the following examples were prepared in a similar fashion.

Example 376 (General Procedure (G))

[1569] N-Acetyl-7-{[(4-bromophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid

422

[1570] HPLC-MS (Method C): m/z: 414 (M+1); Rt=3.76 min.

Example 377 (General Procedure (G))

[1571] N-Acetyl-7-{[(2,3-dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid

423

[1572] HPLC-MS (Method C): m/z: 392 (M+1); Rt=3.26 min.

Example 378 (General Procedure (G))

[1573] N-Acetyl-7-{[(4-chlorobenzyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic Acid

424

[1574] HPLC-MS (Method C): m/z: 384 (M+1); Rt=3.67 min.

Example 379

[1575] 5-(3-(Naphthalen-2-yloxymethyl)-phenyl)-1H-tetrazole

425

[1576] 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.

[1577] 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.

[1578] 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%.

[1579] 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.

[1580] 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%.

[1581]

1
H 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).

Example 380

[1582] N-(3-(Tetrazol-5-yl)phenyl)-2-naphtoic Acid Amide

426

[1583] 2-Naphtoic acid (10 g, 58 mmol) was dissolved in dichloromethane (100 mL) and N,N-dimethylformamide (0.2 mL) was added followed by thionyl chloride (5.1 ml, 70 mmol). The mixture was heated at reflux temperature for 2 hours. After cooling to room temperature, the mixture was added dropwise to a mixture of 3-aminobenzonitril (6.90 g, 58 mmol) and triethyl amine (10 mL) in dichloromethane (75 mL). The resulting mixture was stirred at room temperature for 30 minutes. Water (50 mL) was added and the volatiles was exaporated in vacuo. The resulting mixture was filtered and the filter cake was washed with water followed by heptane (2×25 mL). Drying in vacuo at 50° C. for 16 hours afforded 15.0 g (95%) of N-(3-cyanophenyl)-2-naphtoic acid amide.

[1584] M.p.138-140° C.

[1585] 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.

[1586]

1
H NMR (200 MHz, DMSOD6): δH 7.58-7.70 (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%.

Example 381

[1587] 5-[3-(Biphenyl4-yloxymethyl)phenyl]-1H-tetrazole

427

[1588] 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 5020 C. for 18 hours affording crude 17.39 g of 3-(biphenyl-4-yloxymethyl)benzonitrile as a solid.

[1589]

1
H NMR (200 MHz, CDCl3) δH 5.14 (s, 2H), 7.05 (m, 2H), 7.30-7.78 (m, 11H).

[1590] 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 12520 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 5020 C. for 18 hours affording 8.02 g (70%) of the title compound.

[1591]

1
H NMR (200 MHz, DMSO-d6) δH 5.31 (s, 2H), 7.19 (m, 2H), 7.34 (m, 1H), 7.47 (m, 2H), 7.69 (m, 6H), 8.05 (dt, 1H), 8.24 (s,1H).

Example 382

[1592] 5-(3-Phenoxymethyl)-phenyl)-tetrazole

428

[1593] 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.

[1594] TLC: Rf=0.38 (Ethyl acetate/heptane=1:4)

[1595] 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 aqeous mixture was washed with ethyl acetate (200 mL). The pH of the aqueous phase was adjusted to 1 with 5 N hydrochloric acid and stirred at room temperature for 30 minutes. Filtration, washing with water and drying In vacuo at 50° C. afforded 2.06 g (33%) of the title compound as a solid.

[1596]

1
H 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).

Example 383

[1597] 5-[3-(Biphenyl-4-ylmethoxy)phenyl]-1H-tetrazole

429

[1598] 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.

[1599] 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.

[1600]

1
H NMR (200 MHz, DMSO-d6) δ 5.29 (s, 2H), 7.31 (dd, 1H), 7.37-7.77 (m, 12H).

Example 384

[1601] 5-[4-(Biphenyl-4-ylmethoxy)-3-methoxyphenyl]-1H-tetrazol

430

[1602] This compound was made similarly as described in example 383.

Example 385

[1603]

431

Example 386

[1604] 5-(2-Naphtylmethyl)-1H-tetrazole

432

[1605] This compound was prepared similarly as described in example 379, step 2.

Example 387

[1606] 5-(1-Naphtylmethyl)-1H-tetrazole

433

[1607] This compound was prepared similarly as described in example 379, step 2.

Example 388

[1608] 5-[4-(Biphenyl-4-yloxymethyl)phenyl]-1H-tetrazole

434

[1609] 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-(biphenyl4-yloxymethyl)benzonitrile as a solid.

[1610] The above benzonitrile (3.00 g, 10.5 mmol) was dissolved in N,N-dimethylformamide (50 mL), and sodium azide (1.03 g, 15.8 mmol) and ammonium chloride (0.84 g, 15.8 mmol) were added and the mixture was stirred 16 hours at 125° C. The mixture was cooled to room temperature and water (50 mL) was added. The suspension was stirred overnight, filtered, washed with water and dried in vacuo at 50° C. for 3 days to give crude 3.07 g (89%) of the title compound. From the mother liquor crystals were colected and washed with water, dried by suction to give 0.18 g (5%) of the title compound as a solid.

[1611]

1
H 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%.

Example 389

[1612]

435

[1613] This compound was prepared similarly as described in example 383.

Example 390

[1614]

436

Example 391

[1615]

437

Example 392

[1616]

438

Example 393

[1617] 5-(3-(Biphenyl4-yloxymethyl)-benzyl)-1H-tetrazole

439

Example 394

[1618] 5-(1-Naphthyl)-1H-tetrazole

440

[1619] This compound was prepared similarly as described in example 379, step 2.

Example 395

[1620] 5-[3-Methoxy-4-(4-methylsulfonylbenzyloxy)phenyl]-1H-tetrazole

441

[1621] This compound was made similarly as described in example 383.

Example 396

[1622] 5-(2-Naphthyl)-1H-tetrazole

442

[1623] This compound was prepared similarly as described in example 379, step 2.

Example 397

[1624] 2-Amino-N-(1H-tetrazol-5-yl)-benzamide

443

Example 398

[1625] 5-(4-Hydroxy-3-methoxyphenyl)-1H-tetrazole

444

[1626] This compound was prepared similarly as described in example 379, step 2.

Example 399

[1627] 4-(2H-Tetrazol-5-ylmethoxy)benzoic Acid

445

[1628] 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.

[1629] 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.

[1630] 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.

[1631]

1
H-NMR (DMSO-d6): 4.70 (2H, s), 7.48 (2H, d), 7.73 (2H, d), 13 (1H, bs).

Example 400

[1632] 4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoic Acid

446

[1633] 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.

[1634] 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%).

[1635]

1
H-NMR (DMSO-d6): 5.58 (2H, s), 7.15 (2H, d), 7.93 (2H, d), 12.7 (1H, bs).

Example 401

[1636] 3-(2H-Tetrazol-5-yl)-9H-carbazole

447

[1637] 3-Bromo9H-carbazole was prepared as described by Smith et al. in Tetrahedron 1992, 48, 7479-7488.

[1638] 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.

[1639] 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.

[1640]

1
H-NMR (DMSO-d6): δ 11.78 (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.

[1641] The following commercially available tetrazoles do all bind to the His B10 Zn2+ site of the insulin hexamer:

Example 402

[1642] 5-(3-Tolyl)-1H-tetrazole

448

Example 403

[1643] 5-(2-Bromophenyl)tetrazole

449

Example 404

[1644] 5-(4-Ethoxalylamino-3-nitrophenyl)tetrazole

450

Example 405

[1645]

451

Example 406

[1646]

452

Example 407

[1647]

453

Example 408

[1648]

454

Example 409

[1649] Tetrazole

455

Example 410

[1650] 5-Methyltetrazole

456

Example 411

[1651] 5-Benzyl-2H-tetrazole

457

Example 412

[1652] 4-(2H-Tetrazol-5-yl)benzoic Acid

458

Example 413

[1653] 5-Phenyl-2H-tetrazole

459

Example 414

[1654] 5-(4-Chlorophenylsulfanylmethyl)-2H-tetrazole

460

Example 415

[1655] 5-(3-Benzyloxyphenyl)-2H-tetrazole

461

Example 416

[1656] 2-Phenyl-6-(1 H-tetrazol-5-yl)-chromen-4-one

462

Example 417

[1657]

463

Example 418

[1658]

464

Example 419

[1659]

465

Example 420

[1660]

466

Example 421

[1661]

467

Example 422

[1662] 5-(4-Bromo-phenyl)-1H-tetrazole

468

Example 423

[1663]

469

Example 424

[1664]

470

Example 425

[1665]

471

Example 426

[1666]

472

Example 427

[1667]

473

Example 428

[1668]

474

Example 429

[1669]

475

Example 430

[1670]

476

Example 431

[1671]

477

Example 432

[1672]

478

Example 433

[1673]

479

Example 434

[1674]

480

Example 435

[1675]

481

Example 436

[1676]

482

Example 437

[1677]

483

Example 438

[1678]

484

Example 439

[1679]

485

Example 440

[1680]

486

Example 441

[1681]

487

Example 442

[1682]

488

Example 443

[1683]

489

Example 444

[1684]

490

Example 445

[1685]

491

Example 446

[1686]

492

Example 447

[1687]

493

Example 448

[1688]

494

Example 449

[1689]

495

Example 450

[1690]

496

Example 451

[1691]

497

Example 452

[1692]

498

[1693] General procedure (H) for preparation of compounds of general formula I7:

499

[1694] wherein A1, AR1, and AR2 are as defined above.

[1695] 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.

[1696] When the reductive alkylation is complete, the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art.

[1697] The general procedure (H) is further illustrated in the following example 453:

Example 453 (General Procedure (H))

[1698] Biphenyl-4-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine

500

[1699] A solution of 5-(3-aminophenyl)-2H-tetrazole (example 589, 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.

[1700] HPLC-MS (Method C): m/z: 328 (M+1), 350 (M+23); Rt=4.09 min.

Example 454 (General Procedure (H))

[1701] Benzyl-[3-(2H-tetrazol-5-yl)phenyl]amine

501

[1702] HPLC-MS (Method D): m/z: 252 (M+1); Rt=3,74 min.

Example 455 (General Procedure (H))

[1703] (4-Methoxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

502

[1704] HPLC-MS (Method D): m/z: 282,2 (M+1); Rt=3,57min.

Example 456 (General Procedure (H))

[1705] 4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenol

503

[1706] HPLC-MS (Method D): m/z: 268,4 (M+1); Rt=2,64 min.

Example 457 (General Procedure (H))

[1707] (4-Nitrobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

504

[1708] HPLC-MS (Method D): m/z: 297,4 (M+1); Rt=3,94 min.

Example 458 (General Procedure (H))

[1709] (4-Chlorobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

505

[1710] HPLC-MS (Method D): m/z: 287,2 (M+1); Rt=4.30 min.

Example 459 (General Procedure (H))

[1711] (2-Chlorobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

506

[1712] HPLC-MS (Method D): m/z: 286 (M+1); Rt=4.40 min.

Example 460 (General Procedure (H))

[1713] (4-Bromobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

507

[1714] HPLC-MS (Method D): m/z:332 (M+1); Rt=4.50 min.

Example 461 (General Procedure (H))

[1715] (3-Benzyloxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

508

[1716] HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.94 min.

Example 462 (General Procedure (H))

[1717] Naphthalen-1-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine

509

[1718] HPLC-MS (Method D): m/z: 302 (M+1); Rt=4,70 min.

Example 463 (General Procedure (H))

[1719] Naphthalen-2-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine

510

[1720] HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.60 min.

Example 464 (General Procedure (H))

[1721] 4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic Acid

511

[1722] HPLC-MS (Method D): m/z: 296 (M+1); Rt=3,24 min.

Example 465 (General Procedure (H))

[1723] [3-(2H-Tetrazol-5-yl)-phenyl]-[3-(3-trifluoromethyl-phenoxy)benzyl]amine

512

[1724] HPLC-MS (Method D): m/z: 412 (M+1); Rt=5,54 min.

Example 466 (General Procedure (H))

[1725] (3-Phenoxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

513

[1726] HPLC-MS (Method D): m/z: 344 (M+1); Rt=5,04 min.

Example 467 (General Procedure (H))

[1727] (4-Phenoxy-benzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

514

[1728] HPLC-MS (Method D): m/z: 344 (M+1); Rt=5,00 min.

Example 468 (General Procedure (H))

[1729] (4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenoxy)acetic Acid

515

[1730] HPLC-MS (Method D): m/z: 326 (M+1); Rt=3,10 min.

Example 469 (General Procedure (H))

[1731] (4-Benzyloxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

516

[1732] HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.97 min.

Example 470 (General Procedure (H))

[1733] 3-(4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenyl)acrylic Acid

517

[1734] HPLC-MS (Method D): m/z: 322 (M+1); Rt=3,60 min.

Example 471 (General Procedure (H))

[1735] Dimethyl-(4-{[3-(2H-tetrazol-5-yl)phenylamino]methyl}naphthalen-1-yl)amine

518

[1736] HPLC-MS (Method D): m/z: 345 (M+1); Rt=3,07 min.

Example 472 (General Procedure (H))

[1737] (4′-Methoxybiphenyl-4-ylmethyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

519

[1738] HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.97 min.

Example 473 (General Procedure (H))

[1739] (2′-Chlorobiphenyl-4-ylmethyl)-[3-(2H-tetrazol-5-yl)phenyl]amine

520

[1740] HPLC-MS (Method D): m/z: 362 (M+1); Rt=5,27 min.

Example 474 (General Procedure (H))

[1741] Benzyl-[4-(2H-tetrazol-5-yl)phenyl]amine

521

[1742] For preparation of starting material, see example 590.

[1743] HPLC-MS (Method D): m/z: 252 (M+1); Rt=3,97 min.

Example 475 (General Procedure (H))

[1744] (4-Methoxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

522

[1745] HPLC-MS (Method D): m/z: 282 (M+1); Rt=3,94 min.

Example 476 (General Procedure (H))

[1746] 4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenol

523

[1747] HPLC-MS (Method D): m/z: 268 (M+1); Rt=3,14 min.

Example 477 (General Procedure (H))

[1748] (4-Nitrobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

524

[1749] HPLC-MS (Method D): m/z: (M+1); Rt=3,94 min.

Example 478 (General Procedure (H))

[1750] (4-Chlorobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

525

[1751] HPLC-MS (Method D): m/z: (M+1); Rt=4.47 min.

Example 479 (General Procedure (H))

[1752] (2-Chlorobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

526

[1753] HPLC-MS (Method D): m/z: 286 (M+1); Rt=4,37 min.

Example 480 (General Procedure (H))

[1754] (4-Bromobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

527

[1755] HPLC-MS (Method D): m/z: 331 (M+1); Rt=4.57 min.

Example 481 (General Procedure (H))

[1756] (3-Benzyloxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

528

[1757] HPLC-MS (Method D): m/z: 358 (M+1); Rt=5,07 min.

Example 482 (General Procedure (H))

[1758] Naphthalen-1-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine

529

[1759] HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.

Example 483 (General Procedure (H))

[1760] Naphthalen-2-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine

530

[1761] HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.

Example 484 (General Procedure (H))

[1762] Biphenyl-4-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine

531

[1763] HPLC-MS (Method D): m/z: 328 (M+1); Rt=5,07 min.

Example 485 (General Procedure (H))

[1764] 4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic Acid

532

[1765] HPLC-MS (Method D): m/z: 296 (M+1); Rt=3,34 min.

Example 486 (General Procedure (H))

[1766] [4-(2H-Tetrazol-5-yl)phenyl]-[3-(3-trifluoromethylphenoxy)benzyl]amine

533

[1767] HPLC-MS (Method D): m/z: 412 (M+1); Rt=5,54 min.

Example 487 (General Procedure (H))

[1768] (3-Phenoxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

534

[1769] HPLC-MS (Method D): m/z: 344 (M+1); Rt=5,07 min.

Example 488 (General Procedure (H))

[1770] (4-Phenoxybenzyl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine

535

[1771] HPLC-MS (Method D): m/z: 344 (M+1); Rt=5,03 min.

Example 489 (General Procedure (H))

[1772] 3-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic Acid

536

[1773] HPLC-MS (Method D): m/z: 286 (M+1); Rt=3,47 min.

Example 490 (General Procedure (H))

[1774] (4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenoxy)acetic Acid

537

[1775] HPLC-MS (Method D): m/z: 326 (M+1); Rt=3,40 min.

Example 491 (General Procedure (H))

[1776] (4-Benzyloxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine

538

[1777] HPLC-MS (Method D): m/z: 358 (M+1); Rt=5,14 min.

Example 492 (General Procedure (H))

[1778] 3-(4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenyl)acrylic Acid

539

[1779] HPLC-MS (Method D): m/z: 322 (M+1); Rt=3,66 min.

Example 493 (General Procedure (H))

[1780] Dimethyl-(4-{[4-(2H-tetrazol-5-yl)phenylamino]methyl}naphthalen-1-yl)amine

540

[1781] HPLC-MS (Method D): m/z: 345 (M+1); Rt=3,10 min.

Example 494 (General Procedure (H))

[1782] (4′-Methoxybiphenyl-4-ylmethyl)-[4-(2H-tetrazol-5yl)phenyl]amine

541

[1783] HPLC-MS (Method D): m/z: 358 (M+1); Rt=5,04 min.

Example 495 (General Procedure (H))

[1784] (2′-Chlorobiphenyl-4-ylmethyl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine

542

[1785] HPLC-MS (Method D): m/z: 362 (M+1); Rt=5,30 min.

[1786] General procedure (I) for preparation of compounds of general formula I8:

543

[1787] wherein A1, AR1, and AR2 are as defined above.

[1788] 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.

[1789] The general procedure (I) is further illustrated in the following example 496:

Example 496 (General Procedure (I))

[1790] 4-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic Acid

544

[1791] To a solution of 4-(2H-tetrazol-5-yl)benzoic acid (example 412, 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 alquot of this HOAt-ester solution (0.45 mL) was mixed with 0.25 mL of a solution of 4-aminobenzoic acid (1.2 mmol in 1 mL DMF). (Anilines as hydrochlorides can also be utilised, a slight excess of triethylamine was added to the hydrochloride suspension in DMF prior to mixing with the HOAt-ester.) The resulting mixture was shaken for 3 days at room temperature. 1N hydrochloric acid (2 mL) was added and the mixture was shaken for 16 hours at room temperature. The solid was isolated by centrifugation (alternatively by filtration or extraction) and was washed with water (3 mL). Drying in vacuo at 40° C. for 2 days afforded the title compound.

[1792] HPLC-MS (Method D): m/z: 310 (M+1); Rt=2.83 min.

Example 497 (General Procedure (I))

[1793] 3-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic Acid

545

[1794] HPLC-MS (Method D): m/z: 310 (M+1); Rt=2.89 min.

Example 498 (General Procedure (I))

[1795] 3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}acrylic Acid

546

[1796] HPLC-MS (Method D): m/z: 336 (M+1); Rt=3.10 min.

Example 499 (General Procedure (I))

[1797] 3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}propionic Acid

547

[1798] HPLC-MS (Method D): m/z: 338 (M+1); Rt=2.97 min.

Example 500 (General Procedure (I))

[1799] 3-Methoxy-4-[4-(2H-tetrazol-5-yl)benzoylamino]benzoic Acid

548

[1800] HPLC-MS (Method D): m/z: 340 (M+1); Rt=3.03 min.

Example 501 (General Procedure (I))

[1801] N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-yl)benzamide

549

[1802] HPLC-MS (Method D): m/z: 372 (M+1); Rt=4.47 min.

Example 502 (General Procedure (I))

[1803] N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-yl)benzamide

550

[1804] HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.50 min.

Example 503 (General Procedure (I))

[1805] N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-yl)benzamide

551

[1806] HPLC-MS (Method D): m/z: 354 (M+1); Rt=4.60 min.

Example 504 (General Procedure (I))

[1807] N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-yl)benzamide

552

[1808] HPLC-MS (Method D): m/z: 383 (M+1); Rt=4.60 min.

Example 505 (General Procedure (I))

[1809] N-Phenyl-4-(2H-tetrazol-5-yl)benzamide

553

[1810] HPLC-MS (Method D): m/z: 266 (M+1); Rt=3.23 min.

Example 506 (General Procedure (I))

[1811] 4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic Acid

554

[1812] The starting material was prepared as described in example 399.

[1813] HPLC-MS (Method D): m/z: 340 (M+1); Rt=2.83 min.

Example 507 (General Procedure (I))

[1814] 3-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic Acid

555

[1815] HPLC-MS (Method D): m/z: 340 (M+1); Rt=2.90 min.

Example 508 (General Procedure (I))

[1816] 3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}acrylic Acid

556

[1817] HPLC-MS (Method D): m/z: 366 (M+1); Rt=3.07 min.

Example 509 (General Procedure (I))

[1818] 3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}propionic Acid

557

[1819] HPLC-MS (Method D): m/z: 368 (M+1); Rt=2.97 min.

Example 510 (General Procedure (I))

[1820] 3-Methoxy-4-[4-(2H-tetrazol-5-ylmethoxy)benzoylamino]benzoic Acid

558

[1821] HPLC-MS (Method D): m/z: 370 (M+1); Rt=3.07 min.

Example 511 (General Procedure (I))

[1822] N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

559

[1823] HPLC-MS (Method D): m/z: 402 (M+1); Rt=4.43 min.

Example 512 (General Procedure (I))

[1824] N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

560

[1825] HPLC-MS (Method D): m/z: 388 (M+1); Rt=4.50 min.

Example 513 (General Procedure (I))

[1826] N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

561

[1827] HPLC-MS (Method D): m/z: 384 (M+1); Rt=4.57 min.

Example 514 (General Procedure (I))

[1828] N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

562

[1829] HPLC-MS (Method D): m/z: 413 (M+1); Rt=4.57 min.

Example 515 (General Procedure (I))

[1830] N-Phenyl-4-(2H-tetrazol-5-ylmethoxy)benzamide

563

[1831] HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.23 min.

Example 516 (General Procedure (I))

[1832] 4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic Acid

564

[1833] The starting material was prepared as described in example 400.

[1834] HPLC-MS (Method D): m/z: 356 (M+1); Rt=2.93 min.

Example 517 (General Procedure (I))

[1835] 3-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic Acid

565

[1836] HPLC-MS (Method D): m/z: 356 (M+1); Rt=3.00 min.

Example 518 (General Procedure (I))

[1837] 3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}acrylic Acid

566

[1838] HPLC-MS (Method D): m/z: 382 (M+1); Rt=3.26 min.

Example 519 (General Procedure (I))

[1839] 3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}propionic Acid

567

[1840] HPLC-MS (Method D): m/z: 384 (M+1); Rt=3.10 min.

Example 520 (General Procedure (I))

[1841] 3-Methoxy-4-[4-(2H-tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic Acid

568

[1842] HPLC-MS (Method D): m/z: 386 (M+1); Rt=3.20 min.

Example 521 (General Procedure (I))

[1843] N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

569

[1844] HPLC-MS (Method D): m/z: 418 (M+1); Rt=4.57 min.

Example 522 (General Procedure (I))

[1845] N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

570

[1846] HPLC-MS (Method D): m/z: 404 (M+1); Rt=4.60 min.

Example 523 (General Procedure (I))

[1847] N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

571

[1848] HPLC-MS (Method D): m/z: 400 (M+1); Rt=4.67 min.

Example 524 (General Procedure (I))

[1849] N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

572

[1850] HPLC-MS (Method D): m/z: 429 (M+1); Rt=4.67 min.

Example 525 (General Procedure (I))

[1851] N-Phenyl-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

573

[1852] HPLC-MS (Method D): m/z: 312 (M+1); Rt=3.40 min.

[1853] General procedure (J) for solution phase preparation of amides of general formula I9:

574

[1854] wherein AR2 is as defined above.

[1855] This general procedure (J) is further illustrated in the following example.

Example 526 (General Procedure (J)).

[1856] 9-(3-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

575

[1857] 3-(2H-Tetrazol-5-yl)-9H-carbazole (example 401, 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.

[1858]

1
H-NMR (CDCl3): δ 8.87 (1H, d), 8.28 (1H, bs), 8.22 (1H, dd), 8.13 (1H, d), 7.49 (1H, d), 7.47-7.19 (18H, m); HPLC-MS (Method C): m/z: 243 (triphenylmethyl); Rt=5.72 min.

[1859] 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.

[1860]

1
H-NMR (DMSO-6): δ 8.89 (1H, d), 8.29 (1H, d), 8.12 (1 H, 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.

[1861] The compounds in the following examples were prepared in a similar fashion. Optionally, the compounds can be further purified by recrystallization from e.g. aqueous sodium hydroxide (1 N) or by chromatography.

Example 527 (General Procedure (J))

[1862] 9-(4-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

576

[1863] HPLC-MS (Method C): m/z: 360 (M+1); Rt=4.31 min.

Example 528 (General Procedure (J))

[1864] 9-(4-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

577

[1865] HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.26 min.

Example 529 (General Procedure (J))

[1866] 3-(2H-Tetrazol-5-yl)-9-(4-trifluoromethylbenzyl)-9H-carbazole

578

[1867] HPLC-MS (Method C): m/z: 394 (M+1); Rt=4.40 min.

Example 530 (General Procedure (J))

[1868] 9-(4-Benzyloxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

579

[1869] HPLC-MS (Method C): m/z: 432 (M+1); Rt=4.70 min.

Example 531 (General Procedure (J))

[1870] 9-(3-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

580

[1871] HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.25 min.

Example 532 (General Procedure (J))

[1872] 9-Benzyl-3-(2H-tetrazol-5-yl)-9H-carbazole

581

[1873]

1
H-NMR (DMSO-6): δ 8.91 (1H, dd), 8.30 (1H, d), 8.13 (1H, dd), 7.90 (1H, d), 7.73 (1H, d), 7.53 (1 H, t), 7.36-7.20 (6H, m), 5.77 (2H, s).

Example 533 (General Procedure (J))

[1874] 9-(4-Phenylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

582

[1875]

1
H-NMR (DMSO-d6): δ 8.94 (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).

Example 534 (General Procedure (J))

[1876] 9-(3-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

583

[1877] HPLC-MS (Method C): m/z: 356 (M+1); Rt=3.99 min.

Example 535 (General Procedure (J))

[1878] 9-(Naphthalen-2-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

584

[1879] HPLC-MS (Method C): m/z: 376 (M+1); Rt=4.48 min.

Example 536 (General Procedure (J))

[1880] 9-(3-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

585

[1881] HPLC-MS (Method C): m/z: 404 (M+1); Rt=4.33 min.

Example 537 (General Procedure (J))

[1882] 9-(Biphenyl-2-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

586

[1883] HPLC-MS (Method C): m/z: 402 (M+1); Rt=480 min.

Example 538 (General Procedure (J))

[1884] 3-(2H-Tetrazol-5-yl)-9-[4-(1,2,3-thiadiazol-4-yl)benzyl]-9H-carbazole

587

Example 539 (General Procedure (J))

[1885] 9-(2′-Cyanobiphenyl4-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

588

[1886]

1
H-NMR (DMSO-d6): δ 8.91 (1H, d), 8.31 (1H, d), 8.13 (1H, dd), 7.95 (1H, d), 7.92 (1H, d), 7.78 (1 H, d), 7.75 (1 H, dt), 7.60-7.47 (5H, m), 7.38-7.28 (3H, m), 5.86 (2H, s); HPLC-MS (Method C): m/z: 427 (M+1); Rt=4.38 min.

Example 540 (General Procedure (J))

[1887] 9-(4-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

589

[1888] HPLC-MS (Method C): m/z: 452 (M+1); Rt=4.37 min.

Example 541 (General Procedure (J))

[1889] 9-(3,5-Bis(trifluoromethyl)benzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

590

[1890] HPLC-MS (Method C): m/z: 462 (M+1); Rt=4.70 min.

Example 542 (General Procedure (J))

[1891] 9-(4-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

591

[1892]

1
H-NMR (DMSO-d6): δ 8.89 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.88 (1H, d), 7.70 (1H, d), 7.52 (1H, t), 7.49 (2H, d), 7.31 (1H, t), 7.14 (2H, d), 5.74 (2H, s); HPLC-MS (Method C): m/z: 404 (M+1); Rt=4.40 min.

Example 543 (General Procedure (J))

[1893] 9-(Anthracen-9-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

592

[1894] HPLC-MS (Method C): m/z: 426 (M+1); Rt=4.78 min.

Example 544 (General Procedure (J))

[1895] 9-(4-Carboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

593

[1896] 3.6 fold excess sodium hydride was used.

[1897]

1
H-NMR (DMSO-d6): δ 12.89 (1H, bs), 8.89 (1H, d), 8.30 (1H, d), 8.10 (1H, dd), 7.87 (1H, d), 7.86 (2H, d), 7.68 (1H, d), 7.51 (1H, t), 7.32 (1H, t), 7.27 (2H, d), 5.84 (2H, s); HPLC-MS (Method C): m/z: 370 (M+1); Rt=3.37 min.

Example 545 (General Procedure (J))

[1898] 9-(2-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

594

[1899] HPLC-MS (Method B): m/z: 360 (M+1); Rt=5.30 min.

Example 546 (General Procedure (J))

[1900] 9-(4-Fluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

595

[1901]

1
H-NMR (DMSO-d6): δ 8.88 (1H, d), 8.28 (1H, d), 8.10 (1H, dd), 7.89 (1H, d), 7.72 (1H, d), 7.52 (1H, t), 7.31 (1H, t), 7.31-7.08 (4H, m), 5.74 (2H, s); HPLC-MS (Method C): m/z: 344 (M+1); Rt=4.10 min.

Example 547 (General Procedure (J))

[1902] 9-(3-Fluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

596

[1903]

1
H-NMR (DMSO-d6): δ 8.89 (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.

Example 548 (General Procedure (J))

[1904] 9-(2-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

597

[1905] HPLC-MS (Method C): m/z: 452 (M+1); Rt=4.58 min.

Example 549 (General Procedure (J))

[1906] 9-(3-Carboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

598

[1907] 3.6 fold excess sodium hydride was used.

[1908]

1
H-NMR (DMSO-d6): δ 12.97 (1H, bs), 8.90 (1H, bs), 8.30 (1H, d), 8.12 (1H, bd), 7.8 (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.

Example 550 (General Procedure (J))

[1909] 9-[4-(2-Propyl)benzyl]-3-(2H-tetrazol-5-yl)-9H-carbazole

599

[1910]

1
H-NMR (DMSO-d6): δ 8.87 (1H, d), 8.27 (1H, d), 8.10 (1H, dd), 7.87 (1H, d), 7.71 (1H, d), 7.51 (1H, t), 7.31 (1H, t), 7.15 (2H, d), 7.12 (2H, d), 5.69 (2H, s), 2.80 (1H, sept), 1.12 (6H, d); HPLC-MS (Method C): m/z: 368 (M+1); Rt=4.73 min.

Example 551 (General Procedure (J))

[1911] 9-(3,5-Dimethoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

600

[1912] HPLC-MS (Method C): m/z: 386 (M+1); Rt=4.03 min.

Example 552 (General Procedure (J))

[1913] 3-(2H-Tetrazol-5-yl)-9-(2,4,5-trifluorobenzyl)-9H-carbazole

601

[1914] HPLC-MS (Method B): m/z: 380 (M+1); Rt=5.00 min.

Example 553 (General Procedure (J))

[1915] N-Methyl-N-phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

602

[1916] HPLC-MS (Method B): m/z: 383 (M+1); Rt=4.30 min.

Example 554 (General Procedure (J))

[1917] 9-(4-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

603

[1918]

1
H-NMR (DMSO-d6): δ 8.86 (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.

Example 555 (General Procedure (J))

[1919] 9-(2-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

604

[1920]

1
H-NMR (DMSd): δ 8.87 (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.

Example 556 (General Procedure (J))

[1921] 9-(4-Cyanobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

605

[1922] HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.74 min.

Example 557 (General Procedure (J))

[1923] 9-(3-Cyanobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

606

[1924] HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.73 min.

Example 558 (General Procedure (J))

[1925] 9-(5-Chloro-2-methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

607

[1926]

1
H-NMR (DMSO-d6): δ 8.87 (1H, d), 8.35 (1H, d), 8.10 (1H, dd), 7.73 (1H, d), 7.59 (1H, d), 7.49 (1H, t), 7.29 (1H, t), 7.27 (1H, dd), 7.11 (1H, d), 6.51 (1H, d), 5.63 (2H, s), 3.88 (3H, s); HPLC-MS (Method C): m/z: 390 (M+1); Rt=4.37 min.

Example 559 (General Procedure (J))

[1927] N-Phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

608

[1928]

1
H-NMR (DMSO-d6): δ 10.54 (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.

Example 560 (General Procedure (J))

[1929] N-Butyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

609

[1930]

1
H-NMR (DMSO-d6): δ 8.85 (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.

Example 561 (General Procedure (J))

[1931] 9-(2,4-Dichlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

610

[1932]

1
H-NMR (DMSO-d6): δ 8.92 (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.

Example 562 (General Procedure (J))

[1933] 9-(2-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

611

[1934]

1
H-NMR (DMSO-d6): δ 8.92 (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.

Example 563 (General Procedure (J))

[1935] 9-(3-Nitrobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

612

[1936] HPLC-MS (Method C): m/z: 371 (M+1); Rt=3.78 min.

Example 564 (General Procedure (J))

[1937] 9-(3,4-Dichlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

613

[1938] HPLC-MS (Method B): m/z: 394 (M+1); Rt=5.62 min.

Example 565 (General Procedure (J))

[1939] 9-(2,4-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

614

[1940]

1
H-NMR (DMSO-d6): δ 8.89 (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.

Example 566 (General Procedure (J))

[1941] 9-(3,5-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

615

[1942]

1
H-NMR (DMSO-d6): δ 8.90 (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.

Example 567 (General Procedure (J))

[1943] 9-(3,4-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

616

[1944]

1
H-NMR (DMSO-d6): δ 8.89 (1H, bs), 8.29 (1H, d), 8.12 (1H, bd), 7.92 (1H, d), 7.74 (1H, d), 7.54 (1H, t), 7.42-7.25 (3H, m), 6.97 (1H, bm), 5.75 (2H, s); HPLC-MS (Method B): m/z: 362 (M+1); Rt=5.17 min.

Example 568 (General Procedure (J))

[1945] 9-(3-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

617

[1946] HPLC-MS (Method B): m/z: 452 (M+1); Rt=5.50 min.

Example 569 (General Procedure (J))

[1947] 3-(2H-Tetrazol-5-yl)-9-[3-(trifluoromethyl)benzyl]-9H-carbazole

618

[1948]

1
H-NMR (DMSO-d6): δ 8.89 (1H, d), 8.30 (1H, d), 8.11 (1H, dd), 7.90 (1H, d), 7.72 (1H, d), 7.67 (1H, bs), 7.62 (1H, bd), 7.53 (1H, t), 7.50 (1H, bt), 7.33 (1H, bd), 7.32 (1H, t), 5.87 (2H, s); HPLC-MS (Method B): m/z: 394 (M+1); Rt=5.40 min.

Example 570 (General Procedure (J))

[1949] N-(4-Carboxyphenyl)-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

619

[1950] 3.6 fold excess sodium hydride was used.

[1951] HPLC-MS (Method B): m/z: 413 (M+1); Rt=3.92 min.

Example 571 (General Procedure (J))

[1952] N-(2-Propyl)-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

620

[1953] HPLC-MS (Method B): m/z: 335 (M+1); Rt=3.70 min.

Example 572 (General Procedure (J))

[1954] N-Benzyl-N-phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

621

[1955] HPLC-MS (Method B): m/z: 459 (M+1); Rt=5.37 min.

Example 573 (General Procedure (J))

[1956] N-[4-(2-Methyl-2-propyl)phenyl]-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

622

[1957] HPLC-MS (Method B): m/z: 425 (M+1); Rt=5.35 min.

Example 574 (General Procedure (J))

[1958] N-Phenethyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide

623

[1959] HPLC-MS (Method C): m/z: 397 (M+1); Rt=3.43 min.

Example 575 (General Procedure (J))

[1960] 3-(2H-Tetrazol-5-yl)-9-[2-(trifluoromethyl)benzyl]-9H-carbazole

624

[1961] HPLC-MS (Method C): m/z: 394 (M+1); Rt=4.44 min.

Example 576 (General Procedure (J))

[1962] 9-[2-Fluoro-6-(trifluoromethyl)benzyl]-3-(2H-tetrazol-5-yl)-9H-carbazole

625

[1963] HPLC-MS (Method C): m/z: 412 (M+1); Rt=4.21 min.

Example 577 (General Procedure (J))

[1964] 9-[2,4-Bis(trifluoromethyl)benzyl)]-3-(2H-tetrazol-5-yl)-9H-carbazole

626

[1965] HPLC-MS (Method C): m/z: 462 (M+1); Rt=4.82 min.

Example 578 (General Procedure (J))

[1966] 3-(2H-Tetrazol-5-yl)-9-(2,4,6-trimethylbenzyl)-9H-carbazole

627

[1967] HPLC-MS (Method C): m/z: 368 (M+1); Rt=4.59 min.

Example 579 (General Procedure (J))

[1968] 9-(2,3,5,6-Tetramethylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

628

[1969] HPLC-MS (Method C): m/z: 382 (M+1); Rt=4.47 min.

Example 580 (General Procedure (J))

[1970] 9-[(Naphthalen-1-yl)methyl]-3-(2H-tetrazol-5-yl)-9H-carbazole

629

[1971] HPLC-MS (Method C): m/z: 376 (M+1); Rt=4.43 min.

[1972] Further preferred compounds of the invention that may be prepared according to general procedure (J) includes:

630

631

[1973] The following preferred compounds of the invention may be prepared eg. from 9-(4-bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole (example 542) or from 9-(3-bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole (example 536) 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).

632

[1974] General procedure (K) for preparation of compounds of general formula I10:

633

[1975] wherein AR2 is as defined above.

[1976] The general procedure (K) is further illustrated by the following example:

Example 581 (General Procedure (K))

[1977] 1-Benzyl-5-(2H-tetrazol-5-yl)-1H-indole

634

[1978] 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.

[1979] HPLC-MS (Method C): m/z: 233 (M+1); Rt=4.17 min.

[1980] 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 401. Purification was done by flash chromatography on silica gel eluting with dichloromethane/methanol=9:1.

[1981] HPLC-MS (Method C): m/z: 276 (M+1); Rt=3.35 min.

[1982] The compounds in the following examples were prepared by the same procedure.

Example 582 (General Procedure (K))

[1983] 1-(4-Bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole

635

[1984] HPLC-MS (Method C): m/z: 354 (M+1); Rt=3.80 min.

Example 583 (General Procedure (K))

[1985] 1-(4-Phenylbenzyl)-5-(2H-tetrazol-5-yl)-1H-indole

636

[1986]

1
H-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.

Example 584 (General Procedure (K))

[1987] 5-(2H-Tetrazol-5-yl)-1H-indole

637

[1988] 5-(2H-Tetrazol-5-yl)-1H-indole was prepared from 5-cyanoindole according to the method described in example 401.

[1989] HPLC-MS (Method C): m/z: 186 (M+1); Rt=1.68 min.

Example 585 (General Procedure (K))

[1990] 1-Benzyl-4-(2H-tetrazol-5-yl)-1H-indole

638

[1991] 1-Benzyl-1H-indole-4-carbonitrile was prepared from 4-cyanoindole according to the method described in example 581.

[1992] HPLC-MS (Method C): m/z: 233 (M+1); Rt=4.24 min.

[1993] 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 401.

[1994] HPLC-MS (Method C): m/z: 276 (M+1); Rt=3.44 min.

[1995] Further preferred compounds of the invention that may be prepared according to general procedure (K) includes:

639

640

641

[1996] The following preferred compounds of the invention may be prepared eg. from 1-(4-bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole (example 532) 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).

642

[1997] General procedure (L) for preparation of compounds of general formula I11:

643

[1998] The general procedure (L) is further illustrated by the following example:

Example 586 (General Procedure (L))

[1999] 1-Benzoyl-5-(2H-tetrazol-5-yl)-1H-indole

644

[2000] 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.

[2001] HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.07 min.

[2002] 1-Benzoyl-1H-indole-5-carbonitrile was transformed into 1-benzoyl-5-(2H-tetrazol-5-yl)-1H-indole by the method described in example 401.

[2003] HPLC (Method C): Rt=1.68 min.

[2004] The compound in the following example was prepared by the same procedure.

Example 587 (General Procedure (L))

[2005] 1-Benzoyl-4-(2H-tetrazol-5-yl)-1H-indole

645

[2006] 1-Benzoyl-1H-indole4-carbonitrile was prepared from 4-cyanoindole according to the method described in example 586.

[2007] HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.24 min.

[2008] 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 401.

[2009] HPLC (Method C); Rt=1.56 min.

[2010] The following known and commercially available compounds do all bind to the His B10 Zn2+ site of the insulin hexamer:

Example 588

[2011] 1-(4-Fluorophenyl)-5-(2H-tetrazol-5-yl)-1H-indole

646

Example 589

[2012] 1-Amino-3-(2H-tetrazol-5-yl)benzene

647

Example 590

[2013] 1-Amino-4-(2H-tetrazol-5-yl)benzene

648

[2014] 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.

[2015]

1
H 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.

Example 591

[2016] 1-Nitro-4-(2H-tetrazol-5-yl)benzene

649

Example 592

[2017] 1-Bromo-4-(2H-tetrazol-5-yl)benzene

650

[2018] General procedure (M) for solution phase preparation of amides of general formula I12:

651

[2019] wherein A, B1, B2 are as defined above, R is hydrogen, optionally substituted aryl or C1-8-alkyl and R′ is hydrogen or C1-4-alkyl.

[2020] A—B1—B2—CO2H may be prepared eg by general procedure (D) or by other similar procedures described herein, or may be commercially available.

[2021] The procedure is further illustrated in the following example 593:

Example 593 (General Procedure (M))

[2022] N-(4-Chlorobenzyl)-2-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)-1H-indol-1-yl]acetamide

652

[2023] [3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]acetic acid (example 300, 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 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.

[2024] HPLC-MS (Method C): m/z: 426 (M+1); Rt.=3.79 min.

Example 594 (General Procedure (M))

[2025] 1H-Benzotriazole-5-carboxylic acid 4-chlorobenzylamide

653

[2026] HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.40 min.

Example 595 (General Procedure (M))

[2027] N-(4-Chlorobenzyl)-4-[2-chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide

654

[2028] HPLC-MS (Method A): m/z: 465 (M+1); Rt=4.35 min.

Example 596 (General Procedure (M))

[2029] N-(4-Chlorobenzyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide

655

[2030] HPLC-MS (Method A): m/z: 431 (M+1); Rt=3.68 min.

Example 597 (General Procedure (M))

[2031] 2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]-N-(4-chlorobenzyl)acetamide

656

[2032] HPLC-MS (Method A): m/z: 483 (M+1); Rt=4.06 min.

Example 598 (General Procedure (M))

[2033] N-(4-Chlorobenzyl)-2-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetamide

657

[2034] HPLC-MS (Method A): m/z: 403 (M+1); Rt=4.03 min.

Example 599 (General Procedure (M))

[2035] N-(4-Chlorobenzyl)-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]acrylamide

658

[2036] HPLC-MS (Method A): m/z: 399 (M+1); Rt=3.82.

[2037] N-(4-Chlorobenzyl)-4-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide

659

[2038] HPLC-MS (Method A): m/z: 431 (M+1); Rt=3.84 min.

Example 601 (General Procedure (M))

[2039] 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]-N-(4-chlorobenzyl)butyramide

660

[2040] HPLC-MS (Method A ): m/z: 511 (M+1)); Rt=4.05 min.

Example 602 (General Procedure (M))

[2041] 4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-phenoxy]-N-(4-chlorobenzyl)-butyramide

661

[2042] HPLC-MS (Method A): m/z: 527 (M+1); Rt=4.77 min.

Example 603 (General Procedure (M))

[2043] N-(4-Chlorobenzyl)-2-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetamide

662

[2044] HPLC-MS (Method C): m/z: 431 (M+1); Rt.=4.03 min.

Example 604 (General Procedure (M))

[2045] N-(4-Chlorobenzyl)-3-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)-1H-indol-1-yl]propionamide

663

[2046] HPLC-MS (Method C): m/z: 440 (M+1); Rt.=3.57 min.

Example 605 (General Procedure (M))

[2047] N-(4-Chlorobenzyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyramide

664

[2048] HPLC-MS (Method C): m/z: 481 (M+1); Rt=4.08 min.

Example 606 (General Procedure (M))

[2049] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-N-hexylbutyramide

665

[2050] HPLC-MS (Method C): m/z: 441 (M+1); Rt=4.31 min.

Example 607 (General Procedure (M))

[2051] N-(4-Chlorobenzyl)-4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzamide

666

[2052] HPLC-MS (Method C): m/z:493 (M+1); Rt=4.19 min.

Example 608 (General Procedure (M))

[2053] N-(4-Chlorobenzyl)-3-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzamide

667

[2054] HPLC-MS (Method C): m/z: 493 (M+1); Rt=4.20 min.

Example 609

[2055] 4-({[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carbonyl]amino}methyl)benzoic Acid Methyl Ester

668

[2056] HPLC-MS (Method C): m/z: 436 (M+1); Rt.=3.55 min.

[2057] The commercially available compounds in the following examples do all bind to the HisB10 Zn2+ site:

Example 610

[2058] 1-(4-Bromo-3-methylphenyl)-1,4-dihydrotetrazole-5-thione

669

Example 611

[2059] 1-(4-Iodophenyl)-1,4-dihydrotetrazole-5-thione

670

Example 612

[2060] 1-(2,4,5-Trichlorophenyl)-1H-tetrazole-5-thiol

671

Example 613

[2061] 1-(2,6-Dimethylphenyl)-1,4-dihydrotetrazole-5-thione

672

Example 614

[2062] 1-(2,4,6-Trimethylphenyl)-1,4-dihydrotetrazole-5-thione

673

Example 615

[2063] 1-(4-Dimethylaminophenyl)-1H-tetrazole-5-thiol

674

Example 616

[2064] 1-(3,4-Dichlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione

675

Example 617

[2065] 1-(4-Propylphenyl)-1,4-dihydro-1H-tetrazole-5-thione

676

Example 618

[2066] 1-(3-Chlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione

677

Example 619

[2067] 1-(2-Fluorophenyl)-1,4-dihydro-1H-tetrazole-5-thione

678

Example 620

[2068] 1-(2,4-Dichlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione

679

Example 621

[2069] 1-(4-Trifluoromethoxyphenyl)-1,4-dihydro-1H-tetrazole-5-thione

680

Example 622

[2070] N-[4-(5-Mercaptotetrazol-1-yl)-phenyl]-acetamide

681

Example 623

[2071] 1-(4-Chlorophenyl)-1,4-dihydrotetrazole-5-thione

682

Example 624

[2072] 1-(4-Methoxyphenyl)-1,4-dihydrotetrazole-5-thione

683

Example 625

[2073] 1-(3-Fluoro4-pyrrolidin-1-ylphenyl)-1,4-dihydrotetrazole-5-thione

684

[2074] Preparation of 1-aryl-1,4-dihydrotetrazole-5-thiones (or the tautomeric 1-aryltetrazole-5-thiols) is described in the literature (eg. by Kauer & Sheppard, J. Org. Chem., 32, 3580-92 (1967)) and is generally performed eg. by reaction of aryl-isothiocyanates with sodium azide followed by acidification

[2075] 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:

685

[2076] 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 303.

[2077] 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.

[2078] 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.

[2079] Step 4 is a conversion to mercaptotetrazole as described above.

[2080] Preferred compounds of the invention includes:

686

Example 626

[2081] 4-(4-Hydroxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

687

[2082] 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.

[2083] HPLC-MS (Method C): m/z: 286 (M+1); Rt.=3.56 min.

[2084] A mixture of 2-benzenesulfonyl-3-(4-hydroxyphenyl)acrylonitrile (2.08 g, 7.3 mmol) and sodium azide (0.47g, 7.3 mmol) in DMF (50 mL) was heated at reflux temperature 2 hours. After cooling, the mixture was poured on ice. The mixture was evaporated in vacuo to almost dryness and toluene was added. After filtration, the organic phase was evaporated in vacuo. The residue was purified by silicagel chromatography eluting with a mixture of ethyl acetate and heptane (1:2). This afforded 1.2 g (76%) of the title compound.

[2085]

1
H 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

[2086] The compounds in the following examples are commercially available and may be prepared using a similar methodology:

Example 627

[2087] 4-(4-Trifluoromethoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

688

Example 628

[2088] 4-Benzo[1,3]dioxol-5-yl-1H-[1,2,3]triazole-5-carbonitrile

689

Example 629

[2089] 4-(3-Trifluoromethylphenyl)-1H-[1,2,3]triazole-5-carbonitrile

690

Example 630

[2090] 4-Pyridin-3-yl-1H-[1,2,3]triazole-5-carbonitrile

691

Example 631

[2091] 4-(2,6-Dichlorophenyl)-1H-[1,2,3]triazole-5-carbonitrile

692

Example 632

[2092] 4-Thiophen-2-yl-1H-[1,2,3]triazole-5-carbonitrile

693

Example 633

[2093] 3,5-Dimethylisoxazole-4-carboxylic acid 4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester

694

Example 634

[2094] 3,3-Dimethyl-butyric acid 4-(5-cyano-1H-[1,2,3]triazol4-yl)phenyl Ester

695

Example 635

[2095] 4-Methyl-[1,2,3]thiadiazole-5-carboxylic acid 4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester

696

Example 636

[2096] 4-Chlorobenzoic acid 4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester

697

Example 637

[2097] 4-(3-Phenoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

698

Example 638

[2098] 4-(5-Bromo-2-methoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

699

Example 639

[2099] 4-(2-Chloro-6-fluorophenyl)-1H-[1,2,3]triazole-5-carbonitrile

700

[2100] The following cyanotriazoles are also preferred compounds of the invention:

[2101]

4
-(2-Chloro-6-fluorophenyl)-1H-[1,2,3]triazole-5-carbonitrile.

[2102] Terephthalic acid mono[4-(5-cyano-1H-[1,2,3]triazol4-yl)phenyl] ester.

[2103] N-[4-(5-cyano-1H-[1,2,3]triazol-4-yl)-phenyl]terephthalamic acid

[2104] 4-(4-Octyloxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile

[2105] 4-(Styrylphenyl))-1H-[1,2,3]triazole-5-carbonitrile.

[2106] 4-(4′-Trifluoromethylbiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.

[2107] 4-(4′-Chlorobiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.

[2108] 4-(4′-Methoxybiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.

[2109] 4-(1-Naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.

[2110] 4-(9-Anthranyl)-1H-[1,2,3]triazole-5-carbonitrile.

[2111] 4-(4-Methoxy-1-naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.

[2112] 4-(4-Aminophenyl)-1H-[1,2,3]triazole-5-carbonitrile.

[2113] 4-(2-Naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.

[2114] General procedure (N) for preparation of compounds of general formula I13:

701

[2115] wherein

[2116] n is 1 or 3-20,

[2117] AR1 is as defined above,

[2118] 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.

[2119] This procedure is very similar to general procedure (D), steps 1 and 2 are identical.

[2120] Steps 3 and 4 are described in the literature (eg Beck & Gûnther, Chem. Ber., 106, 2758-66 (1973))

[2121] 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.

[2122] The following compounds may be prepared according to this general procedure (N):

[2123] 4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyric acid:

702

[2124] 2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetic acid:

703

[2125] 4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyric acid ethyl ester

[2126] 5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoic acid

[2127] 8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoic acid

[2128] 10-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)decanoic acid

[2129] 12-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)dodecanoic acid

[2130] General procedure (O) for preparation of compounds of general formula I7:

704

[2131] wherein PS is polymeric support, a Tentagel S RAM resin, n is 1-20, m is 0-5, and p is 0 or 1.

[2132] The compounds of the invention of general formula (I2) can be prepared by means of standard peptide chemistry (General procedure H), e.g. in 0.5 mmol scale, using Fmoc strategy and HOAt or HOBT activated amino acids. The compounds prepared in the following examples according to General procedure (O) were all isolated as the TFA salts. This procedure is further illustrated in the following:

[2133] Typically, 2 gram of Fmoc Tentagel S RAM resin (Rapp Polymere, Tubingen) with substitution 0,25 mmol/g was washed with NMP then treated with 25% piperidine in NMP for 30 min followed by wash with NMP which renders the resin ready for coupling.

[2134] Step wise coupling of Fmoc-Arginine (Fmoc-Arg(Pmc)-OH), Fmoc-Glycine (Fmoc-Gly-OH) and Fmoc-4-aminobenzoic acid (Fmoc-4-Abz-OH):

[2135] To 2 mmol of Fmoc-L-Arg(Pmc)-OH (Novabiochem) was added 3,33 ml 0,6M HOAt in NMP (Perseptives) or 0,6M HOBT in NMP (Novabiochem) containing 0,2% bromphenolblue as indicator and added 330 μl of diisopropylcarbodiimide DIC (Fluka) and the solution was then added to the resin. After coupling for minimum 1 hour, or when the blue colour disappeared, the resin was washed with NMP and the Fmoc group was deprotected with 25% piperidine in NMP for 20 minutes followed by wash with NMP. This stepwise assembling of the arginine residues was repeated to give 3, 4, 5 or 6 arginines on the resin. The Fmoc-Glycine (Novabiochem) and Fmoc4-aminobenzoic acid (Fluka and Neosystems) were coupled using the same procedure as described for Fmoc-Arg(Pmc)-OH.

[2136] Coupling of A-OH, e.g. 1H-benzotriazole-5-carboxylic acid on Gly.

[2137] When A-OH, e.g. 1H-benzotriazole-5-carboxylic acid (Aldrich) was coupled on a glycine or arginine residue the coupling procedure was as described above.

[2138] Coupling of A-OH, e.g. 1H-benzotriazole-5-carboxylic acid on Abz or 4-Apac:

[2139] Due to the lower nucleophilicity of the amino group in Abz the following procedure was necessary. To 4 mmol of A-OH, e.g. 1H-benzotriazole-5-carboxylic acid was added 6,66 ml of a solution of 0,6M HOAt, 0,2 mmol dimethylaminopyridine (DMAP) and 4-mmol DIC and was then added to the resin and allowed to react overnight.

[2140] Introduction of fragment 4-Apac instead of 4-Abz:

[2141] 4-Nitrophenoxyacetic acid may be coupled on a glycine or arginine residue using DIC and HOBT/HOAt as described above. Subsequent reduction of the nitro group may be done using SnCl2 in NMP or DMF e.g. as described by Tumelty et al. (Tet. Lett., (1998) 7467-70).

[2142] Cleavage of the peptides from the resin.

[2143] After synthesis the resin was washed extensively with diethyl ether and dried. To 1 gram of the peptidyl resin was added 25 ml of a TFA solution containing 5% thioanisole, 5% ethanol, 5% phenol and 2% triisopropylsilane and allowed to react for 2 hours. The TFA solution was filtered and concentrated with argon flow for approximately 30 minutes. Then diethylether ca. 5-7 times the residual volume of TFA was added and the peptide precipitate was extracted in 10% AcOH and washed 5 times with diethyl ether and lyophilized.

[2144] RP-HPLC analysis and purification: The crude products were analysed on RP-HPLC Cl 8 column (4,6×250 mm) using one of two gradients (see table 1 and 2), temperature 25° C., wavelength 214 nm and flow rate 1 ml/min with A-buffer 0,15% (w/w) TFA in H2O and B-Buffer (87,5% (w/w) MeCN, 0,13% (w/w) TFA in H2O)

[2145] The products were purified on preparative RP-HPLC C18 column (2×25 cm) using a gradient (variable, see e.g examples 640 to 643643643), temperature 25° C., wavelength 214 nm and flow rate 6 ml/min with A-buffer 0,15% (w/w) TFA in H2O and B-Buffer (87,5% (w/w) MeCN, 0,13% (w/w) TFA in H2O) and verified by mass spectrometry (MALDI).

7TABLE 1Time (min.)Flow (ml/min) (% A% B01.0095.05.030.001.0080.020.035.001.000.0100.040.001.000.0100.045.001.0095.05.0

[2146]

8

TABLE 2

Time (min.)
Flow (ml/min)
% A
% B

0
1.00
95.0
5.0

30.00
1.00
40.0
60.0

31.00
1.00
0.00
100.0

35.00
1.00
0.00
100.0

36.00
1.00
95.0
5.0

[2147] The following examples were prepared using this general procedure (0).

Example 640 (General Procedure (O))

[2148] Benzotriazol-5-ylcarbonyl-Gly2-Arg3-NH2 (BT-G2R3).

705

[2149] MS (MALDI): m/z: 746.7 g/mol; calculated: 744.2 g/mol. HPLC gradient:

9Time (min)Flow (ml/min)% A% B0.006.0090.010.0120.006.0090.010.0121.000.1090.010.0

Example 641 (General Procedure (O))

[2150] Benzotriazol-5-ylcarbonyl-Gly2-Arg4-NH2 (BT-G2R4).

706

[2151] MS (MALDI): m/z: 903.0 g/mol; calculated: 900.6 g/mol. HPLC gradient:

10Time (min)Flow (ml/min)% A% B0.006.0095.05.030.006.0080.020.035.006.000.0100.040.006.000.0100.045.006.0095.05.064.006.0095.05.0

Example 642 (General Procedure (O))

[2152] Benzotriazol-5-ylcarbonyl-Gly2-Arg5-NH2 (BT-G2R5).

707

[2153] MS (MALDI): m/z: 1060.8 g/mol; calculated: 1057 g/mol. HPLC gradient

11Time (min)Flow (ml/min)% A% B0.006.0088.012.0120.006.0088.012.0121.000.1088.012.0

Example 643 (General Procedure (O))

[2154] Benzotriazol-5-ylcarbonyl-Gly2-Arg6-NH2 (BT-G2R6).

708

[2155] MS (MALDI): m/z: 1214.8 g/mol; calculated: 1213.4 g/mol. HPLC gradient:

12Time (min)Flow (ml/min)% A% B0.006.0088.012.0120.006.0088.012.0121.000.1088.012.0

Example 644 (General Procedure (O))

[2156] Benzotriazol-5-ylcarbonyl4-Abz-Gly2-Arg5-NH2 (BT-4-Abz-G2R5).

709

[2157] MS (MALDI): m/z: 1176.7 g/mol; calculated: 1177.9 g/mol. HPLC gradient:

13Time (min)Flow (ml/min)% A% B0.006.0095.05.040.006.0060.040.045.006.0060.040.050.006.000.0100.055.006.000.0100.060.006.0095.05.0

Example 645 (General Procedure (O))

[2158] Benzothiazol-5-ylcarbonyl-4-Abz-Gly-Arg5-NH2 (BT-4-Abz-GR5).

710

[2159] MS (MALDI): m/z: 1122 g/mol; calculated: 1120.4 g/mol. HPLC gradient:

14Time (min)Flow (ml/min)% A% B0.006.0095.05.040.006.0060.040.045.006.0060.040.050.006.000.0100.055.006.000.0100.060.006.0095.05.0

Example 646 (General Procedure (O))

[2160] Benzotriazol-5-ylcarbonyl-4-Abz-Arg5-NH2 (BT4-Abz-R5)

711

[2161] MS (MALDI): m/z: 1064.3 g/mol; calculated: 1063.2 g/mol. HPLC gradient:

15Time (min)Flow (ml/min)% A% B0.006.0095.05.040.006.0060.040.045.006.0060.040.050.006.000.0100.055.006.000.0100.060.006.0095.05.0

[2162] General procedure (P) for preparation of compounds of general formula I8:

712

[2163] wherein X, Y, R10, E, B1, B2 are as defined above,

[2164] p is 0 or 1,

[2165] m is 0-5 and

[2166] n is 1-20.

[2167] This general procedure is very similar to General procedure (O), where benzotriazole-5-carboxylic acid in the last step before cleavage from the resin is replaced with compounds optionally prepared according to general procedure (D):

713

Example 647 (General Procedure (P))

[2168] 4-{2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetylamino}benzoyl-Gly2-Arg5-NH2

714

Example 648 (General Procedure (P))

[2169] 3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg5-NH2

715

[2170] MS (MALDI): m/z: 1057.3 g/mol; calculated: 1055.3 g/mol.

Example 649 (General Procedure (P))

[2171] 3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg4-NH2

716

[2172] MS (MALDI): m/z: 899.1 g/mol; calculated: 901.6 g/mol.

Example 650 (General Procedure (P))

[2173] 3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg3-NH2

717

[2174] MS (MALDI): m/z: 746.2 g/mol; calculated: 742.9 g/mol.

Example 651 (General Procedure (P))

[2175] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH2

718

[2176] MS (MALDI): m/z: 1088.7 g/mol; calculated: 1087 g/mol.

Example 652 (General Procedure (P))

[2177] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH2

719

[2178] MS (MALDI): m/z: 933.0 g/mol; calculated: 931 g/mol.

Example 653 (General Procedure (P))

[2179] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2

720

[2180] MS (MALDI): m/z: 776.9 g/mol; calculated: 774.0 g/mol.

Example 654 (General Procedure (P))

[2181] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg12-NH2

721

[2182] MS (MALDI): m/z: 2232.9.4 g/mol; calculated: 2230.3 g/mol.

Example 655 (General Procedure (P))

[2183] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg8-NH2

722

[2184] MS (MALDI): m/z: 1607.4 g/mol; calculated: 1605.5 g/mol.

Example 656 (General Procedure (P))

[2185] [4(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg5-NH2

723

[2186] MS (MALDI): m/z: 1141.9 g/mol; calculated: 1137.4 g/mol.

Example 657 (General Procedure (P))

[2187] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg4-NH2

724

[2188] MS (MALDI): m/z: 985.4 g/mol; calculated: 981.2 g/mol.

Example 658 (General Procedure (P))

[2189] 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg3-NH2

725

[2190] MS (MALDI): m/z: 828.5 g/mol; calculated: 825.0 g/mol.

[2191] The following compounds were prepared according to the methodology described in general procedure (O) and (P):

Example 659

[2192] 4-(2H-Tetrazol-5-yl)benzoyl4-Abz-Gly2-Arg5-NH2

726

[2193] MS (MALDI): m/z: 1203.8 g/mol; calculated: 1203.8 g/mol.

Example 660

[2194] 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg5-NH2

727

[2195] MS (MALDI): m/z: 1152.5 g/mol; calculated: 1149.3 g/mol.

Example 661

[2196] 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg8-NH2

728

[2197] MS (MALDI): m/z: 1621.0 g/mol; calculated: 1617.5 g/mol.

Example 662

[2198] 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg12-NH2

729

[2199] MS (MALDI): m/z: 2247.9 g/mol; calculated: 2242.3 g/mol.

[2200] Other preferred compounds of the invention that may be prepared according to general procedure (O) and/or general procedure (P) includes:

16Building block from example 291:4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg10-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg9-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg8-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg7-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg11-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg12-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg13-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg14-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg15-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg16-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg17-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg18-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg19-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg20-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys6-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys5-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys4-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys3-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys7-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys8-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys9-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys10-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys11-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys12-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys13-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys14-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys15-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys16-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys17-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys18-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys19-NH24-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys20-NH2Building block from example 292:5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg6-NH25-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg5-NH25-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg4-NH25-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg3-NH2Building bCock from page 164:6-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg3-NH26-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg4-NH26-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg5-NH2Building block from page 164:7-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg3-NH27-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg4-NH27-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg5-NH2Building block from page 164:8-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg3-NH28-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg4-NH28-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg5-NH2Building block from page 164:10-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg3-NH210-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg4-NH210-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg5-NH2Building block from page 164:11-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg3-NH211-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg4-NH211-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg5-NH2Building block from page 164:12-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg3-NH212-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg4-NH212-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg5-NH2Building block from page 164:15-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg3-NH215-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg4-NH215-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg5-NH2Building block from example 298:2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg6-NH22-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg5-NH22-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg4-NH22-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg3-NH2Building block from example 302:2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetyl-Arg6-NH22-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetyl-Arg5-NH22-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetyl-Arg4-NH22-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetyl-Arg3-NH24-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg6-NH24-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg5-NH24-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg4-NH24-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg3-NH215-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg6-NH215-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg5-NH215-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg4-NH215-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg3-NH25-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg6-NH25-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg5-NH25-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg4-NH25-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg3-NH2Building block from example 284:3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg6-NH2Building block from example 295:2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg6-NH22-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg5-NH22-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg4-NH22-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg3-NH28-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg6-NH28-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg5-NH28-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg4-NH28-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg3-NH26-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg6-NH26-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg5-NH26-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg4-NH26-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg3-NH2Building block from example 288:4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH24-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH24-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH24-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2Building block from example 282:4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH2Building block from example 289:4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH24-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH24-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH24-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH211-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg6-NH211-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg5-NH211-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg4-NH211-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg3-NH24-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg6-NH24-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg5-NH24-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg4-NH24-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg3-NH2Building block from example 286:4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg6-NH24-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg5-NH24-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg4-NH24-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg3-NH2Building block from example 285:2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg6-NH22-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg5-NH22-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg4-NH22-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg3-NH2Building block from example 283:2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg6-NH22-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg5-NH22-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg4-NH22-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg3-NH2Building block from example 296:4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH24-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH24-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH24-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2Building block from example 290:4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH24-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH24-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH24-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2Building block from example 544:4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg6-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg4-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg3-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg7-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg8-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg9-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg10-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg11-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg12-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg13-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg14-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg15-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg16-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg17-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg18-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg19-NH24-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg20-NH2Building block from page 251:4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg6-NH24′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg5-NH24′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg4-NH24′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg3-NH2Building block from example 549:3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg6-NH23-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg5-NH23-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg4-NH23-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg3-NH2Building block from page 252:4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg6-NH24′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg5-NH24′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg4-NH24′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg3-NH2Building block from example 412:4-(2H-Tetrazol-5-yl)benzoyl-Gly2-Arg6-NH24-(2H-Tetrazol-5-yl)benzoyl-Gly2-Arg5-NH24-(2H-Tetrazol-5-yl)benzoyl-Gly2-Arg4-NH24-(2H-Tetrazol-5-yl)benzoyl-Gly2-Arg3-NH2Building block from example 355:[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg6-NH2[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg5-NH2[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg4-NH2[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg3-NH2Building block from example 342:(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg6-NH2(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg5-NH2(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg4-NH2(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg3-NH2Building block from example 342:4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg6-NH24-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg5-NH24-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg4-NH24-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg3-NH24-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg6-NH24-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg5-NH24-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg4-NH24-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg3-NH24-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg6-NH24-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg5-NH24-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg4-NH24-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg3-NH24-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg6-NH24-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg5-NH24-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg4-NH24-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg3-NH2Benzotriazol-5-ylcarbonyl-4-Abz-Gly2-Arg6-NH2Benzotriazol-5-ylcarbonyl-4-Abz-Gly2-Arg4-NH2Benzotriazol-5-ylcarbonyl-4-Abz-Gly2-Arg3-NH24-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg3-NH24-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg4-NH24-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg5-NH23′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg3-NH23′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg4-NH23′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg5-NH22-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg3-NH22-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg4-NH22-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg5-NH24-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg5-NH24-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg4-NH24-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg3-NH25-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg5-NH25-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg4-NH25-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg3-NH28-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg5-NH28-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg4-NH28-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg3-NH24-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg6-NH24-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg5-NH24-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg4-NH24-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg3-NH2N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg5-NH2N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg4-NH2N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg3-NH2

Example 663

[2201] Equilibrium Solubility. For pH-solubility profiles, a 0.6 mM human insulin stock solution containing 0.2 mM Zn2+, 30 mM phenol, 0.2 mM mannitol, 2 mM phosphate and Zn2+-binding ligand as required 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 samples were incubated at 23 C. for 24 hours. After centrifugation (20,000 g in 20 min at 23 C.) of each sample, pH was measured and the solubility was determined by quantitation of insulin contents in the supernatant by SEC HPLC analysis The effect of various concentration of the ligand BTG2R5 on the pH-dependence of insulin solubility is illustrated in FIG. 1.

Example 664

[2202] The effect of increasing concentrations of the ligand BTG2R4 on the pH-dependence of insulin solubility is illustrated in FIG. 2. The solubility was determined as in example 663. Solution conditions: 0.6 mM human insulin, 0.2 mM mM Zn2+, 30 mM phenol, 0.2 M mannitol, 2 mM phosphate, 23 C.

Example 665

[2203] The slow release (prolonged action) properties of certain formulations of the present invention was characterized by the disappearance rate from the subcutaneous depot following subcutaneous injections in pigs. T50% is the time when 50% of the A14 Tyr(125I) insulin has disappeared from the site of injection as measured with an external y-counter (Ribel et al., The Pig as a Model for Subcutaneous Absorption in Man. In: M. Serrano-Rtios and P. J. Lefebre (Eds): Diabetes (1985) Proceedings of the 12th congress of the International Diabetes Federation, Madrid, Spain, 1985 (Excerpta Medica, Amsterdam (1986), 891-896). The composition of a series of protracted formulations is given in the table below together with the T50% values. The disappearance curves are illustrated in FIG. 3a-d. For comparison, the T50% for the corresponding insulin preparations formulated without the ligands would be about 2 hours.

[2204] The induction of slow release by addition of exogenous ligands of the invention affords further advantages in terms of versatility regarding the choice of insulin species and release patterns. Consequently, human or mutant insulins such as AspB28, LysB28ProB29, or GlyA21LysB3GluB29 may be formulated as slow- or dual-release preparations by adding variable amounts of HisB10 Zn2+-site ligand. This is illustrated below for AspB28 human insulin employing two different levels of the ligand TZD-Abz-G2R5 (example 647). As shown in the table and in FIG. 3 panels e-f, addition of this ligand in slight excess of the Zn2+ concentration produces a slow release preparation with T50% about 14.8. In contrast, when the ligand is added in concentrations lower than that of Zn2+, a distinctly dual-release formulation results.

17125I-Prep. 1125I-Prep. 2125I-Prep. 3125I-Prep. 4125I-Prep. 5125I-Prep. 6Insulin (mM)0.6 human0.6 human0.6 human0.6 human0.6 AspB280.6 AspB28insulininsulininsulininsulininsulininsulinZn2+ (mM)0.30.30.30.30.30.3Phenolic30 mM30 mM30 mM30 mM 7-30 mM30 mMligandphenolphenolphenolhydroxyindolephenolphenolZn2+ ligand6 mM BTG2R46 mM BTG2R62 mM BT-2 mM BT-0.4 mM TZD-0.15 mM TZD-(Ex. 641)(Ex. 643)AbzG2R5AbzG2R5AbzG2R5AbzG2R5(Ex. 644)(Ex. 644)(Ex. 647)(Ex. 647)Mannitol (mM)112112150150154176Phosphate222222buffer (mM)pH7.47.47.47.47.47.4T50% (hrs)10.210.3>2220.214.8biphasic

ANALYTICAL METHODS

[2205] Assays to quantify the binding affinity of ligands to the metal site of the insulin R6 hexamers:

[2206] 4H3N-Assay:

[2207] The binding affinity of ligands to the metal sit& of insulin R6 hexamers are measured in a UV/vis based displacement assay. The UV/vis spectrum of 3-hydroxy4-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, Bio-chemistry 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.

[2208] 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−.

[2209] 1000 μL of 2,1 mM human insulin in buffer

[2210] 66.7 μL of 10 mM Zn-acetate in buffer

[2211] 800 μL of 500 mM phenol in H2O

[2212] 201 μL of 4H3N in H2O

[2213] 7.93 ml buffer

[2214] The ligand is dissolved in DMSO to a concentration of 20 mM. The ligand solution is titrated to a cuvette containing 2 mL stock solution and after each addition the UV/vis spectrum is measured. The titration points are listed in Table 3 below.

18TABLE 3ligand additionligand conc.dilution(μl)(mM)factor10.0101.000510.0201.001010.0301.001520.0501.002550.1001.0050100.1981.0100200.3921.0200200.5831.0300200.7691.0400200.9521.0500

[2215] The UV/vis spectra resulting from a titration of the compound 3-hydroxy-2-naphthoic acid is shown in FIG. 5. Inserted in the upper right corner is the absorbance at 444 nm vs. the concentration of ligand.

[2216] 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)

[2217] The observed dissociation constant is recalculated to obtain the apparent dissociation constant

K

D
(app)=KD(obs)/(1+[4H3N]/K4H3N)

[2218] The value of K4H3N=50 μM is taken from Huang et al., 1997, Biochemistry 36,9878-9888.

[2219] TZD-Assay:

[2220] 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.

[2221] Preparation

[2222] 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 □M.

[2223] Measurements

[2224] 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.

[2225] Data Analysis

[2226] This equation is fitted to the datapoints

ΔF(455 nm))=ΔFmax*[ligand]free/(KD(app)*(1+[TZD]/KTZD)+[ligand]free))

[2227] 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

[2228] Two different fitting-procedures can be used. One in which both parameters, KD(app) and

[2229] 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
PA 2001 01337	Sep 2001	DK
PA 2002 01066	Jul 2002	DK

Novel ligands for the hisb10 zn2+ sites of the r-state insulin hexamer

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