# Patent

## Title
Ligands for the HisB10 Zn2 + sites of the R-state insulin hexamer

## Abstract

The present invention relates to novel ligands for the HisB10 Zn2+ sites of the R-state insulin hexamer having the formula A-B-C-D-X (III), wherein: A is a chemical group which reversibly binds to a HisB10 Zn2+ sites of an insulin hexamer; B is a linker; C is a fragment consisting of 0 to 5 neutral amino acids; D is a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidino groups; and X is —OH, —NH2 or a diamino group. The present invention also relates to R-state insulin hexamers comprising such ligands, and aqueous insulin preparations comprising such R-state insulin hexamers.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from application serial no. PCT/DK02/00595 filed on Sep. 13, 2002 under 35 U.S.C. 120 and claims priority under 35 U.S.C. 119 of Danish application no. PA 2001 01337 filed on Sep. 14, 2001, Danish application no. PA 2002 01066 filed on Jul. 5, 2002, U.S. application Ser. No. 60/323,925, filed 21 Sep. 2001, U.S. application Ser. No. 60/396,051, filed 10 Jul. 2002, the contents of which are fully incorporated herein by reference.


FIELD OF THE INVENTION
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
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),




  
    
      
        
          
            T
            6
          
          ⁢
          
            ⟷
            
              L
              0
              A
            
          
          ⁢
          
            T
            3
          
        
        ⁢
        
          
            R
            3
          
          ⁢
          
            ⟷
            
              L
              0
              B
            
          
          ⁢
          
            R
            6
          
        
      
    
    
      
        (
        1
        )
      
    
  




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.

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.
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.
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 chloride.
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).
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.
Another approach involves the use of insulin derivatives where the net charge is increased to shift the isoelectric point, and hence the pH of minimum solubility, from about 5.5 to the physiological range. Such preparations may be injected as clear solutions at slightly acidic pH. The subsequent adjustment of the pH to neutral induces crystallization/precipitation in the subcutaneous depot and dissolution again becomes rate-limiting for the absorption. GlyA21ArgB31ArgB32 human insulin belongs to this category of insulin analogues.
Most recently, a series of soluble insulin derivatives with a hydrophobic moiety covalently attached to the side chain of LysB29 have been synthesized. These derivatives may show prolonged action profile due to various mechanisms including albumin binding (e.g. B29-Nε-myristoyl-des(B30) human insulin), extensive protein self-association and/or stickiness (e.g. B29-Nε-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin) induced by the attached hydrophobic group.
SUMMARY OF THE INVENTION
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
The following is a detailed definition of the terms used to describe the invention:
“Halogen” designates an atom selected from the group consisting of F, Cl, Br and I.
The term “C1-C6-alkyl” as used herein represents a saturated, branched or straight hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl and the like.
The term “C1-C6-alkylene” as used herein represents a saturated, branched or straight bivalent hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methylene, 1,2-ethylene, 1,3-propylene, 1,2-propylene, 1,4-butylene, 1,5-pentylene, 1,6-hexylene, and the like.
The term “C2-C6-alkenyl” as used herein represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, iso-propenyl, 1,3-butadienyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentynyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 2,4-hexadienyl, 5-hexenyl and the like.
The term “C2-C6-alkynyl” as used herein represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one triple bond. Examples of such groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 2,4-hexadienyl and the like.
The term “C1-C6-alkoxy” as used herein refers to the radical —O—C1-C6-alkyl, wherein C1-C6-alkyl is as defined above. Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.
The term “C3-C8-cycloalkyl” as used herein represents a saturated, carbocyclic group having from 3 to 8 carbon atoms. Representative examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
The term “C4-8-cycloalkenyl” as used herein represents a non-aromatic, carbocyclic group having from 4 to 8 carbon atoms containing one or two double bonds. Representative examples are 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl, 2-cycloheptenyl, 3-cycloheptenyl, 2-cyclooctenyl, 1,4-cyclooctadienyl and the like.
The term “heterocyclyl” as used herein represents a non-aromatic 3 to 10 membered ring containing one or more heteroatoms selected from nitrogen, oxygen and sulphur and optionally containing one or two double bonds. Representative examples are pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, aziridinyl, tetrahydrofuranyl and the like.
The term “aryl” as used herein is intended to include carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9 to 14 membered bi- and tricyclic, carbocyclic, aromatic ring systems. Representative examples are phenyl, biphenylyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, indenyl, azulenyl and the like. Aryl is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and the like.
The term “arylene” as used herein is intended to include divalent, carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9 to 14 membered bi- and tricyclic, divalent, carbocyclic, aromatic ring systems. Representative examples are phenylene, biphenylylene, naphthylene, anthracenylene, phenanthrenylene, fluorenylene, indenylene, azulenylene and the like. Arylene is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 1,2,3,4-tetrahydronaphthylene, 1,4-dihydronaphthylene and the like.
The term “aryloxy” as used herein denotes a group —O-aryl, wherein aryl is as defined above.
The term “aroyl” as used herein denotes a group —C(O)-aryl, wherein aryl is as defined above.
The term “heteroaryl” as used herein is intended to include aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as 5 to 7 membered monocyclic and 8 to 14 membered bi- and tricyclic aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur. Representative examples are furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl, thiazolidinyl, 2-thiooxothiazolidinyl and the like. Heteroaryl is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.
The term “heteroarylene” as used herein is intended to include divalent, aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as 5 to 7 membered monocyclic and 8 to 14 membered bi- and tricyclic aromatic, heterocyclic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur. Representative examples are furylene, thienylene, pyrrolylene, oxazolylene, thiazolylene, imidazolylene, isoxazolylene, isothiazolylene, 1,2,3-triazolylene, 1,2,4-triazolylene, pyranylene, pyridylene, pyridazinylene, pyrimidinylene, pyrazinylene, 1,2,3-triazinylene, 1,2,4-triazinylene, 1,3,5-triazinylene, 1,2,3-oxadiazolylene, 1,2,4-oxadiazolylene, 1,2,5-oxadiazolylene, 1,3,4-oxadiazolylene, 1,2,3-thiadiazolylene, 1,2,4-thiadiazolylene, 1,2,5-thiadiazolylene, 1,3,4-thiadiazolylene, tetrazolylene, thiadiazinylene, indolylene, isoindolylene, benzofurylene, benzothienylene, indazolylene, benzimidazolylene, benzthiazolylene, benzisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene, quinazolinylene, quinolizinylene, quinolinylene, isoquinolinylene, quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene, azepinylene, diazepinylene, acridinylene and the like. Heteroaryl is also intended to include the partially hydrogenated derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydrobenzofuranylene, pyrrolinylene, pyrazolinylene, indolinylene, oxazolidinylene, oxazolinylene, oxazepinylene and the like.
“Aryl-C1-C6-alkyl”, “heteroaryl-C1-C6-alkyl”, “aryl-C2-C6-alkenyl” etc. is intended to mean C1-C6-alkyl or C2-C6-alkenyl as defined above, substituted by an aryl or heteroaryl as defined above, for example:




The term “optionally substituted” as used herein means that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent the substituents may be the same or different.
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.
Certain of the above defined terms may occur more than once in the structural formulae, and upon such occurrence each term shall be defined independently of the other.
Furthermore, when using the terms “independently are” and “independently selected from” it should be understood that the groups in question may be the same or different.
The term “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.
The term “fragment” as used herein is intended to mean a bivalent chemical group
The term “Neutral amino acid” as used herein is intended to mean any natural (codable) and non-natural amino acid, including α- or β-aminocarboxylic acids, including D-isomers of these (when applicable) without charges at physiologically relevant pH in the side chain, such as glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, aspargine, glutamine, cysteine, methionine, 3-aminobenzoic acid, 4-aminobenzoic acid or the like.
The term “positively charged group” as used herein is intended to mean any pharmaceutically acceptable group that contains a positive charge at physiologically relevant pH, such as amino (primary, secondary and tertiary), ammonium and guanidino groups.
The term “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.
The term “β amino acid” as used herein is intended to mean any β-aminocarboxylic acid, such as β-alanine, isoserine or the like.
When in the specification or claims mention is made of groups of compounds such as carboxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, triazoles, 4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thiazolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, naphthoic acids and salicylic acids, these groups of compounds are intended to include also derivatives of the compounds from which the groups take their name.
The term 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.
By “analogue of human insulin” as used herein (and related expressions) is meant human insulin in which one or more amino acids have been deleted and/or replaced by other amino acids, including non-codeable amino acids, or human insulin comprising additional amino acids, i.e. more than 51 amino acids, such that the resulting analogue possesses insulin activity.
The term “phenolic compound” or similar expressions as used herein refers to a chemical compound in which a hydroxyl group is bound directly to a benzene or substituted benzene ring. Examples of such compounds include, but are not limited to, phenol, o-cresol, m-cresol and p-cresol.
The term “physiologically relevant pH” as used herein is intended to mean a pH of about 7.1 to 7.9.
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.










Abbreviations:
















4H3N
4-hydroxy-3-nitrobenzoic acid


Abz
Aminobenzoic acid


AcOH
acetic acid


BT
Benzotriazol-5-oyl


DMF
N,N-Dimethylformamide


DMSO
Dimethylsulfoxide


DIC
Diisopropylcarbodiimide


EDAC
1-ethyl-3-(3′-dimethylamino-propyl)carbodiimide, hydrochloride


Fmoc
9H-Fluorene-9-ylmethoxycarbonyl


G, Gly
Glycine


HOAt
1-hydroxy-7-azabenzotriazole


HOBT
1-Hydroxybenzotriazole


K, Lys
Lysine


NMP
N-methyl-2-pyrrolidone


Pbf
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl


Pmc
2,2,5,7,8-pentamethylchroman-6-sulfonyl


R, Arg
Arginine


TFA
Trifluoroacetic acid









Abbreviations for non-natural amino acid residues:







BRIEF DESCRIPTION OF DRAWINGS
FIG. 1: Effect of BTG2R5—NH2 (SEQ ID NO: 1) on pH-solubility profile of an insulin preparation.
FIG. 2: Effect of BTG2R4—NH2 (SEQ ID NO: 2) on the pH-solubility profile of an insulin preparation.
FIG. 3: Disappearance from the subcutaneous depot (pig model) of insulin preparations in the presence of BT-AbzG2R5—NH2 (SEQ ID NO: 3) with phenol and 7-hydroxy indole (a-b); and BT-G2R5—NH2 (SEQ ID NO: 1) and BT-G2R4 (SEQ ID NO: 2) 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 (SEQ ID NO: 4)



DESCRIPTION OF THE INVENTION
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.
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.
The anions currently used in insulin formulations as allosteric ligands for the R-state hexamers (notably chloride ion) bind only weakly to the HisB10 anion site. The present invention, which is based on the discovery of suitable higher affinity ligands for these anion sites, provides ligands which are extended to modify timing via changes in hexamer solubility as outlined above.
Most ligand binding sites in proteins are highly asymmetric. Because the 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.
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.
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.
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).
Thus, from the structure-activity relationship (SAR) information obtained from the binding assay(s) it will be apparent for those skilled in the art to modify the starter compounds in question by introduction of a chemical group that will allow for coupling to a peptide containing e.g. one or more arginine or lysine residues. These chemical groups include carboxylic acid (amide bond formation with the peptide), carbaldehyde (reductive alkylation of the peptide), sulfonyl chloride (sulphonamide formation with the peptide) or the like.
The decision where and how to introduce this chemical group can be made in various ways. For example: From the SAR of a series of closely related starter compounds, a suitable position in the starter compound can be identified and the chemical group can be attached to this position, optionally using a spacer group, using synthesis procedures known to those skilled in the art.
Alternatively, this chemical group can be attached (optionally using a spacer group using and synthesis procedures known to those skilled in the art) to a position on the starter compound remote from the Zn2+-binding functionality
The zinc-binding ligands of the present invention are characterised by the following formula (I):

A-B-C-D-X  (I)

wherein:

    
    
        A is a functionality capable of reversibly coordinating to a HisB10 Zn2+ site of an insulin hexamer;
        B is a valence bond or a non-naturally occurring amino acid residue containing an aromatic ring;
        C is a valence bond or a fragment consisting of 1 to 5 neutral α- or β-amino acids;
        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
        X is OH, NH2 or a diamino group.
    
    


The length of the zinc-binding ligand should be such that it extends from the HisB10 Zn2+ site to beyond the hexamer surface.
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.
More preferably, A comprises a benzotriazole, a 3-hydroxy 2-napthoic acid, a salicylic acid, a tetrazole or a thiazolidinedione structure.
A is advantageously selected from one of the following chemical structures:







wherein

    
    
        R1 is hydrogen, fluoro, chloro, bromo or iodo,
        m is 0 or 1.
        B is preferably a valence bond or one of the following amino acid residues:
    
    






    
    
        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.
    
    


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.
        X is preferably OH, NH2 or
    
    






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

    Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 5)
    Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 1)
    Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 2)
    Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-NH2 (SEQ ID NO: 6)
    Benzotriazol-5-ylcarbonyl-Gly-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 7)
    Benzotriazol-5-ylcarbonyl-Gly-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 8)
    Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 9)
    Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 10)
    Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 11)
    Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-NH2 (SEQ ID NO: 12)
    Benzotriazol-5-ylcarbonyl-4-Abz-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 13)
    Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 14)
    Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 15)
    Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-NH2 (SEQ ID NO: 16)
    Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 17)
    Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 18)
    Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-NH2 [4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 19)
    [3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 19)
    4-(2H-Tetrazol-5-yl)benzoyl-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH2 (SEQ ID NO: 19)


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

A-B-C-D-X  (II)

wherein:

    
    
        A is a chemical group which reversibly binds to a HisB10 Zn2+ site of an insulin hexamer;
        B is a linker selected from
        
            A valence bond
            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—;
            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)—;
            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;
            R6 and R7 are independently H, C1-C4-alkyl;
        
        
        C is a fragment consisting of 1 to 5 neutral α- or β-amino acids
        D is a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidine groups; and
        X is —OH, —NH2 or a diamino group,

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.

    
    


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.
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
In another embodiment A is one of the following structures:




wherein

    
    
        X is ═O, ═S or ═NH
        Y is —S—, —O— or —NH—
        R8 and R11 are independently hydrogen or C1-C6-alkyl,
        R9 is hydrogen or C1-C6-alkyl or aryl, R8 and R9 may optionally be combined to form a double bond,
        R10 and R12 are independently hydrogen, aryl, C1-C6-alkyl, or —C(O)NR16R17 
        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.
        E and R10 may be connected through one or two valence bonds, G and R12 may be connected through one or two valence bonds;
        R13, R14 and R15 are independently selected from
        
            hydrogen, halogen, —CN, —CH2CN, —CHF2, —CF3, —OCF3, —OCHF2, —OCH2CF3, —OCF2CHF2, —S(O)2CF3, —OS(O)2CF3, —SCF3, —NO2, —OR6, —NR16R17, —SR16, —NR16S(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,
            C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
            which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17 
            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,
            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,
        
        
        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
    
    


In another embodiment X is ═O or ═S
In another embodiment X is ═O
In another embodiment X is ═S
In another embodiment Y is —O— or —S—
In another embodiment Y is —O—
In another embodiment Y is —S—
In another embodiment E is arylene optionally substituted with up to three substituents R13, R14 and R15.
In another embodiment E is phenylene or naphtylene optionally substituted with up to three substituents R13, R14 and R15.
In another embodiment E is heteroarylene optionally substituted with up to three substituents R13, R14 and R15.
In another embodiment E is indolylene optionally substituted with up to three substituents R13, R14 and R15.
In another embodiment R8 is hydrogen.
In another embodiment R9 is hydrogen.
In another embodiment R8 and R9 are combined to form a double bond.
In another embodiment R10 is C1-C6-alkyl.
In another embodiment R10 is methyl.
In another embodiment G is phenylene optionally substituted with up to three substituents R13, R14 and R15.
In another embodiment R11 is hydrogen.
In another embodiment R12 is hydrogen.
In another embodiment R13, R14 and R15 are independently selected from

    
    
        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,
        C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17 
        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,
        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.
    
    


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

    
    
        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)R16, —C(O)OR16, or —C2-C6-alkenyl-C(═O)R16,
        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 
        aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl,
        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.
    
    


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

    
    
        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,
        C1-C6-alkyl or C1-C6-alkenyl which may optionally be substituted with one or more substituents selected from halogen, —CF3, —OR16, and —NR16R17 
        aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl,
        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.
    
    


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

    
    
        hydrogen, halogen, —OR16, —OC1-C6-alkyl-C(O)OR16, or —C(O)OR16,
        C1-C6-alkyl which may optionally be substituted with one or more substituents selected from halogen, —OR16, and —NR16R17 
        aryl, aryloxy, aryl-C1-C6-alkoxy,
        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.
    
    


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
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.
In another embodiment A is one of the following structures




wherein

    
    
        R20 is hydrogen or C1-C6-alkyl,
        R21 is hydrogen or C1-C6-alkyl,
        U and V are a valence bond or C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl independently,
        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,
        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,
        R18, R19, R22, R23, R24, R25, R26 and R27 are independently selected from
        
            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,
            C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
            which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
            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,
            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,
        
        
        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
    
    


In another embodiment U is a valence bond
In another embodiment U is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl
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 
In another embodiment J is arylene optionally substituted with up to three substituents R22, R23 and R24 
In another embodiment J is phenylene optionally substituted with up to three substituents R22, R23 and R24 
In another embodiment R22, R23 and R24 are independently selected from

    
    
        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,
        C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        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,
        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
    
    


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

    
    
        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,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        aryl, aryloxy, aroyl, 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
    
    


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

    
    
        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,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, or —CF3 
        aryl, aryloxy, aroyl, 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)OH, —CN, —CF3, —NO2, or C1-C6-alkyl
    
    


In another embodiment R20 is hydrogen or methyl
In another embodiment R20 is hydrogen
In another embodiment R28 is hydrogen, C1-C6-alkyl or aryl
In another embodiment R28 is hydrogen or C1-C6-alkyl
In another embodiment R29 is hydrogen or C1-C6-alkyl
In another embodiment V is a valence bond
In another embodiment V is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl
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 
In another embodiment L is C1-C6-alkyl
In another embodiment L is phenylene optionally substituted with up to three substituents R25, R26 and R27 
In another embodiment R25, R26 and R27 are independently selected from

    
    
        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,
        C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        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,
        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
    
    


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

    
    
        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,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        aryl, aryloxy, aroyl, 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
    
    


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

    
    
        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,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, or —CF3 
        aryl, aryloxy, aroyl, 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)OH, —CN, —CF3, —NO2, or C1-C6-alkyl
    
    


In another embodiment R21 is hydrogen or methyl
In another embodiment R21 is hydrogen
In another embodiment R28 is Hydrogen, C1-C6-alkyl or aryl
In another embodiment R28 is Hydrogen or C1-C6-alkyl
In another embodiment R29 is Hydrogen or C1-C6-alkyl
In another embodiment R18 and R19 are independently selected from

    
    
        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.
        C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        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
    
    


In another embodiment R18 and R19 are independently selected from

    
    
        hydrogen, halogen, —CN, —CF3, —NO2, —OR28, —NR28R29, or —C(O)OR28 
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        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
    
    


In another embodiment A is a compound of the form M-Q-T-

    
    
        wherein M is one of the following structures
    
    






    
    
        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
        Q is selected from the following:
        
            a valence bond
            —CH2N(R30)— or —SO2N(R31)—
            A compound of the formula
        
        
    
    






    
    
        
        
             wherein Z1 is S(O)2 or CH2, Z2 is N, —O— or —S—, and n is 1 or 2;
            T is
            A valence bond
            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 
            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,
        
        
        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.
        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,
    
    


In another embodiment M is one of the following structures




In another embodiment M is




In another embodiment M is




In another embodiment the salicylic acid moiety is of the formula




In another embodiment the napthoic acid moiety is of the formula




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

    
    
        A valence bond
        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 
        Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70
    
    


In another embodiment T is

    
    
        A valence bond
        Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70
    
    


In another embodiment T is phenylene or naphthalene
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
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
In another embodiment the cyclic moiety in T is optionally substituted by halogen, —C(O)OR32 or —OR32 
In another embodiment T is a valence bond
In another embodiment R30 and R31 are independently hydrogen or C1-C6-alkyl
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 
In another embodiment R34 is hydrogen, halogen, —CF3, —NO2, —OR32, —NR32R33, —SR32, —NR32C(O)R33, or —C(O)OR32 
In another embodiment R34 is hydrogen, halogen, —CF3, —NO2, —OR32, —NR32R33, or —NR32C(O)R33 
In another embodiment R34 is hydrogen, halogen, or —OR32 
In another embodiment R32 and R33 independently are hydrogen, C1-C6-alkyl, or aryl
In another embodiment R32 and R33 independently are hydrogen or C1-C6-alkyl
In another embodiment C consists of 1-5 neutral amino acids independently selected from the group consisting of Gly, Ala, Thr, and Ser
In another embodiment C consists of 1-5 Gly
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
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
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
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
In another embodiment GB is of the formula —B1—B2—C(O)— or —B1—B2—SO2—, wherein B1 and B2 are as defined above
In another embodiment GB is of the formula —B1—B2—C(O)— or —B1—B2—CH2—, wherein B1 and B2 are as defined above
In another embodiment GB is of the formula —B1—B2—C(O)— or —B1—B2—NH—, wherein B1 and B2 are as defined above
In another embodiment GB is of the formula —B1—B2—CH2— or —B1—B2—SO2—, wherein B1 and B2 are as defined above
In another embodiment GB is of the formula —B1—B2—NH— or —B1—B2—SO2—, wherein B1 and B2 are as defined above
In another embodiment GB is of the formula —B1—B2—CH2— or —B1—B2—NH—, wherein B1 and B2 are as defined above
In another embodiment GB is of the formula —B1—B2—C(O)—
In another embodiment GB is of the formula —B1—B2—CH2—
In another embodiment GB is of the formula —B1—B2—SO2—
In another embodiment GB is of the formula —B1—B2—NH—
In another embodiment B1 is a valence bond, —O—, or —S—
In another embodiment B1 is a valence bond, —O—, or —N(R6)—
In another embodiment B1 is a valence bond, —S—, or —N(R6)—
In another embodiment B1 is —O—, —S— or —N(R6)—
In another embodiment B1 is a valence bond or —O—
In another embodiment B1 is a valence bond or —S—
In another embodiment B1 is a valence bond or —N(R6)—
In another embodiment B1 is —O— or —S—
In another embodiment B1 is —O— or —N(R6)—
In another embodiment B1 is —S— or —N(R6)—
In another embodiment B1 is a valence bond
In another embodiment B1 is —O—
In another embodiment B1 is —S—
In another embodiment B1 is —N(R6)—
In another embodiment B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-O—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-S—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-NR6—C1-C18-alkyl-C(═O)—; and the alkylene and arylene moieties are optionally substituted as defined above
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
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
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
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
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
In another embodiment B2 is a valence bond or C1-C18-alkylene, and the alkylene moiety is optionally substituted as defined above
In another embodiment D comprises 1 to 16 positively charged groups
In another embodiment D comprises 1 to 12 positively charged groups
In another embodiment D comprises 1 to 10 positively charged groups
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.
In another embodiment the basic amino acid is Arg
In another embodiment X is —OH or —NH2 
In another embodiment X is —NH2 
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.
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
In another embodiment the insulin is an analogue of human insulin selected from the group consisting of

    
    
        i. An analogue wherein position B28 is Asp, Lys, Leu, Val, or Ala and position B29 is Lys or Pro; and
        ii. des(B28-B30), des(B27) or des(B30) human insulin.
    
    


In another embodiment the insulin is an analogue of human insulin wherein position B28 is Asp or Lys, and position B29 is Lys or Pro.
In another embodiment the insulin is des(B30) human insulin.
In another embodiment the insulin is a derivative of human insulin having one or more lipophilic substituents.
In another embodiment the insulin derivative is selected from the group consisting of B29-Nε-myristoyl-des(B30) human insulin, B29-Nε-palmitoyl-des(B30) human insulin, B29-Nε-myristoyl human insulin, B29-Nε-palmitoyl human insulin, B28-Nε-myristoyl LysB28 ProB29 human insulin, B28-Nε-palmitoyl LysB28 ProB29 human insulin, B30-Nε-myristoyl-ThrB29LysB30 human insulin, B30-Nε-palmitoyl-ThrB29LysB30 human insulin, B29-Nε-(N-palmitoyl-γ-glutamyl)-des(B30) human insulin, B29-Nε-(N-lithocholyl-γ-glutamyl)-des(B30) human insulin, B29-Nε-(ω-carboxyheptadecanoyl)-des(B30) human insulin and B29-Nε-(ω-carboxyheptadecanoyl) human insulin.
In another embodiment the insulin derivative is B29-Nε-myristoyl-des(B30) human insulin.
In another embodiment the insulin hexamer of the invention further comprises at least 3 phenolic molecules.
In another embodiment the invention provides an insulin preparation comprising R-state insulin hexamers as defined above
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.
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.
In another embodiment the ratio between precipitated insulin and dissolved insulin is in the range from 95:5 to 5:95
In another embodiment the ratio between precipitated insulin and dissolved insulin is in the range from 80:20 to 20:80
In another embodiment the ratio between precipitated insulin and dissolved insulin is in the range from 70:30 to 30:70.
In another embodiment the invention provides a zinc-binding ligand of the following general formula (III)

A-B-C-D-X  (III)

wherein:

    
    
        A is a chemical group which reversibly binds to a HisB10 Zn2+ site of an insulin hexamer;
        B is a linker selected from
        
            A valence bond
            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—,
            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)—;
            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;
            R6 and R7 are independently H, C1-C4-alkyl;
        
        
        C is a fragment consisting of 0 to 5 neutral amino acids, wherein the individual neutral amino acids are the same or different
        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
        X is —OH, —NH2 or a diamino group,

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.

    
    


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.
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.
In another embodiment of the invention A is




wherein

    
    
        X is ═O, ═S or ═NH
        Y is —S—, —O— or —NH—
        R8 and R11 are independently hydrogen or C1-C6-alkyl,
        R9 is hydrogen or C1-C6-alkyl or aryl, R8 and R9 may optionally be combined to form a double bond,
        R10 and R12 are independently hydrogen, aryl, C1-C6-alkyl, or —C(O)NR16R17 
        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 
        E and R10 may be connected through one or two valence bonds, G and R12 may be connected through one or two valence bonds;
        R13, R14, R15 and R15A are independently selected from
        
            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)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-C6-alkenyl-C(═O)OR16, —C(O)OR16, or —C2-C6-alkenyl-C(═O)R16, ═O, or —C2-C6-alkenyl-C(═O)NR16R17,
            C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
            which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17 
            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,
            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,
        
        
        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
    
    


In another embodiment of the invention X is ═O or ═S
In another embodiment of the invention X is ═O
In another embodiment of the invention X is ═S
In another embodiment of the invention Y is —O— or —S—
In another embodiment of the invention Y is —O—
In another embodiment of the invention Y is —S—
In another embodiment of the invention E is arylene optionally substituted with up to four substituents, R13, R14, R15, and R15A.
In another embodiment of the invention E is phenylene or naphtylene optionally substituted with up to four substituents, R13, R14, R15, and R15A.
In another embodiment of the invention E is




In another embodiment of the invention E is




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




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




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




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




In another embodiment of the invention R8 is Hydrogen.
In another embodiment of the invention R9 is Hydrogen.
In another embodiment of the invention R8 and R9 are combined to form a double bond.
In another embodiment of the invention R10 is C1-C6-alkyl.
In another embodiment of the invention R10 is methyl.
In another embodiment of the invention G is phenylene optionally substituted with up to four substituents, R13, R14, R15, and R15A.
In another embodiment of the invention R11 is Hydrogen.
In another embodiment of the invention R12 is Hydrogen.
In another embodiment of the invention R13, R14, R15 and R15A are independently selected from

    
    
        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)OR16, or —C2-C6-alkenyl-C(═O)R16,
        C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR16, and —NR16R17 
        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,
        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.
    
    


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

    
    
        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,
        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 
        aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl,
        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.
    
    


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

    
    
        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,
        C1-C6-alkyl or C1-C6-alkenyl which may optionally be substituted with one or more substituents selected from halogen, —CF3, —OR16, and —NR16R17 
        aryl, aryloxy, aroyl, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, heteroaryl
        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.
    
    


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

    
    
        hydrogen, halogen, —OR6, —OC1-C6-alkyl-C(O)OR16, or —C(O)OR16,
        C1-C6-alkyl which may optionally be substituted with one or more substituents selected from halogen, —OR16, and —NR16R17 
        aryl, aryloxy, aryl-C1-C6-alkoxy,
        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.
    
    


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
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.
In another embodiment of the invention A is




wherein

    
    
        R20 is hydrogen or C1-C6-alkyl,
        R21 is hydrogen or C1-C6-alkyl,
        U and V are a valence bond or C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl independently,
        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,
        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,
        R18, R19, R22, R23R24, R25, R26 and R27 are independently selected from
        
            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,
            C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
            which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
            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,
            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,
        
        
        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
    
    


In another embodiment of the invention U is a valence bond
In another embodiment of the invention U is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl
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 
In another embodiment of the invention J is arylene optionally substituted with up to three substituents R22, R23 and R24 
In another embodiment of the invention J is phenylene optionally substituted with up to three substituents R22, R23 and R24 
In another embodiment of the invention J is




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

    
    
        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,
        C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        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,
        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
    
    


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

    
    
        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,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        aryl, aryloxy, aroyl, 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
    
    


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

    
    
        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,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, or —CF3 
        aryl, aryloxy, aroyl, 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)OH, —CN, —CF3, —NO2, or C1-C6-alkyl
    
    


In another embodiment of the invention R20 is hydrogen or methyl
In another embodiment of the invention R20 is hydrogen
In another embodiment of the invention R28 is Hydrogen, C1-C6-alkyl or aryl
In another embodiment of the invention R28 is Hydrogen or C1-C6-alkyl
In another embodiment of the invention R29 is Hydrogen or C1-C6-alkyl
In another embodiment of the invention V is a valence bond
In another embodiment of the invention V is C1-C6-alkylene optionally substituted with one or more hydroxy, C1-C6-alkyl, or aryl
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 
In another embodiment of the invention L is C1-C6-alkylene
In another embodiment of the invention L is phenylene optionally substituted with up to three substituents R25, R26 and R27 
In another embodiment of the invention R25, R26 and R27 are independently selected from

    
    
        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,
        C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        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,
        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
    
    


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

    
    
        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,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        aryl, aryloxy, aroyl, 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
    
    


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

    
    
        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,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, or —CF3 
        aryl, aryloxy, aroyl, 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)OH, —CN, —CF3, —NO2, or C1-C6-alkyl
    
    


In another embodiment of the invention R21 is hydrogen or methyl
In another embodiment of the invention R21 is hydrogen
In another embodiment of the invention R28 is Hydrogen, C1-C6-alkyl or aryl
In another embodiment of the invention R28 is Hydrogen or C1-C6-alkyl
In another embodiment of the invention R29 is Hydrogen or C1-C6-alkyl
In another embodiment of the invention R18 and R19 are independently selected from

    
    
        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,
        C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        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
    
    


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

    
    
        hydrogen, halogen, —CN, —CF3, —NO2, —OR28, —NR28R29, or —C(O)OR28,
        C1-C6-alkyl optionally substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR28, and —NR28R29 
        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
    
    


In another embodiment of the invention A is




In another embodiment of the invention A is of the form M-Q-T-

    
    
        wherein M is
    
    






    
    
        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
        Q is selected from the following:
        
            a valence bond
            —CH2N(R30)— or —SO2N(R31)—
            A compound of the formula
        
        
    
    






    
    
        
        
             wherein Z1 is S(O)2 or CH2, Z2 is N, —O— or —S—, and n is 1 or 2;
        
        
        T is
        
            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 
            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,
            A valence bond
        
        
        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.
        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,
    
    


In another embodiment of the invention M is




In another embodiment of the invention M is




In another embodiment of the invention M is




In another embodiment of the invention M is




In another embodiment of the invention M is




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

    
    
        A valence bond
        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 
        Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70
    
    


In another embodiment of the invention T is

    
    
        A valence bond
        Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70
    
    


In another embodiment of the invention T is phenylene or naphthalene
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
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
In another embodiment of the invention the cyclic moiety in T is optionally substituted by halogen, —C(O)OR32 or —OR32 
In another embodiment of the invention T is a valence bond
In another embodiment of the invention R30 and R31 are independently hydrogen or C1-C6-alkyl
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 
In another embodiment of the invention R34 is hydrogen, halogen, —CF3, —NO2, —OR32, —NR32R33, —SR32, —NR32C(O)R33, or —C(O)OR32 
In another embodiment of the invention R34 is hydrogen, halogen, —CF3, —NO2, —OR32, —NR32R33, or —NR32C(O)R33 
In another embodiment of the invention R34 is hydrogen, halogen, or —OR32 
In another embodiment of the invention R32 and R33 independently are hydrogen, C1-C6-alkyl, or aryl
In another embodiment of the invention R32 and R33 independently are hydrogen or C1-C6-alkyl
In another embodiment of the invention A is




    
    
        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;
        A2 is a valence bond, C1-C6-alkylene, C1-C6-alkenylene, or —C1-C6-alkyl-O—;
        R1A is C1-C6-alkyl, aryl, wherein the alkyl or aryl moieties are optionally substituted by one or more halogen, cyano, nitro, amino;
        AR1 is a valence bond, arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted by one or more R1B independently
        R1B is selected from
        
            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
            C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
            which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR1C, and —NR1CR1D 
            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,
            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,
        
        
        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,
        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
        R1E and R1F are independently selected from C1-C6-alkyl, aryl optionally substituted by one or more halogen, —COOH;
        AR2 is
        
            a valence bond
            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;
            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;
        
        
        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)
        R2B and R2C are independently selected from hydrogen and C1-C6-alkyl
    
    


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 
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 
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 
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 
In another embodiment of the invention A1 is a valence bond
In another embodiment of the invention A2 is a valence bond or —C1-C6-alkyl-O—
In another embodiment of the invention A2 is a valence bond
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
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
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
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
In another embodiment of the invention AR1 is phenylene optionally substituted by one or more R1B independently
In another embodiment of the invention AR1 is indolylene
In another embodiment of the invention AR1 is carbazolylene
In another embodiment of the invention AR1 is




In another embodiment of the invention AR1 is




In another embodiment of the invention R1B is selected from

    
    
        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
        C1-C6-alkyl or C2-C6-alkenyl
        which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR1C, and —NR1CR1D 
        aryl, aryloxy, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, heteroaryl, heteroaryl-C1-C6-alkyl, or heteroaryl-C2-C6-alkenyl

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

    
    


In another embodiment of the invention R1B is selected from

    
    
        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
        C1-C6-alkyl
    
    


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
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
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
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)—
In another embodiment of the invention R1E and R1F are independently selected from C1-C6-alkyl
In another embodiment of the invention AR2 is

    
    
        a valence bond
        C1-C6-alkylene, wherein the alkyl is optionally substituted by one or more R2A independently
        arylene, aryl-C1-C6-alkyl, heteroarylene, wherein the aryl and heteroaryl moieties are optionally substituted by one or more R2A independently
    
    


In another embodiment of the invention AR2 is

    
    
        a valence bond
        C1-C6-alkylene, wherein the alkyl is optionally substituted by one or more R2A independently
        phenyl, phenyl-C1-C6-alkyl, wherein the phenyl moieties are optionally substituted by one or more R2A independently
    
    


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




    
    
        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
        R3A is independently selected from
        
            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,
            C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
            which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR3B, and —NR3BR3C 
            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,
            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,
        
        
        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
        R3D is C1-C6-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl optionally substituted with one or more C1-C6-alkyl.
    
    


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
In another embodiment of the invention AR3 is arylene optionally substituted with one or more R3A independently
In another embodiment of the invention AR3 is phenylene, naphthalene or anthranylene optionally substituted with one or more R3A independently
In another embodiment of the invention AR3 is phenylene optionally substituted with one or more R3A independently
In another embodiment of the invention R3A is independently selected from

    
    
        halogen, —CN, —CF3, —NO2, —OR3B, —NR3BR3C, —SR3B, —OC1-C6-alkyl-C(O)OR3B or —C(O)OR3B 
        C1-C6-alkyl optionally substituted with one or more substituents selected from halo gen, —CN, —CF3, —OCF3, —OR3B, and —NR3BR3C 
        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
    
    


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
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
In another embodiment of the invention A is




    
    
        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
        R4A is independently selected from
        
            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,
            C1-C6-alkyl, C2-C6-alkenyl or C2-C6-alkynyl,
            which may optionally be substituted with one or more substituents selected from halogen, —CN, —CF3, —OCF3, —OR4B, and —NR4BR4C 
            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,
            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,
        
        
        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; R4B and 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
        R4D is C1-C6-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl optionally substituted with one or more C1-C6-alkyl.
    
    


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
In another embodiment of the invention AR4 is arylene or heteroarylene optionally substituted with one or more R4A independently
In another embodiment of the invention AR4 is phenylene, naphtylene, anthrylene, thienylene, pyridylene, or benzodioxylene optionally substituted with one or more R4A independently
In another embodiment of the invention AR4 is phenylene optionally substituted with one or more R4A independently
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 
In another embodiment of the invention R4B and R4C are independently hydrogen, CF3, C1-C12-alkyl, —C(═O)—R4D, or aryl
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
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
In another embodiment of the invention C consists of 0 to 5 Gly
In another embodiment of the invention C consists of 0 Gly
In another embodiment of the invention C consists of 1 Gly
In another embodiment of the invention C consists of 2 Gly
In another embodiment of the invention C consists of 3 Gly
In another embodiment of the invention C consists of 4 Gly
In another embodiment of the invention C consists of 5 Gly
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
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
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
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
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
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
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
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
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
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
In another embodiment of the invention GB is of the formula —B1—B2—C(O)—
In another embodiment of the invention GB is of the formula —B1—B2—CH2—
In another embodiment of the invention GB is of the formula —B1—B2—SO2—
In another embodiment of the invention GB is of the formula —B1—B2—NH—
In another embodiment of the invention B1 is a valence bond, —O—, or —S—
In another embodiment of the invention B1 is a valence bond, —O—, or —N(R6)—
In another embodiment of the invention B1 is a valence bond, —S—, or —N(R6)—
In another embodiment of the invention B1 is —O—, —S— or —N(R6)—
In another embodiment of the invention B1 is a valence bond —O—
In another embodiment of the invention B1 is a valence bond or —S—
In another embodiment of the invention B1 is a valence bond or —N(R6)—
In another embodiment of the invention B1 is —O— or —S—
In another embodiment of the invention B1 is —O— or —N(R6)—
In another embodiment of the invention B1 is —S— or —N(R6)—
In another embodiment of the invention B1 is a valence bond
In another embodiment of the invention B1 is —O—
In another embodiment of the invention B1 is —S—
In another embodiment of the invention B1 is —N(R6)—
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
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
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
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
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
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
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
In another embodiment of the invention D comprises 1 to 16 positively charged groups
In another embodiment of the invention D comprises 1 to 12 positively charged groups
In another embodiment of the invention D comprises 1 to 10 positively charged groups
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.
In another embodiment of the invention the basic amino acid is Arg
In another embodiment of the invention X is —OH or —NH2 
In another embodiment of the invention X is —NH2 
The invention furthermore provides an R-state insulin hexamer comprising:

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


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

    
    
        iii. An analogue wherein position B28 is Asp, Lys, Leu, Val, or Ala and position B29 is Lys or Pro; and
        iv. des(B28-B30), des(B27) or des(B30) human insulin.
    
    


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.
In another embodiment of the invention the insulin is des(B30) human insulin.
In another embodiment of the invention the insulin is a derivative of human insulin having one or more lipophilic substituents.
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.
In another embodiment of the invention the insulin derivative is B29-Nε-myristoyl-des(B30) human insulin.
In another embodiment of the invention the insulin hexamer as defined above further comprises at least 3 phenolic molecules.
The invention furthermore provides an aqueous insulin preparation comprising R-state insulin hexamers as defined above
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.
In another embodiment of the invention the ratio between precipitated insulin and dissolved insulin is in the range from 99:1 to 1:99.
In another embodiment of the invention the ratio between precipitated insulin and dissolved insulin is in the range from 95:5 to 5:95
In another embodiment of the invention the ratio between precipitated insulin and dissolved insulin is in the range from 80:20 to 20:80
In another embodiment of the invention the ratio between precipitated insulin and dissolved insulin is in the range from 70:30 to 30:70
The invention furthermore provides a method of preparing a zinc-binding ligand as defined above comprising the steps of

    
    
        Identifying starter compounds that are able to displace a ligand from the R-state HisB10-Zn2+ site
        optionally attaching a fragment consisting of 0 to 5 neutral α- or β-amino acids
        attaching a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidino groups
    
    


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.
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.
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.
The present invention also encompasses pharmaceutically acceptable salts of the present compounds. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulphuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, picric, pyruvic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, which is incorporated herein by reference. Examples of metal salts include lithium, sodium, potassium, magnesium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methyl-, dimethyl-, trimethyl-, ethyl-, hydroxyethyl-, diethyl-, n-butyl-, sec-butyl-, tert-butyl-, tetramethylammonium salts and the like.
Also intended as pharmaceutically acceptable acid addition salts are the hydrates, which the present compounds, are able to form.
Furthermore, the pharmaceutically acceptable salts comprise basic amino acid salts such as lysine, arginine and ornithine.
The acid addition salts may be obtained as the direct products of compound synthesis. In the alternative, the free base may be dissolved in a suitable solvent containing the appropriate acid, and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.
The compounds of the present invention may form solvates with standard low molecular weight solvents using methods well known to the person skilled in the art. Such solvates are also contemplated as being within the scope of the present invention.
Pharmaceutical Compositions
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.
In one embodiment of the invention the insulin preparation comprises 60 to 3000 nmol/ml of insulin.
In another embodiment of the invention the insulin preparation comprises 240 to 1200 nmol/ml of insulin.
In another embodiment of the invention the insulin preparation comprises about 600 nmol/ml of insulin.
Zinc ions may be present in an amount corresponding to 10 to 40 μg Zn/100 U insulin, more preferably 10 to 26 μg Zn/100 U insulin.
Insulin formulations of the invention are usually administered from multi-dose containers where a preservative effect is desired. Since phenolic preservatives also stabilize the R-state hexamer the formulations may contain up to 50 mM of phenolic molecules. The phenolic molecules in the insulin formulation may be selected from the group consisting of phenol, m-cresol, chloro-cresol, thymol, 7-hydroxyindole or any mixture thereof.
In one embodiment of the invention 0.5 to 4.0 mg/ml of phenolic compound may be employed.
In another embodiment of the invention 0.6 to 4.0 mg/ml of m-cresol may be employed.
In another embodiment of the invention 0.5 to 4.0 mg/ml of phenol may be employed.
In another embodiment of the invention 1.4 to 4.0 mg/ml of phenol may be employed.
In another embodiment of the invention 0.5 to 4.0 mg/ml of a mixture of m-cresol or phenol may be employed.
In another embodiment of the invention 1.4 to 4.0 mg/ml of a mixture of m-cresol or phenol may be employed.
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.
The action of insulin may further be slowed down in vivo by the addition of physiologically acceptable agents that increase the viscosity of the pharmaceutical preparation. Thus, the pharmaceutical preparation according to the invention may furthermore comprise an agent which increases the viscosity, such as polyethylene glycol, polypropylene glycol, copolymers thereof, dextrans and/or polylactides.
In a particular embodiment the insulin preparation of the invention comprises between 0.001% by weight and 1% by weight of anon-ionic surfactant, for example tween 20 or Polox 188.
A nonionic detergent can be added to stabilise insulin against fibrillation during storage and handling.
The 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
The following examples and general procedures refer to intermediate compounds and final products identified in the specification and in the synthesis schemes. The preparation of the compounds of the present invention is described in detail using the following examples, but the chemical reactions described are disclosed in terms of their general applicability to the preparation of compounds of the invention. Occasionally, the reaction may not be applicable as described to each compound included within the disclosed scope of the invention. The compounds for which this occurs will be readily recognised by those skilled in the art. In these cases the reactions can be successfully performed by conventional modifications known to those skilled in the art, that is, by appropriate protection of interfering groups, by changing to other conventional reagents, or by routine modification of reaction conditions. Alternatively, other reactions disclosed herein or otherwise conventional will be applicable to the preparation of the corresponding compounds of the invention. In all preparative methods, all starting materials are known or may easily be prepared from known starting materials. All temperatures are set forth in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight when referring to yields and all parts are by volume when referring to solvents and eluents.
HPLC-MS (Method A)
The following instrumentation was used:

    
    
        Hewlett Packard series 1100 G1312A Bin Pump
        Hewlett Packard series 1100 Column compartment
        Hewlett Packard series 1100 G13 15A DAD diode array detector
        Hewlett Packard series 1100 MSD
    
    


The instrument was controlled by HP Chemstation software.
The HPLC pump was connected to two eluent reservoirs containing:

    
    
        A: 0.01% in water
        B: 0.01% TFA in acetonitrile
    
    


The analysis was performed at 40° C. by injecting an appropriate volume of the sample (preferably 1 μL) onto the column, which was eluted with a gradient of acetonitrile.
The HPLC conditions, detector settings and mass spectrometer settings used are given in the following table.













Column
Waters Xterra MS C-18 × 3 mm id


Gradient
10%-100% acetonitrile lineary during 7.5 min at 1.0 mL/min


Detection
UV: 210 nm (analog output from DAD)


MS
Ionisation mode: API-ES



Scan 100-1000 amu step 0.1 amu









HPLC-MS (Method B)

The following instrumentation was used:

    
    
        Sciex API 100 Single quadropole mass spectrometer
        Perkin Elmer Series 200 Quard pump
        Perkin Elmer Series 200 autosampler
        Applied Biosystems 785A UV detector
        Sedex 55 evaporative light scattering detector
        A Valco column switch with a Valco actuator controlled by timed events from the pump.
    
    


The Sciex Sample control software running on a Macintosh PowerPC 7200 Computer was used for the instrument control and data acquisition.
The HPLC pump was connected to four eluent reservoirs containing:

    
    
        A: acetonitrile
        B: water
        C: 0.5% TFA in water
        D: 0.02 M ammonium acetate
    
    


The requirements for samples are that they contain approximately 500 μg/mL of the compound to be analysed in an acceptable solvent such as methanol, ethanol, acetonitrile, THF, water and mixtures thereof. (High concentrations of strongly eluting solvents will interfere with the chromatography at low acetonitrile concentrations.)
The analysis was performed at room temperature by injecting 20 μL of the sample solution on the column, which was eluted with a gradient of acetonitrile in either 0.05% TFA or 0.002 M ammonium acetate. Depending on the analysis method varying elution conditions were used.
The eluate from the column was passed through a flow splitting T-connector, which passed approximately 20 μL/min through approx. 1 m. 75μ fused silica capillary to the API interface of API 100 spectrometer.
The remaining 1.48 mL/min was passed through the UV detector and to the ELS detector.
During the LC-analysis the detection data were acquired concurrently from the mass spectrometer, the UV detector and the ELS detector.
The LC conditions, detector settings and mass spectrometer settings used for the different methods are given in the following table.













Column
YMC ODS-A 120 Å s - 5μ 3 mm × 50 mm id


Gradient
5%-90% acetonitrile in 0.05% TFA linearly during 7.5 min at



1.5 mL/min









Detection
UV: 214 nm
ELS: 40° C.











MS
Experiment:
Start: 100 amu
Stop: 800 amu
Step:






0.2 amu



Dwell:
0.571 msec



Method:
Scan 284 times =




9.5 min









HPLC-MS (Method C)

The Following Instrumentation is Used:

    
    
        Hewlett Packard series 1100 G1312A Bin Pump
        Hewlett Packard series 1100 Column compartment
        Hewlett Packard series 1100 G1315A DAD diode array detector
        Hewlett Packard series 1100 MSD
        Sedere 75 Evaporative Light Scattering detector
    
    


The instrument is controlled by HP Chemstation software.
The HPLC pump is connected to two eluent reservoirs containing:













A
0.01% TFA in water


B
0.01% TFA in acetonitrile









The analysis is performed at 40° C. by injecting an appropriate volume of the sample (preferably 1 μl) onto the column which is eluted with a gradient of acetonitrile.
The HPLC conditions, detector settings and mass spectrometer settings used are given in the following table.













Column
Waters Xterra MS C-18 × 3 mm id 5 μm


Gradient
50%-100% acetonitrile linear during 7.5 min at 1.5 ml/min


Detection
210 nm (analogue output from DAD)



ELS (analogue output from ELS)


MS
ionisation mode API-ES



Scan 100-1000 amu step 0.1 amu









After the DAD the flow is divided yielding approximately 1 ml/min to the ELS and 0.5 ml/min to the MS.
HPLC-MS (Method D)
The following instrumentation was used:

    
    
        Sciex API 150 Single Quadropole mass spectrometer
        Hewlett Packard Series 1100 G1312A Bin pump
        Gilson 215 micro injector
        Hewlett Packard Series 1100 G1315A DAD diode array detector
        Sedex 55 evaporative light scattering detector
        A Valco column switch with a Valco actuator controlled by timed events from the pump.
    
    


The Sciex Sample control software running on a Macintosh Power G3 Computer was used for the instrument control and data acquisition.
The HPLC pump was connected to two eluent reservoirs containing:

    
    
        A: Acetonitrile containing 0.05% TFA
        B: Water containing 0.05% TFA
    
    


The requirements for the samples are that they contain approximately 500 μg/ml of the compound to be analysed in an acceptable solvent such as methanol, ethanol, acetonitrile, THF, water and mixtures thereof. (High concentrations of strongly eluting solvents will interfere with the chromatography at low acetonitrile concentrations.)
The analysis was performed at room temperature by injecting 20 μl of the sample solution on the column, which was eluted with a gradient of acetonitrile in 0.05% TFA The eluate from the column was passed through a flow splitting T-connector, which passed approximately 20 μl/min through approx. 1 m 75μ fused silica capillary to the API interface of API 150 spectrometer.
The remaining 1.48 ml/min was passed through the UV detector and to the ELS detector. During the LC-analysis the detection data were acquired concurrently from the mass spectrometer, the UV detector and the ELS detector.
The LC conditions, detector settings and mass spectrometer settings used for the different methods are given in the following table.













Column
Waters X-terra C18 5μ 3 mm × 50 mm id


Gradient
5%-90% acetonitrile in 0.05% TFA linearly during 7.5 min at



1.5 ml/min









Detection
UV: 214 nm
ELS: 40° C.











MS
Experiment:
Start: 100 amu
Stop: 800 amu
Step:






0.2 amu



Dwell:
0.571 msec



Method:
Scan 284 times =




9.5 min









EXAMPLES
Example 1
1H-Benzotriazole




Example 2
5,6-Dimethyl-1H-benzotriazole




Example 3
1H-Benzotriazole-5-carboxylic acid




Example 4
4-Nitro-1H-benzotriazole




Example 5
5-Amino-1H-benzotriazole




Example 6
5-Chloro-1H-benzotriazole




Example 7
5-Nitro-1H-benzotriazole




Example 8
4-[(1H-Benzotriazole-5-carbonyl)amino]benzoic acid




4-[(1H-Benzotriazole-5-carbonyl)amino]benzoic acid methyl ester (5.2 g, 17.6 mmol) was dissolved in THF (60 mL) and methanol (10 mL) was added followed by 1N sodium hydroxide (35 mL). The mixture was stirred at room temperature for 16 hours and then 1N hydrochloric acid (45 mL) was added. The mixture was added water (200 mL) and extracted with ethyl acetate (2×500 mL). The combined organic phases were evaporated in vacuo to afford 0.44 g of 4-[(1H-benzotriazole-5-carbonyl)amino]benzoic acid. By filtration of the aqueous phase a further crop of 4-[(1H-benzotriazole-5-carbonyl)amino]benzoic acid was isolated (0.52 g).
1H-NMR (DMSO-d6): δ 7.97 (4H, s), 8.03 (2H, m), 8.66 (1H, bs), 10.7 (1H, s), 12.6 (1H, bs); HPLC-MS (Method A): m/z: 283 (M+1); Rt=1.85 min.
General Procedure (A) for Preparation of Compounds of General Formula I1:




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.

The carboxylic acid of 1H-benzotriazole-5-carboxylic acid is activated, ie the OH functionality is converted into a leaving group L (selected from eg fluorine, chlorine, bromine, iodine, 1-imidazolyl, 1,2,4-triazolyl, 1-benzotriazolyloxy, 1-(4-aza benzotriazolyl)oxy, pentafluorophenoxy, N-succinyloxy 3,4-dihydro-4-oxo-3-(1,2,3-benzotriazinyl)oxy, benzotriazole 5-COO, or any other leaving group known to act as a leaving group in acylation reactions. The activated benzotriazole-5-carboxylic acid is then reacted with R2—(CH2)n—B′ in the presence of a base. The base can be either absent (i.e. R2—(CH2)n—B′ acts as a base) or triethylamine, N-ethyl-N,N-diisopropylamine, N-methylmorpholine, 2,6-lutidine, 2,2,6,6-tetramethylpiperidine, potassium carbonate, sodium carbonate, caesium carbonate or any other base known to be useful in acylation reactions. The reaction is performed in a solvent such as THF, dioxane, toluene, dichloromethane, DMF, NMP or a mixture of two or more of these. The reaction is performed between 0° C. and 80° C., preferably between 20° C. and 40° C. When the acylation is complete, the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art.
The general procedure (A) is further illustrated in the following example:
Example 9
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid phenylamide




Benzotriazole-5-carboxylic acid (856 mg), HOAt (715 mg) and EDAC (1.00 g) were dissolved in DMF (17.5 mL) and the mixture was stirred at room temperature 1 hour. A 0.5 mL aliquot of this mixture was added to aniline (13.7 μL, 0.15 mmol) and the resulting mixture was vigorously shaken at room temperature for 16 hours. 1N hydrochloric acid (2 mL) and ethyl acetate (1 mL) were added and the mixture was vigorously shaken at room temperature for 2 hours. The organic phase was isolated and concentrated in vacuo to afford the title compound.
HPLC-MS (Method B): m/z: 239 (M+1); Rt=3.93 min.
The compounds in the following examples were similarly made. Optionally, the compounds may be isolated by filtration or by chromatography.
Example 10
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (4-methoxyphenyl)amide




HPLC-MS (Method A): m/z: 269 (M+1) & 291 (M+23); Rt=2.41 min
HPLC-MS (Method B): m/z: 239 (M+1); Rt=3.93 min.
Example 11
General Procedure (A)
{4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}carbamic acid tert-butyl ester




HPLC-MS (Method B): m/z: 354 (M+1); Rt=4.58 min.
Example 12
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (4-acetylaminophenyl)amide




HPLC-MS (Method B): m/z: 296 (M+1); Rt=3.32 min.
Example 13
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (3-fluorophenyl)amide




HPLC-MS (Method B): m/z: 257 (M+1); Rt=4.33 min.
Example 14
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (2-chlorophenyl)amide




HPLC-MS (Method B): m/z: 273 (M+1); Rt=4.18 min.
Example 15
General Procedure (A)
4-[(1H-Benzotriazole-5-carbonyl)amino]benzoic acid methyl ester




HPLC-MS (Method A): m/z: 297 (M+1); Rt: 2.60 min. HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.30 min.
Example 16
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (4-butylphenyl)amide




HPLC-MS (Method B): m/z: 295 (M+1); Rt=5.80 min.
Example 17
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (1-phenylethyl)amide




HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.08 min.
Example 18
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid benzylamide




HPLC-MS (Method B): m/z: 253 (M+1); Rt=3.88 min.
Example 19
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid 4-chlorobenzylamide




HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.40 min.
Example 20
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid 2-chlorobenzylamide




HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.25 min.
Example 21
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid 4-methoxybenzylamide




HPLC-MS (Method B): m/z: 283 (M+1); Rt=3.93 min.
Example 22
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid 3-methoxybenzylamide




HPLC-MS (Method B): m/z: 283 (M+1); Rt=3.97 min.
Example 23
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (1,2-diphenylethyl)amide




HPLC-MS (Method B): m/z: 343 (M+1); Rt=5.05 min.
Example 24
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid 3-bromobenzylamide




HPLC-MS (Method B): m/z: 331 (M+1); Rt=4.45 min.
Example 25
General Procedure (A)
4-{[(1H-Benzotriazole-5-carbonyl)amino]methyl}benzoic acid




HPLC-MS (Method B): m/z: 297 (M+1); Rt=3.35 min.
Example 26
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid phenethylamide




HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.08 min.
Example 27
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid [2-(4-chlorophenyl)ethyl]amide




HPLC-MS (Method B): m/z: 301 (M+1); Rt=4.50 min.
Example 28
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid [2-(4-methoxyphenyl)ethyl]amide




HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.15 min.
Example 29
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid [2-(3-methoxyphenyl)ethyl]amide




HPLC-MS (Method B): m/z: 297 (M+1); Rt=4.13 min.
Example 30
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid [2-(3-chlorophenyl)ethyl]amide




HPLC-MS (Method B): m/z: 301 (M+1); Rt=4.55 min.
Example 31
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (2,2-diphenylethyl)amide




HPLC-MS (Method B): m/z: 343 (M+1); Rt=5.00 min.
Example 32
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (3,4-dichlorophenyl)methylamide




HPLC-MS (Method B): m/z: 321 (M+1); Rt=4.67 min.
Example 33
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid methylphenylamide




HPLC-MS (Method B): m/z: 253 (M+1); Rt=3.82 min.
Example 34
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid benzylmethylamide




HPLC-MS (Method B): m/z: 267 (M+1); Rt=4.05 min.
Example 35
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid [2-(3-chloro-4-methoxyphenyl)ethyl]methyl-amide




HPLC-MS (Method B): m/z: 345 (M+1); Rt=4.37 min.
Example 36
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid methylphenethylamide




HPLC-MS (Method B): m/z 281 (M+1); Rt=4.15 min.
Example 37
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid [2-(3,4-dimethoxyphenyl)ethyl]methylamide




HPLC-MS (Method B): m/z: 341 (M+1); Rt=3.78 min;
Example 38
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (2-hydroxy-2-phenylethyl)methylamide




HPLC-MS (Method B): m/z: 297 (M+1); Rt=3.48 min.
Example 39
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (3-bromophenyl)amide




HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.19 min.
Example 40
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (4-bromophenyl)amide




HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.18 min.
Example 41
General Procedure (A)
{4-[(1H-Benzotriazole-5-carbonyl)amino]benzoylamino}acetic acid




HPLC-MS (Method A): m/z: 340 (M+1); Rt=1.71 min.
Example 42
General Procedure (A)
{4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acetic acid




HPLC-MS (Method A): m/z: 297 (M+1); Rt=2.02 min.
Example 43
General Procedure (A)
3-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acrylic acid




HPLC-MS (Method A): m/z: 309 (M+1); Rt=3.19 min.
Example 44
General Procedure (A)
{3-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}acetic acid




HPLC-MS (Method A): m/z: 297 (M+1); Rt=2.10 min.
Example 45
General Procedure (A)
2-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenoxy}-2-methylpropionic acid




HPLC-MS (Method A): m/z: 341 (M+1); Rt=2.42 min.
Example 46
General Procedure (A)
3-{4-[(1H-Benzotriazole-5-carbonyl)amino]benzoylamino}propionic acid




HPLC-MS (Method A): m/z: 354 (M+1); Rt=1.78 min.
Example 47
General Procedure (A)
3-{4-[(1H-Benzotriazole-5-carbonyl)amino]phenyl}propionic acid




HPLC-MS (Method A): m/z: 311 (M+1); Rt=2.20 min.
Example 48
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (4-benzyloxyphenyl)amide




HPLC-MS (Method A): m/z: 345 (M+1); Rt=3.60 min.
Example 49
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (3-chloro-4-methoxyphenyl)amide




HPLC-MS (Method A): m/z: 303 (M+1); Rt=2.88 min.
Example 50
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (4-phenoxyphenyl)amide




HPLC-MS (Method A): m/z: 331 (M+1); Rt=3.62 min.
Example 51
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (4-butoxyphenyl)amide




HPLC-MS (Method A): m/z: 311 (M+1); Rt=3.59 min.
Example 52
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (3-bromo-4-trifluoromethoxyphenyl)amide




HPLC-MS (Method A): m/z: 402 (M+1); Rt=3.93 min.
Example 53
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid (3,5-dichloro-4-hydroxyphenyl)amide




HPLC-MS (Method A): m/z: 323 (M+1); Rt=2.57 min.
Example 54
General Procedure (A)
4-{[(1H-Benzotriazole-5-carbonyl)amino]methyl}benzoic acid




HPLC-MS (Method A): m/z: 297 (M+1); Rt=1.86 min.
Example 55
General Procedure (A)
{4-[(1H-Benzotriazole-5-carbonyl)amino]phenylsulfanyl}acetic acid




HPLC-MS (Method A): m/z: 329 (M+1); Rt=2.34 min.
Example 56
N-(1H-Benzotriazol-5-yl)acetamide




HPLC-MS (Method A): m/z: 177 (M+1); Rt=0.84 min.
Example 57
General Procedure (A)
1H-Benzotriazole-5-carboxylic acid 4-nitrobenzylamide




General Procedure (B) for Preparation of Compounds of General Formula I2:





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.

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.
This general procedure (B) is further illustrated in the following example:
Example 58
General Procedure (B)
5-(3-Phenoxybenzylidene)thiazolidine-2,4-dione




A solution of thiazolidine-2,4-dione (90%, 78 mg, 0.6 mmol) and ammonium acetate (92 mg, 1.2 mmol) in acetic acid (1 mL) was added to 3-phenoxybenzaldehyde (52 μL, 0.6 mmol) and the resulting mixture was shaken at 115° C. for 16 hours. After cooling, the mixture was concentrated in vacuo to afford the title compound.
HPLC-MS (Method A): m/z: 298 (M+1); Rt=4.54 min.
The compounds in the following examples were similarly prepared. Optionally, the compounds can be further purified by filtration and washing with water, ethanol and/or heptane instead of concentration in vacuo. Also optionally the compounds can be purified by washing with ethanol, water and/or heptane, or by chromatography, such as preparative HPLC.
Example 59
General Procedure (B)
5-(4-Dimethylaminobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 249 (M+1); Rt=4.90 min
Example 60
General Procedure (B)
5-Naphthalen-1-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.16 min.
Example 61
General Procedure (B)
5-Benzylidene-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 206 (M+1); Rt=4.87 min.
Example 62
General Procedure (B)
5-(4-Methoxy-benzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 263 (M+1); Rt=4.90 min.
Example 63
General Procedure (B)
5-(4-Chloro-benzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 240 (M+1); Rt=5.53 min.
Example 64
General Procedure (B)
5-(4-Nitro-benzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 251 (M+1); Rt=4.87 min.
Example 65
General Procedure (B)
5-(4-Hydroxy-3-methoxy-benzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 252 (M+1); Rt=4.07 min.
Example 66
General Procedure (B)
5-(4-Methylsulfanyl-benzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 252 (M+1); Rt=5.43 min.
Example 67
General Procedure (B)
5-(3-Fluoro-4-methoxy-benzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 354 (M+1); Rt=4.97 min.
Example 68
General Procedure (B)
5-(4-tert-Butylbenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 262 (M+1); Rt=6.70 min.
Example 69
General Procedure (B)
N-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acetamide




HPLC-MS (Method A): m/z: 263 (M+1); Rt=3.90 min.
Example 70
General Procedure (B)
5-Biphenyl-4-ylmethylene-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 282 (M+1); Rt=4.52 min.
Example 71
General Procedure (B)
5-(4-Phenoxy-benzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 298 (M+1); Rt=6.50 min.
Example 72
General Procedure (B)
5-(3-Benzyloxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 312 (M+1); Rt=6.37 min.
Example 73
General Procedure (B)
5-(3-p-Tolyloxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 312 (M+1); Rt=6.87 min.
Example 74
General Procedure (B)
5-Napthalen-2-ylmethylene-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.15 min.
Example 75
General Procedure (B)
5-Benzo[1,3]dioxol-5-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 250 (M+1), Rt=3.18 min.
Example 76
General Procedure (B)
5-(4-Chlorobenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 256 (M+1); Rt=4.51 min.
Example 77
General Procedure (B)
5-(4-Dimethylaminobenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 265 (M+1); Rt=5.66 min.
Example 78
General Procedure (B)
5-(4-Nitrobenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 267 (M+1); Rt=3.94 min.
Example 79
General Procedure (B)
5-(4-Methylsulfanylbenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 268 (M+1); Rt=6.39 min.
Example 80
General Procedure (B)
5-(3-Fluoro-4-methoxybenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 270 (M+1); Rt=5.52 min.
Example 81
General Procedure (B)
5-Naphthalen-2-ylmethylene-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 272 (M+1); Rt=6.75 min.
Example 82
General Procedure (B)
5-(4-Diethylaminobenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 293 (M+1); Rt=5.99 min.
Example 83
General Procedure (B)
5-Biphenyl-4-ylmethylene-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 298 (M+1); Rt=7.03 min.
Example 84
General Procedure (B)
5-(3-Phenoxybenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 314 (M+1); Rt=6.89 min.
Example 85
General Procedure (B)
5-(3-Benzyloxybenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 328 (M+1); Rt=6.95 min.
Example 86
General Procedure (B)
5-(4-Benzyloxybenzylidene)-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 328 (M+1); RT=6.89 min.
Example 87
General Procedure (B)
5-Naphthalen-1-ylmethylene-2-thioxothiazolidin-4-one




HPLC-MS (Method A): m/z: 272 (M+1); Rt=6.43 min.
Example 88
General Procedure (B)
5-(3-Methoxybenzyl)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 236 (M+1); Rt=3.05 min.
Example 89
General Procedure (D)
4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid ethyl ester




HPLC-MS (Method A): m/z: 392 (M+23), Rt=4.32 min.
Example 90
General Procedure (D)
4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenoxy]-butyric acid




HPLC-MS (Method A): m/z: 410 (M+23); Rt=3.35 min.
Example 91
General Procedure (B)
5-(3-Bromobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 285 (M+1); Rt=4.01 min.
Example 92
General Procedure (B)
5-(4-Bromobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 285 (M+1); Rt=4.05 min.
Example 93
General Procedure (B)
5-(3-Chlorobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 240 (M+1); Rt=3.91 min.
Example 94
General Procedure (B)
5-Thiophen-2-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 212 (M+1); Rt=3.09 min.
Example 95
General Procedure (B)
5-(4-Bromothiophen-2-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 291 (M+1); Rt=3.85 min.
Example 96
General Procedure (B)
5-(3,5-Dichlorobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 274 (M+1); Rt=4.52 min.
Example 97
General Procedure (B)
5-(1-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 259 (M+1); Rt=3.55 min.
Example 98
General Procedure (B)
5-(1H-Indol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 245 (M+1); Rt=2.73 min.
Example 99
General Procedure (B)
5-Fluoren-9-ylidenethiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 280 (M+1); Rt=4.34 min.
Example 100
General Procedure (B)
5-(1-Phenylethylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 220 (M+1); Rt=3.38 min.
Example 101
General Procedure (B)
5-[1-(4-Methoxyphenyl)-ethylidene]-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 250 (M+1); Rt=3.55 min.
Example 102
General Procedure (B)
5-(1-Naphthalen-2-yl-ethylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 270 (M+1); Rt=4.30 min.
Example 103
General Procedure (B)
5-[1-(4-Bromophenyl)-ethylidene]-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.18 min.
Example 104
General Procedure (B)
5-(2,2-Diphenylethylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 296 (M+1); Rt=4.49 min.
Example 105
General Procedure (B)
5-[1-(3-Methoxyphenyl)-ethylidene]-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 250 (M+1); Rt=3.60 min.
Example 106
General Procedure (B)
5-[1-(6-Methoxynaphthalen-2-yl)-ethylidene]-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.26 min.
Example 107
General Procedure (B)
5-[1-(4-Phenoxyphenyl)-ethylidene]-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 312 (M+1); Rt=4.68 min.
Example 108
General Procedure (B)
5-[1-(3-Fluoro-4-methoxyphenyl)ethylidene]thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 268 (M+1); Rt=3.58 min.
Example 109
General Procedure (B)
5-[1-(3-Bromophenyl)-ethylidene]-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 300 (M+1); Rt=4.13 min.
Example 110
General Procedure (B)
5-Anthracen-9-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 306 (M+1); Rt=4.64 min.
Example 111
General Procedure (B)
5-(2-Methoxynaphthalen-1-ylmethylene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.02 min.
Example 112
General Procedure (B)
5-(4-Methoxynaphthalen-1-ylmethylene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.31 min.
Example 113
General Procedure (B)
5-(4-Dimethylaminonaphthalen-1-ylmethylene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 299 (M+1); Rt=4.22 min.
Example 114
General Procedure (B)
5-(4-Methylnaphthalen-1-ylmethylene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 270 (M+1); Rt=4.47 min.
Example 115
General Procedure (B)
5-Pyridin-2-ylmethylene-thiazolidine-2,4-dione




Example 116
5-Pyridin-2-ylmethyl-thiazolidine-2,4-dione




5-Pyridin-2-ylmethylene-thiazolidine-2,4-dione (5 g) in tetrahydrofuran (300 ml) was added 10% Pd/C (1 g) and the mixture was hydrogenated at ambient pressure for 16 hours. More 10% Pd/C (5 g) was added and the mixture was hydrogenated at 50 psi for 16 hours. After filtration and evaporation in vacuo, the residue was purified by column chromatography eluting with a mixture of ethyl acetate and heptane (1:1). This afforded the title compound (0.8 g, 16%) as a solid.
TLC: Rf=0.30 (SiO2; EtOAc:heptane 1:1)
Example 117
General Procedure (B)
5-(1H-Imidazol-4-ylmethylene)-thiazolidine-2,4-dione




Example 118
General Procedure (B)
5-(4-Benzyloxy-benzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 6.43 min; 99% (2A)
Example 119
General Procedure (B)
5-[4-(4-Fluorobenzyloxy)benzylidene]-2-thioxothiazolidin-4-one




Example 120
General Procedure (B)
5-(4-Butoxybenzylidene)-2-thioxothiazolidin-4-one




Example 121
General Procedure (B)
5-(3-Methoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 236 (M+1); Rt=4.97 min
Example 122
General Procedure (B)
5-(3-Methoxybenzylidene)imidazolidine-2,4-dione




HPLC-MS (Method A): m/z: 219 (M+1); Rt=2.43 min.
Example 123
General Procedure (B)
5-(4-Methoxybenzylidene)imidazolidine-2,4-dione




HPLC-MS (Method A): m/z: 219 (M+1); Rt=2.38 min.
Example 124
General Procedure (B)
5-(2,3-Dichlorobenzylidene)thiazolidine-2,4-dione




Example 125
General Procedure (B)
5-Benzofuran-7-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.57 min.
Example 126
General Procedure (B)
5-Benzo[1,3]dioxol-4-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 250 (M+1); Rt=4.00 min.
Example 127
General Procedure (B)
5-(4-Methoxy-2,3-dimethylbenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 264 (M+1); Rt=5.05 min.
Example 128
General Procedure (B)
5-(2-Benzyloxy-3-methoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 342 (M+1); Rt=5.14 min.
Example 129
General Procedure (B)
5-(2-Hydroxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 222 (M+1); Rt=3.67 min.
Example 130
General Procedure (B)
5-(2,4-Dichlorobenzylidene)thiazolidine-2,4-dione




1H-NMR (DMSO-d6): 7.60 (2H, “s”), 7.78 (1H, s), 7.82 (1H, s).
Example 131
General Procedure (B)
5-(2-Chlorobenzylidene)thiazolidine-2,4-dione




1H-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)
5-(2-Bromobenzylidene)thiazolidine-2,4-dione




1H-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)
5-(2,4-Dimethoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 266 (M+1) Rt=4.40 min.
Example 134
General Procedure (B)
5-(2-Methoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 236 (M+1); Rt=4.17 min.
Example 135
General Procedure (B)
5-(2,6-Difluorobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 242 (M+1); Rt=4.30 min.
Example 136
General Procedure (B)
5-(2,4-Dimethylbenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 234 (M+1); Rt=5.00 min.
Example 137
General Procedure (B)
5-(2,4,6-Trimethoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 296 (M+1); Rt=4.27 min.
Example 138
General Procedure (B)
5-(4-Hydroxy-2-methoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 252 (M+1); Rt=3.64 min.
Example 139
General Procedure (B)
5-(4-Hydroxynaphthalen-1-ylmethylene)thiazolidine-2,4-dione




1H-NMR (DMSO-d6): δ=7.04 (1H, d), 7.57 (2H, m), 7.67 (1H, t), 8.11 (1H, d), 8.25 (1H, d), 8.39 (1H, s) 11.1 (1H, s), 12.5 (1H, bs). HPLC-MS (Method C): m/z: 272 (M+1); Rt=3.44 min.
Example 140
General Procedure (B)
5-(2-Trifluoromethoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 290 (M+1); Rt=4.94 min.
Example 141
General Procedure (B)
5-Biphenyl-2-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 282 (M+1); Rt=5.17 min.
Example 142
General Procedure (B)
5-(2-Benzyloxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 312 (M+1); Rt=5.40 min.
Example 143
General Procedure (B)
5-Adamantan-2-ylidenethiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 250 (M+1); Rt=4.30 min.
General Procedure (C) for Preparation of Compounds of General Formula I2:




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.

This general procedure (C) is quite similar to general procedure (B) and is further illustrated in the following example:
Example 144
General Procedure (C)
5-(3,4-Dibromobenzylidene)thiazolidine-2,4-dione




A mixture of thiazolidine-2,4-dione (90%, 65 mg, 0.5 mmol), 3,4-dibromobenzaldehyde (132 mg, 0.5 mmol), and piperidine (247 μL, 2.5 mmol) was shaken in acetic acid (2 mL) at 110° C. for 16 hours. After cooling, the mixture was concentrated to dryness in vacuo. The resulting crude product was shaken with water, centrifuged, and the supernatant was discarded. Subsequently the residue was shaken with ethanol, centrifuged, the supernatant was discarded and the residue was further evaporated to dryness to afford the title compound.
1H NMR (Acetone-d6): δH 7.99 (d, 1H), 7.90 (d, 1H), 7.70 (s, 1H), 7.54 (d, 1H); HPLC-MS (Method A): m/z: 364 (M+1); Rt=4.31 min.
The compounds in the following examples were similarly prepared. Optionally, the compounds can be further purified by filtration and washing with water instead of concentration in vacuo. Also optionally the compounds can be purified by washing with ethanol, water and/or heptane, or by preparative HPLC.
Example 145
General Procedure (C)
5-(4-Hydroxy-3-iodo-5-methoxybenzylidene)thiazolidine-2,4-dione




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)
5-(4-Hydroxy-2,6-dimethylbenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z 250 (M+1); Rt.=2.45 min.
Example 147
General Procedure (C)
4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-2-yloxymethyl]-benzoic acid




HPLC-MS (Method C): m/z: 506 (M+23); Rt.=4.27 min.
Example 148
General Procedure (C)
5-(4-Bromo-2,6-dichlorobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 354 (M+1); Rt.=4.36 min.
Example 149
General Procedure (C)
5-(6-Hydroxy-2-naphthylmethylene)thiazolidine-2,4-dione




Mp 310-314° C., 1H NMR (DMSO-d6): δH=12.5 (s, broad, 1H), 8.06 (d, 1H), 7.90-7.78 (m, 2H), 7.86 (s, 1H), 7.58 (dd, 1H), 7.20 7.12 (m, 2H). 13C NMR (DMSO-d6): δC=166.2, 165.8, 155.4, 133.3, 130.1, 129.1, 128.6, 125.4, 125.3, 125.1, 124.3, 120.0, 117.8, 106.8; HPLC-MS (Method A): m/z: 272 (M+1); Rt=3.12 min.
Preparation of the Starting Material, 6-hydroxy-2-naphtalenecarbaldehyde
6-Cyano-2-naphthalenecarbaldehyde (1.0 g, 5.9 mmol) was dissolved in dry hexane (15 mL) under nitrogen. The solution was cooled to −60° C. and a solution of diisobutyl aluminium hydride (DIBAH) (15 mL, 1M in hexane) was added dropwise. After the addition, the solution was left at room temperature overnight. Saturated ammonium chloride solution (20 mL) was added and the mixture was stirred at room temperature for 20 min, subsequently aqueous H2SO4 (10% solution, 15 mL) was added followed by water until all salt was dissolved. The resulting solution was extracted with ethyl acetate (3×), the combined organic phases were dried with MgSO4, evaporated to dryness to afford 0.89 g of 6-hydroxy-2-naphtalenecarbaldehyde.
Mp.: 153.5-156.5° C.; HPLC-MS (Method A): m/z: 173 (M+1); Rt=2.67 min; 1H NMR (DMSO-d6): δH=10.32 (s, 1H), 8.95 (d, 1H), 10.02 (s, 1H), 8.42 (s, broad, 1H), 8.01 (d, 1H), 7.82-7.78 (m, 2H), 7.23-7.18 (m, 2H).
Alternative Preparation of 6-hydroxy-2-naphtalenecarbaldehyde
To a stirred cooled mixture of 6-bromo-2-hydroxynaphthalene (25.3 g, 0.113 mol) in THF (600 mL) at −78° C. was added n-BuLi (2.5 M, 100 mL, 0.250 mol) dropwise. The mixture turned yellow and the temperature rose to −64° C. After ca 5 min a suspension appeared. After addition, the mixture was maintained at −78° C. After 20 minutes, a solution of DMF (28.9 mL, 0.373 mol) in THF (100 mL) was added over 20 minutes. After addition, the mixture was allowed to warm slowly to 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)
5-(3-Iodo-4-methoxybenzylidene)thiazolidiene-2,4-dione




1H NMR (DMSO-d6): δH 12.55 (s, broad, 1H), 8.02 (d, 1H), 7.72 (s, 1H) 7.61 (d, 1H), 7.18 (d, 1H), 3.88 (s, 3H); 13C NMR (DMSO-d6); δC 168.1, 167.7, 159.8, 141.5, 132.0, 130.8, 128.0, 122.1, 112.5, 87.5, 57.3. HPLC-MS (Method A): m/z: 362 (M+1); Rt=4.08 min.
Preparation of the Starting Material, 3-iodo-4-methoxybenzaldehyde
4-Methoxybenzaldehyde (0.5 g, 3.67 mmol) and silver trifluoroacetate (0.92 g, 4.19 mmol) were mixed in dichloromethane (25 mL). Iodine (1.19 g, 4.7 mmol) was added in small portions and the mixture was stirred overnight at room temperature under nitrogen. The mixture was subsequently filtered and the residue washed with DCM. The combined filtrates were treated with an aqueous sodium thiosulfate solution (1 M) until the colour disappeared. Subsequent extraction with dichloromethane (3×20 mL) followed by drying with MgSO4 and evaporation in vacuo afforded 0.94 g of 3-iodo-4-methoxybenzaldehyde.
Mp 104-107° C.; HPLC-MS (Method A): m/z: 263 (M+1); Rt=3.56 min.; 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)
5-(1-Bromonaphthalen-2-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z:=336 (M+1); Rt=4.46 min.
Example 152
General Procedure (C)
1-[5-(2,4-Dioxothiazolidin-5-ylidenemethyl)thiazol-2-yl]piperidine-4-carboxylic acid ethyl ester




1H NMR (DMSO-d6): δH=7.88 (s, 1H), 7.78 (s, 1H), 4.10 (q, 2H), 4.0-3.8 (m, 2H), 3.40-3.18 (m, 2H), 2.75-2.60 (m, 1H), 2.04-1.88 (m, 2H), 1.73-1.49 (m, 2H), 1.08 (t, 3H); HPLC-MS (Method A): m/z: 368 (M+1); Rt=3.41 min.
Example 153
General Procedure (C)
5-(2-Phenyl-[1,2,3]triazol-4-ylmethylene)thiazolidine-2,4-dione




1H NMR (DMSO-d6): δH=12.6 (s, broad, 1H), 8.46 (s, 1H), 8.08 (dd, 2H), 7.82 (s, 1H), 7.70-7.45 (m, 3H). HPLC-MS (Method A): m/z: 273 (M+1); Rt=3.76 min.
Example 154
General Procedure (C)
5-(Quinolin-4-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 257 (M+1); Rt=2.40 min.
Example 155
General Procedure (C)
5-(6-Methylpyridin-2-ylmethylene)thiazolidine-2,4-dione




1H 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, 3H); HPLC-MS (Method A): m/z: 221 (M+1); Rt=3.03 min.
Example 156
General Procedure (C)
5-(2,4-dioxothiazolidin-5-ylidenemethyl)-furan-2-ylmethylacetate




1H NMR (DMSO-d6): δH=12.46 (s, broad, 1H), 7.58 (s, 1H), 7.05 (d, 1H), 6.74 (s, 1H), 5.13 (s, 2H), 2.10 (s, 3H). HPLC-MS (Method A): m/z: 208 (M−CH3COO); Rt=2.67 min.
Example 157
General Procedure (C)
5-(2,4-Dioxothiazolidin-5-ylidenemethyl)furan-2-sulfonic acid




HPLC-MS (Method A): m/z: 276 (M+1); Rt=0.98 min.
Example 158
General Procedure (C)
5-(5-Benzyloxy-1H-pyrrolo[2,3-c]pyridin-3-ylmethylene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 352 (M+1); Rt=3.01 min.
Example 159
General Procedure (C)
5-(Quinolin-2-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 257 (M+1); Rt=3.40 min.
Example 160
General Procedure (C)
5-(2,4-Dioxothiazolidin-5-ylidenemethyl)thiophene-2-carboxylic acid




HPLC-MS (Method A): m/z: 256 (M+1); Rt=1.96 min.
Example 161
General Procedure (C)
5-(2-Phenyl-1H-imidazol-4-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 272 (M+1); Rt=2.89 min.
Example 162
General Procedure (C)
5-(4-Imidazol-1-yl-benzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 272 (M+1); Rt=1.38 min.
Example 163
General Procedure (C)
5-(9-Ethyl-9H-carbazol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 323 (M+1); Rt=4.52 min.
Example 164
General Procedure (C)
5-(1,4-Dimethyl-9H-carbazol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 323 (M+1); Rt=4.35 min.
Example 165
General Procedure (C)
5-(2-Methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 259 (M+1); Rt=3.24 min.
Example 166
General Procedure (C)
5-(2-Ethylindol-3-ylmethylene)thiazolidine-2,4-dione




2-Methylindole (1.0 g, 7.6 mmol) dissolved in diethyl ether (100 mL) under nitrogen was treated with n-Butyl lithium (2 M in pentane, 22.8 mmol) and potassium tert-butoxide (15.2 mmol) with stirring at RT for 30 min. The temperature was lowered to −70 C. and methyl Iodide (15.2 mmol) was added and the resulting mixture was stirred at −70 for 2 h. Then 5 drops of water was added and the mixture allowed to warm up to RT. Subsequently, the mixture was poured into water (300 mL), pH was adjusted to 6 by means of 1N hydrochloric acid and the mixture was extracted with diethyl ether. The organic phase was dried with Na2SO4 and evaporated to dryness. The residue was purified by column chromatography on silica gel using heptane/ether (4/1) as eluent. This afforded 720 mg (69%) of 2-ethylindole.
1H NMR (DMSO-d6): δ=10.85 (1H, s); 7.39 (1H, d); 7.25 (1H, d); 6.98 (1H, t); 6.90 (1H, t); 6.10 (1H, s); 2.71 (2H, q); 1.28 (3H, t).
2-Ethylindole (0.5 g, 3.4 mmol) dissolved in DMF (2 mL) was added to a cold (0° C.) premixed (30 minutes) mixture of DMF (1.15 mL) and phosphorous oxychloride (0.64 g, 4.16 mmol). After addition of 2-ethylindole, the mixture was heated to 40° C. for 1 h, water (5 mL) was added and the pH adjusted to 5 by means of 1 N sodium hydroxide. The mixture was subsequently extracted with diethyl ether, the organic phase isolated, dried with MgSO4 and evaporated to dryness affording 2-ethylindole-3-carbaldehyde (300 mg).
HPLC-MS (Method C): m/z: 174 (M+1); Rt.=2.47 min.
2-Ethylindole-3-carbaldehyde (170 mg) was treated with thiazolidine-2,4-dione using the general procedure (C) to afford the title compound (50 mg).
HPLC-MS (Method C): m/z: 273 (M+1); Rt.=3.26 min.
Example 167
General Procedure (C)
5-[2-(4-Bromophenylsulfanyl)-1-methyl-1H-indol-3-ylmethylene]thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 447 (M+1); Rt=5.25 min.
Example 168
General Procedure (C)
5-[2-(2,4-Dichlorobenzyloxy)-naphthalen-1-ylmethylene]thiazolidine-2,4-dione




HPLC-MS (Method A): (anyone 1) m/z: 430 (M+1); Rt=5.47 min.
Example 169
General Procedure (C)
5-{4-[3-(4-Bromophenyl)-3-oxopropenyl]-benzylidene}thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 416 (M+1); Rt=5.02 min.
Example 170
General Procedure (C)
5-(4-Pyridin-2-ylbenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 283 (M+1), Rt=2.97 min.
Example 171
General Procedure (C)
5-(3,4-Bisbenzyloxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 418 (M+1); Rt=5.13 min.
Example 172
General Procedure (C)
5-[4-(4-Nitrobenzyloxy)-benzylidene]thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 357 (M+1); Rt=4.45 min.
Example 173
General Procedure (C)
5-(2-Phenyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 321 (M+1); Rt=3.93 min.
Example 174
General Procedure (C)
5-(5-Benzyloxy-1H-indol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 351 (M+1); Rt=4.18 min.
Example 175
General Procedure (C)
5-(4-Hydroxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A); m/z: 222 (M+1); Rt=2.42 min.
Example 176
General Procedure (C)
5-(1-Methyl-1H-indol-2-ylmethylene)thiazolidine-2,4-dione




1H NMR (DMSO-d6): δH=12.60 (s, broad, 1H), 7.85 (s, 1H), 7.68 (dd, 1H), 7.55 (dd, 1H), 7.38 (dt, 1H), 7.11 (dt, 1H) 6.84 (s, 1H), 3.88 (s, 3H); HPLC-MS (Method A): m/z: 259 (M+1); Rt=4.00 min.
Example 177
General Procedure (C)
5-(5-Nitro-1H-indol-3-ylmethylene)thiazolidine-2,4-dione




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)
5-(6-Methoxynaphthalen-2-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 286 (M+1); Rt=4.27 min.
Example 179
General Procedure (C)
5-(3-Bromo-4-methoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 314 (M+1), Rt=3.96 min.
Example 180
General Procedure (C)
3-{(2-Cyanoethyl)-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]amino}propionitrile




HPLC-MS (Method A): m/z: 327 (M+1); Rt=2.90 min.
Example 181
General Procedure (C)
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methyl ester




HPLC-MS (Method A): m/z: 303 (M+1); Rt=3.22-3.90 min.
Example 182
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid pentyl ester




(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).
HPLC-MS (Method C): m/z: 359 (M+1); Rt.=4.25 min.
Example 183
General Procedure (C)
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carboxylic acid




HPLC-MS (Method A): m/z: 289 (M+1); Rt=2.67 min.
Example 184
General Procedure (C)
5-(1-Benzylindol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 335 (M+1); Rt=4.55 min.
Example 185
General Procedure (C)
5-(1-Benzenesulfonylindol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z:=385 (M+1); Rt=4.59 min.
Example 186
General Procedure (C)
5-(4-[1,2,3]Thiadiazol-4-ylbenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.45 min.
Example 187
General Procedure (C)
5-[4-(4-Nitrobenzyloxy)-benzylidene]thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 357 (M+1); Rt=4.42 min.
Example 188
General Procedure (C)
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-1-carboxylic acid ethyl ester




HPLC-MS (Method A): m/z: 317 (M+1); Rt=4.35 min.
Example 189
General Procedure (C)
5-[2-(4-Pentylbenzoyl)-benzofuran-5-ylmethylene]thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 420 (M+1); Rt=5.92 min.
Example 190
General Procedure (C)
5-[1-(2-Fluorobenzyl)-4-nitroindol-3-ylmethylene]thiazolidine-2,4-dione




HPLC-MS (Method A): (Anyone 1) m/z: 398 (M+1); Rt=4.42 min.
Example 191
General Procedure (C)
5-(4-Benzyloxyindol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 351 (M+1); Rt=3.95 min.
Example 192
General Procedure (C)
5-(4-Isobutylbenzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 262 (M+1); Rt=4.97 min.
Example 193
General Procedure (C)
Trifluoromethanesulfonic acid 4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yl ester




HPLC-MS (Method A): m/z: 404 (M+1); Rt=4.96 min.
Preparation of Starting Material:
4-Hydroxy-1-naphthaldehyde (10 g, 58 mmol) was dissolved in pyridin (50 ml) and the mixture was cooled to 0-5° C. With stirring, trifluoromethanesulfonic acid anhydride (11.7 ml, 70 mmol) was added drop-wise. After addition was complete, the mixture was allowed to warm up to room temperature, and diethyl ether (200 ml) was added. The mixture was washed with water (2×250 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)
5-(4-Nitroindol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 290 (M+1); Rt=3.14 min.
Example 195
General Procedure (C)
5-(3,5-Dibromo-4-hydroxy-benzylidene)thiazolidine-2,4-dione




1H NMR (DMSO-d6): δH=12.65 (broad, 1H), 10.85 (broad, 1H), 7.78 (s, 2H), 7.70 (s, 1H);
HPLC-MS (Method A): m/z: 380 (M+1); Rt=3.56 min.
Example 196
General Procedure (C)




HPLC-MS (Method A): m/z: 385 (M+1); Rt=5.08 min.
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.
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)
4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-ylmethyl]benzoic acid methyl ester




HPLC-MS (Method A): m/z: 393 (M+1); Rt=4.60 min.
Example 198
General Procedure (C)
5-[1-(9,10-Dioxo-9,10-dihydroanthracen-2-ylmethyl)-1H-indol-3-ylmethylene]thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 465 (M+1); Rt=5.02 min.
Example 199
General Procedure (C)
4′-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-ylmethyl]biphenyl-2-carbonitrile




HPLC-MS (Method A): m/z: 458 (M+23); Rt=4.81 min.
Example 200
General Procedure (C)
3-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-methylindol-1-ylmethyl]benzonitrile




2-Methylindole-3-carbaldehyde (200 mg, 1.26 mmol) was added to a slurry of 3-bromomethylbenzenecarbonitrile (1.26 mmol) followed by sodium hydride, 60%, (1.26 mmol) in DMF (2 mL). The mixture was shaken for 16 hours, evaporated to dryness and washed with water and ethanol. The residue was treated with thiazolidine-2,4-dione following the general procedure C to afford the title compound (100 mg).
HPLC-MS (Method C): m/z: 374 (M+1); Rt.=3.95 min.
Example 201
General Procedure (C)
5-(1-Benzyl-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione




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.
HPLC-MS (Method C): m/z: 349 (M+1); Rt.=4.19 min.
Example 202
4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2-methylindol-1-ylmethyl]benzoic acid methyl ester




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.
HPLC-MS (Method C): m/z: 407 (M+1); Rt.=4.19 min.
Example 203
General Procedure (C)
5-(2-Chloro-1-methyl-1H-indol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 293 (M+1); Rt=4.10 min.
Example 204
General Procedure (C)
5-(4-Hydroxy-3,5-diiodo-benzylidene)-thiazolidine-2,4-dione




HPLC-MS (Method A): m/z: 474 (M+1); Rt=6.61 min.
Example 205
General Procedure (C)
5-(4-Hydroxy-3-iodobenzylidene)thiazolidine-2,4-dione




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)
5-(2,3,6-Trichlorobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 309 (M+1); Rt.=4.07 min
Example 207
General Procedure (C)
5-(2,6-Dichlorobenzylidene)thiazolidine-2,4-dione




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)
5-[1-(2,6-Dichloro-4-trifluoromethylphenyl)-2,5-dimethyl-1H-pyrrol-3-ylmethylene]thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 436 (M+1); Rt.=4.81 min
Example 209
General Procedure (C)
5-[1-(3,5-Dichlorophenyl)-5-(4-methanesulfonylphenyl)-2-methyl-1H-pyrrol-3-ylmethylene]-thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 508 (M+1); Rt.=4.31 min
Example 210
General Procedure (C)
5-[1-(2,5-Dimethoxyphenyl)-5-(4-methanesulfonylphenyl)-2-methyl-1H-pyrrol-3-ylmethylene]-thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 499 (M+1); Rt.=3.70 min
Example 211
General Procedure (C)
4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)-2,5-dimethylpyrrol-1-yl]benzoic acid




HPLC-MS (Method C): m/z: 342 (M+1); Rt.=3.19 min
Example 212
General Procedure (C)
5-(4-Hydroxy-2,6-dimethoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 282 (M+1); Rt.=2.56, mp=331-333° C.
Example 213
General Procedure (C)
5-(2,6-Dimethylbenzylidene)thiazolidine-2,4-dione




M.p: 104-105° C.
HPLC-MS (Method C): m/z: 234 (M+1); Rt.=3.58 min,
Example 214
General Procedure (C)
5-(2,6-Dimethoxybenzylidene)thiazolidine-2,4-dione




Mp: 241-242° C.
HPLC-MS (Method C): m/z: 266 (M+1); Rt.=3.25 min;
Example 215
General Procedure (C)
5-[4-(2-Fluoro-6-nitrobenzyloxy)-2,6-dimethoxybenzylidene]thiazolidine-2,4-dione




Mp: 255-256° C.
HPLC-MS (Method C): m/z: 435 (M+1), Rt=4.13 min,
Example 216
General Procedure (C)
5-Benzofuran-2-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 246 (M+1); Rt.=3.65 min, mp=265-266° C.
Example 217
General Procedure (C)
5-[3-(4-Dimethylaminophenyl)allylidene]thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 276 (M+1); Rt.=3.63, mp=259-263° C.
1H-NMR: (DMSO-d6) δ=12.3 (1H, broad); 7.46 (2H, d); 7.39 (1H, d); 7.11 (1H, d); 6.69 (2H, d); 6.59 (1H, dd); 2.98 (3H, s).
Example 218
General Procedure (C)
5-(2-Methyl-3-phenylallylidene)thiazolidine-2,4-dione




Mp: 203-210° C.
HPLC-MS (Method C): m/z: 246 (M+1); Rt=3.79 min.
Example 219
General Procedure (C)
5-(2-Chloro-3-phenylallylidene)thiazolidine-2,4-dione




Mp: 251-254° C.
HPLC-MS (Method C): m/z: 266 (M+1; Rt=3.90 min
Example 220
General Procedure (C)
5-(2-Oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione




Mp: 338-347° C.
HPLC-MS (Method C): m/z: 273 (M+1); Rt.=2.59 min.
Example 221
General Procedure (C)
5-(2,4,6-Tribromo-3-hydroxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 459 (M+1); Rt.=3.65 min.
Example 222
General Procedure (C)
5-(5-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 339 (M+1); Rt=3.37 min.
Example 223
General Procedure (C)
5-(7-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 319 (M+1); Rt=3.48 min.
Example 224
General Procedure (C)
5-(6-Bromoindol-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 325 (M+1); Rt=3.54 min.
Example 225
General Procedure (C)
5-(8-Methyl-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 287 (M+1); Rt=2.86 min.
Example 226
General Procedure (C)
5-(6-Methoxy-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 303 (M+1); Rt=2.65 min.
Example 227
General Procedure (C)
5-Quinolin-3-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 257 (M+1); Rt=2.77 min.
Example 228
General Procedure (C)
5-(8-Hydroxyquinolin-2-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 273 (M+1); Rt=3.44 min.
Example 229
General Procedure (C)
5-Quinolin-8-ylmethylenethiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 257 (M+1); Rt=3.15 min.
Example 230
General Procedure (C)
5-(1-Bromo-6-methoxynaphthalen-2-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 366 (M+1); Rt=4.44 min.
Example 231
General Procedure (C)
5-(6-Methyl-2-oxo-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 287 (M+1); Rt.=2.89 min.
Example 232
General Procedure (D)
5-(2,6-Dichloro-4-dibenzylaminobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method C): m/z: 469 (M+1); Rt=5.35 min.
Other preferred compounds include
3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-carboxylic acid




The following compounds are commercially available and may be prepared using general procedures (B) and/or (C).
Example 233
5-(5-Bromo-1H-indol-3-ylmethylene)thiazolidine-2,4-dione




Example 234
5-Pyridin-4-ylmethylenethiazolidine-2,4-dione




Example 235
5-(3-Bromo-4-methoxybenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A):
Example 236
5-(3-Nitrobenzylidene)thiazolidine-2,4-dione




HPLC-MS (Method A):
Example 237
5-Cyclohexylidene-1,3-thiazolidine-2,4-dione




HPLC-MS (Method A):
Example 238
5-(3,4-Dihydroxybenzylidene)thiazolidine-2,4-dione




Example 239
5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione




Example 240
5-(4-Hydroxy-3-methoxy-5-nitrobenzylidene)thiazolidine-2,4-dione




Example 241
5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione




Example 242
5-(4-Hydroxy-3,5-dimethoxybenzylidene)thiazolidine-2,4-dione




Example 243
5-(3-Bromo-5-ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione




Example 244
5-(3-Ethoxy-4-hydroxy-5-nitrobenzylidene)thiazolidine-2,4-dione




Example 245




Example 246




Example 247




Example 248




Example 249




Example 250




Example 251




Example 252




Example 253




Example 254




Example 255




Example 256




Example 257
5-(3-Hydroxy-5-methyl-phenylamino)-thiazolidine-2,4-dione




Example 258




Example 259




Example 260




Example 261




Example 262




Example 263




Example 264




Example 265




Example 266




Example 267




Example 268




Example 269




Example 270




Example 271




Example 272




Example 273




Example 274




Example 275




Example 276




Example 277




Example 278




Example 279




Example 280




Example 281




General Procedure (D) for Preparation of Compounds of General Formula I3:





    
    
        wherein X, Y, R10 are as defined above,
        n is 1 or 3-20,
        E is arylene or heterarylene (including up to four optional substituents, R13, R14, R15, and R15A as defined above),
        R′ is a standard carboxylic acid protecting group, such as C1-C6-alkyl or benzyl and Lea is a leaving group, such as chloro, bromo, iodo, methanesulfonyloxy, toluenesulfonyloxy or the like.
    
    


Step 1 is an alkylation of a phenol moiety. The reaction is preformed by reacting R10—C(═O)-E-OH with an ω-bromo-alkane-carboxylic acid ester (or a synthetic equivalent) in the presence of a base such as sodium or potassium carbonate, sodium or potassium hydroxide, sodium hydride, sodium or potassium alkoxide in a solvent, such as DMF, NMP, DMSO, acetone, acetonitrile, ethyl acetate or isopropyl acetate. The reaction is performed at 20-160° C., usually at room temperature, but when the phenol moiety has one or more substituents heating to 50° C. or more can be beneficial, especially when the substituents are in the ortho position relatively to the phenol. This will readily be recognised by those skilled in the art.
Step 2 is a hydrolysis of the product from step 1.
Step 3 is similar to general procedure (B) and (C).
This general procedure (D) is further illustrated in the following examples:
Example 282
General Procedure (D)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid




Step 1:
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.
Step 2:
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.
Step 3:
Thiazolidine-2,4-dione (3.55 g, 27.6 mmol), 4-(4-formylphenoxy)butyric acid (5.74 g, 27.6 mmol), anhydrous sodium acetate (11.3 g, 138 mmol) and acetic acid (100 mL) was refluxed for 16 h. After cooling, the mixture was filtered and washed with acetic acid and water. Drying in vacuo afforded 2.74 g (32%) of 4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid as a solid.
1H-NMR (DMSO-d6): δ 1.97 (2H, p), 2.40 (2H, t), 4.07 (2H, t), 7.08 (2H, d), 7.56 (2H, d), 7.77 (1H, s), 12.2 (1H, bs), 12.5 (1H, bs); HPLC-MS (Method A): m/z: 308 (M+1); Rt=2.89 min.
Example 283
General Procedure (D)
[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid




Step 3:
Thiazolidine-2,4-dione (3.9 g, 33 mmol), 3-formylphenoxyacetic acid (6.0 g, 33 mmol), anhydrous sodium acetate (13.6 g, 165 mmol) and acetic acid (100 mL) was refluxed for 16 h. After cooling, the mixture was filtered and washed with acetic acid and water. Drying in vacuo afforded 5.13 g (56%) of [3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid as a solid.
1H-NMR (DMSO-d6): δ 4.69 (2H, s), 6.95 (1H, dd), 7.09 (1H, t), 7.15 (1H, d), 7.39 (1H, t), 7.53 (1H, s); HPLC-MS (Method A): m/z=280 (M+1) (poor ionisation); Rt=2.49 min.
The compounds in the following examples were similarly prepared.
Example 284
General Procedure (D)
3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acrylic acid




1H-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)
[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid




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)
4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid




Example 287
General Procedure (D)
3-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid




1H-NMR (DMSO-d6): δ 7.57 (1H, s), 7.60 (1H, t), 7.79 (1H, dt), 7.92 (1H, dt), 8.14 (1H, t).
Example 288
General Procedure (D)
4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid




1H-NMR (DMSO-d6): δ 2.00 (2H, p), 2.45 (2H, t), 4.17 (2H, t), 7.31 (1H, d), 7.54 (1H, dd), 7.69 (1H, d), 7.74 (1H, s), 12.2 (1H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 364 (M+23); Rt=3.19 min.
Example 289
General Procedure (D)
4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid




1H-NMR (DMSO-d6): δ 1.99 (2H, p), 2.46 (2H, t), 4.17 (2H, t), 7.28 (1H, d), 7.57 (1H, dd), 7.25 (1H, s), 7.85 (1H, d), 12.2 (1H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 410 (M+23); Rt=3.35 min.
Example 290
General Procedure (D)
4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid




1H-NMR (DMSO-d6): δ 1.99 (2H, p), 2.45 (2H, t), 4.18 (2H, t), 7.28 (1H, d), 7.55 (1H, dd), 7.60 (1H, s), 7.86 (1H, d), 12.2 (1H, bs), 13.8 (1H, bs). HPLC-MS (Method A): m/z: 424 (M+23); Rt=3.84 min.
HPLC-MS (Method A): m/z: 424 (M+23); Rt=3.84 min
Example 291
General Procedure (D)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid




1H-NMR (DMSO-d6): δ 2.12 (2H, p), 2.5 (below DMSO), 4.28 (2H, t), 7.12 (1H, d), 7.6-7.7 (3H, m), 8.12 (1H, d), 8.31 (1H, d), 8.39 (1H, s), 12.2 (1H, bs, 12.6 (1H, bs). HPLC-MS (method A): m/z: 380 (M+23); Rt=3.76 min.
Example 292
General Procedure (D)
5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoic acid




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
5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoic acid




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.
HPLC-MS (Method C): m/z: 473 (M+23), Rt.=3.77 min
Example 294
4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid




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.
HPLC-MS (Method C): m/z: 459 (M+23); Rt.=3.59 min.
Example 295
General Procedure (D)
[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetic acid




1H-NMR (DMSO-d6): δ 4.90 (2H, s), 7.12 (1H, d), 7.52 (1H, dd), 7.65 (1H, s) 7.84 (1H, d). HPLC-MS (Method A): m/z: not observed; Rt=2.89 min.
Example 296
General Procedure (D)
4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid




1H-NMR (DMSO-d6): δ 1.98 (2H, p), 2.42 (2H, t), 4.04 (2H, t), 7.05 (1H, dd), 7.15 (2H, m), 7.45 (1H, t), 7.77 (1H, s), 12.1 (1H, bs), 12.6 (1H, bs). HPLC-MS (Method A): m/z: 330 (M+23); Rt=3.05 min.
Example 297
General Procedure (D)
[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-3-methoxyphenoxy]acetic acid




HPLC-MS (Method B): m/z: 310 (M+1); Rt=3.43 min.
Example 298
General Procedure (D)
[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetic acid




HPLC-MS (Method A): m/z: 330 (M+1); Rt=3.25 min.
Example 299
General Procedure (D)
8-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalene-1-carboxylic acid




HPLC-MS (Method A): m/z: 299 (M+1); Rt=2.49 min.
Example 300
General Procedure (D)
[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]acetic acid




HPLC-MS (Method A): m/z: 303 (M+1); Rt=2.90 min.
Preparation of Starting Material:
3-Formylindol (10 g, 69 mmol) was dissolved in N,N-dimethylformamide (100 mL) and under an atmosphere of nitrogen and with external cooling, keeping the temperature below 15° C., sodium hydride (60% in mineral oil, 3.0 g, 76 mmol) was added in portions. Then a solution of ethyl bromoacetate (8.4 mL, 76 mmol) in N,N-dimethylformamide (15 mL) was added dropwise over 30 minutes and the resulting mixture was stirred at room temperature for 16 hours. The mixture was concentrated in vacuo and the residue was partitioned between water (300 mL) and ethyl acetate (2×150 mL). The combined organic extracts were washed with a saturated aqueous solution of ammonium chloride (100 mL), dried (MgSO4) and concentrated in vacuo to afford 15.9 g (quant.) of (3-formylindol-1-yl)acetic acid ethyl ester as an oil.
1H-NMR (CDCl3): δH=1.30 (3H, t), 4.23 (2H, q), 4.90 (2H, s), 7.3 (3H, m), 7.77 (1H, s), 8.32 (1H, d), 10.0 (1H, s).
(3-Formylindol-1-yl)acetic acid ethyl ester (15.9 g 69 mmol) was dissolved in 1,4-dioxane (100 mL) and 1N sodium hydroxide (10 mL) was added and the resulting mixture was stirred at room temperature for 4 days. Water (500 mL) was added and the mixture was washed with diethyl ether (150 mL). The aqueous phase was acidified with 5N hydrochloric acid and extracted with ethyl acetate (250+150 mL). The combined organic extracts were dried (MgSO4) and concentrated in vacuo to afford 10.3 g (73%) of (3-formylindol-1-yl)acetic acid as a solid.
1H-NMR (DMSO-d6): δH=5.20 (2H, s), 7.3 (2H, m), 7.55 (1H, d), 8.12 (1H, d), 8.30 (1H, s), 9.95 (1H, s), 13.3 (1H, bs).
Example 301
General Procedure (D)
3-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yl]propionic acid




HPLC-MS (Method A): m/z: 317 (M+1); Rt=3.08 min.
Preparation of Starting Material:
A mixture of 3-formylindol (10 g, 69 mmol), ethyl 3-bromopropionate (10.5 mL, 83 mmol) and potassium carbonate (28.5 g, 207 mmol) and acetonitrile (100 mL) was stirred vigorously at refux temperature for 2 days. After cooling, the mixture was filtered and the filtrate was concentrated in vacuo to afford 17.5 g (quant.) of 3-(3-formylindol-1-yl)propionic acid ethyl ester as a solid.
1H-NMR (DMSO-d6): δH=1.10 (3H, t), 2.94 (2H, t), 4.02 (2H, q), 4.55 (2H, t), 7.3 (2H, m), 7.67 (1H, d), 8.12 (1H, d), 8.30 (1H, s), 9.90 (1H, s).
3-(3-Formylindol-1-yl)propionic acid ethyl ester (17.5 g 69 mmol) was hydrolysed as described above to afford 12.5 g (83%) of 3-(3-formylindol-1-yl)propionic acid as a solid.
1H-NMR (DMSO-d6): δH=2.87 (2H, t), 4.50 (2H, t), 7.3 (2H, m), 7.68 (1H, d), 8.12 (1H, d), 8.31 (1H, s), 9.95 (1H, s), 12.5 (1H, bs).
Example 302
General Procedure (D)
{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-yl}acetic acid




HPLC-MS (Method A): m/z: 429 (M+23); Rt=3.89 min.
Example 303
General Procedure (D)
6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxyoctanoic acid




HPLC-MS (Method C): m/z: 436 (M+23); Rt.=4.36 min
The intermediate aldehyde for this compound was prepared by a slightly modified procedure: 6-Hydroxynaphthalene-2-carbaldehyde (1.0 g, 5.8 mmol) was dissolved in DMF (10 mL) and sodium hydride 60% (278 mg) was added and the mixture stirred at RT for 15 min. 8-Bromooctanoic acid (0.37 g, 1.7 mmol) was converted to the sodium salt by addition of sodium hydride 60% and added to an aliquot (2.5 mL) of the above naphtholate solution and the resulting mixture was stirred at RT for 16 hours. Aqueous acetic acid (10%) was added and the mixture was extracted 3 times with diethyl ether. The combined organic phases were dried with MgSO4 and evaporated to dryness affording 300 mg of 8-(6-formylnaphthalen-2-yloxy)octanoic acid.
HPLC-MS (Method C): m/z 315 (M+1); Rt.=4.24 min.
Example 304
General Procedure (D)
12-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]dodecanoic acid




HPLC-MS (Method C): m/z: 492 (M+23); Rt.=5.3 min.
The intermediate aldehyde was prepared similarly as described in example 303.
Example 305
General Procedure (D)
11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoic acid




HPLC-MS (Method C): m/z: 478 (M+23); Rt.=5.17 min.
The intermediate aldehyde was prepared similarly as described in example 303.
Example 306
General Procedure (D)
15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoic acid




HPLC-MS (Method C): m/z: 534 (M+23); Rt.=6.07 min.
The intermediate aldehyde was prepared similarly as described in example 303.
Example 307
General Procedure (D)
6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoic acid




HPLC-MS (Method C): m/z: 408 (M+23); Rt.=3.71 min.
Example 308
General Procedure (D)
4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyric acid




HPLC-MS (Method C): m/z: 380 (M+23); Rt.=3.23 min.
Example 309
General Procedure (D)
6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoic acid ethyl ester




HPLC-MS (Method C): m/z: 436 (M+23); Rt.=4.64 min.
Example 310
General Procedure (D)
4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyric acid ethyl ester




HPLC-MS (Method C): m/z: 408 (M+23); Rt.=4.28 min.
Example 311
N-(3-Aminopropyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyramide




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.
HPLC-MS (Method C): m/z: 515 (M+1); Rt=3.79 min.
(3-{4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyrylamino}-propyl)carbamic acid tert-butyl ester (4.9 g, 9.5 mmol) was added dichloromethane (50 mL) and trifluoroacetic acid (50 mL) and the resulting mixture was stirred at room temperature for 45 minutes. The mixture was concentrated in vacuo and co-evaporated with toluene. To the residue was added ethyl acetate (100 mL) and the mixture was filtered and dried in vacuo to afford the title compound as the trifluoroacetic acid salt.
HPLC-MS (Method C): m/z: 414 (M+1); Rt=2.27 min.
Preferred Compounds of the Invention Includes:




The following compounds are commercially available and may be prepared according to general procedure (D):
Example 312




Example 313




Example 314




Example 315




Example 316




Example 317




Example 318




The following salicylic acid derivatives do all bind to the His B10 Zn2+ site of the insulin hexamer:
Example 319
Salicylic Acid




Example 320
Thiosalicylic Acid (or: 2-Mercaptobenzoic acid)




Example 321
2-Hydroxy-5-nitrobenzoic acid




Example 322
3-Nitrosalicyclic acid




Example 323
5,5′-Methylenedisalicylic acid




Example 324
2-Amino-5-trifluoromethylbenzoesyre




Example 325
2-Amino-4-chlorobenzoic acid




Example 326
2-Amino-5-methoxybenzoesyre




Example 327




Example 328




Example 329




Example 330




Example 331




Example 332




Example 333
5-Iodosalicylic acid




Example 334
5-Chlorosalicylic acid




Example 335
1-Hydroxy-2-naphthoic acid




Example 336
3,5-Dihydroxy-2-naphthoic acid




Example 337
3-Hydroxy-2-naphthoic acid




Example 338
3,7-Dihydroxy-2-naphthoic acid




Example 339
2-Hydroxybenzo[a]carbazole-3-carboxylic acid




Example 340
7-Bromo-3-hydroxy-2-naphthoic acid




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
1,6-Dibromo-2-hydroxynaphthalene-3-carboxylic acid




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
7-Formyl-3-hydroxynaphthalene-2-carboxylic acid




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 50° C. for 2 minutes and then allowed to cool to ambient temperature over a period of 30 minutes. The mixture was cooled to −78° C., and butyllithium (1.6 M in hexanes, 53 mL, 85 mmol) was added over a period of 15 minutes. N,N-Dimethylformamide (8.7 mL, 8.2 g, 112 mmol) was added after 90 minutes additional stirring. The cooling was discontinued, and the reaction mixture was stirred at room temperature for 17 hours before it was poured into 1 N hydrochloric acid (aq.) (750 mL). The organic solvents were evaporated in vacuo, and the resulting precipitate was filtered off and rinsed with water (3×100 mL) to yield the crude product (16.2 g). Purification on silica gel (dichloromethane/methanol/acetic acid=90:9:1) furnished the title compound as a solid.
1H-NMR (DMSO-d6): 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
3-Hydroxy-7-methoxy-2-naphthoic acid




Example 344
4-Amino-2-hydroxybenzoic acid




Example 345
5-Acetylamino-2-hydroxybenzoic acid




Example 346
2-Hydroxy-5-methoxybenzoic acid




The following compounds were prepared as described below:
Example 347
4-Bromo-3-hydroxynaphthalene-2-carboxylic acid




3-Hydroxynaphthalene-2-carboxylic acid (3.0 g, 15.9 mmol) was suspended in acetic acid (40 mL) and with vigorous stirring a solution of bromine (817 μL, 15.9 mmol) in acetic acid (10 mL) was added drop wise during 30 minutes. The suspension was stirred at room temperature for 1 hour, filtered and washed with water. Drying in vacuo afforded 3.74 g (88%) of 4-bromo-3-hydroxynaphthalene-2-carboxylic acid as a solid.
1H-NMR (DMSO-d6): δ 7.49 (1H, t), 7.75 (1H, t), 8.07 (2H, “t”), 8.64 (1H, s). The substitution pattern was confirmed by a COSY experiment, showing connectivities between the 3 (4 hydrogen) “triplets”. HPLC-MS (Method A): m/z: 267 (M+1); Rt=3.73 min.
Example 348
3-Hydroxy-4-iodonaphthalene-2-carboxylic acid




3-Hydroxynaphthalene-2-carboxylic acid (0.5 g, 2.7 mmol) was suspended in acetic acid (5 mL) and with stirring iodine monochloride (135 μL, 2.7 mml) was added. The suspension was stirred at room temperature for 1 hour, filtered and washed with water. Drying afforded 0.72 g (85%) of 4-iodo-3-hydroxynaphthalene-2-carboxylic acid as a solid.
1H-NMR (DMSO-d6): δ 7.47 (1H, t), 7.73 (1H, t), 7.98 (1H, d), 8.05 (1H, d), 8.66 (1H, s). HPLC-MS (Method A): m/z: 315 (M+1); Rt=3.94 min.
Example 349
2-Hydroxy-5-[(4-methoxyphenylamino)methyl]benzoic acid




p-Anisidine (1.3 g, 10.6 mmol) was dissolved in methanol (20 mL) and 5-formylsalicylic acid (1.75 g, 10.6 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The solid formed was isolated by filtration, re-dissolved in N-methyl pyrrolidone (20 mL) and methanol (2 mL). To the mixture was added sodium cyanoborohydride (1.2 g) and the mixture was heated to 70° C. for 3 hours. To the cooled mixture was added ethyl acetate (100 mL) and the mixture was extracted with water (100 mL) and saturated aqueous ammonium chloride (100 mL). The combined aqueous phases were concentrated in vacuo and a 2 g aliquot was purified by SepPac chromatography eluting with mixtures of aetonitrile and water containing 0.1% trifluoroacetic acid to afford the title compound.
HPLC-MS (Method A): m/z: 274 (M+1); Rt=1.77 min.
1H-NMR (methanol-d4): δ 3.82 (3H, s), 4.45 (2H, s), 6.96 (1H, d), 7.03 (2H, d), 7.23 (2H, d), 7.45 (1H, dd), 7.92 (1H, d).
Example 350
2-Hydroxy-5-(4-methoxyphenylsulfamoyl)benzoic acid




A solution of 5-chlrosulfonylsalicylic acid (0.96 g, 4.1 mmol) in dichloromethane (20 mL) and triethylamine (1.69 mL, 12.2 mmol) was added p-anisidine (0.49 g, 4.1 mmol) and the resulting mixture was stirred at room temperature for 16 hours. The mixture was added dichloromethane (50 mL) and was washed with water (2×100 mL). Drying (MgSO4) of the organic phase and concentration in vacuo afforded 0.57 g crude product. Purification by column chromatography on silica gel eluting first with ethyl acetate:heptane (1:1) then with methanol afforded 0.1 g of the title compound.
HPLC-MS (Method A): m/z: 346 (M+23); Rt=2.89 min.
1H-NMR (DMSO-d6): δ 3.67 (3H, s), 6.62 (1H, d), 6.77 (2H, d), 6.96 (2H, d), 7.40 (1H, dd), 8.05 (1H, d), 9.6 (1H, bs).
General Procedure (E) for Preparation of Compounds of General Formula I4:




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.

An analogous chemical transformation has previously been described in the literature (Bumagin et al., Tetrahedron, 1997, 53, 14437-14450). The reaction is generally known as the Suzuki coupling reaction and is generally performed by reacting an aryl halide or triflate with an arylboronic acid or a heteroarylboronic acid in the presence of a palladium catalyst and a base such as sodium acetate, sodium carbonate or sodium hydroxide. The solvent can be water, acetone, DMF, NMP, HMPA, methanol, ethanol toluene or a mixture of two or more of these solvents. The reaction is performed at room temperature or at elevated temperature.
The general procedure (E) is further illustrated in the following example:
Example 351
General Procedure (E)
7-(4-Acetylphenyl)-3-hydroxynaphthalene-2-carboxylic acid




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.
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)
3-Hydroxy-7-(3-methoxyphenyl)naphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 295 (M+1); Rt=4.60 min.
Example 353
General Procedure (E)
3-Hydroxy-7-phenylnaphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 265 (M+1); Rt=4.6 min.
Example 354
General Procedure (E)
3-Hydroxy-7-p-tolylnaphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 279 (M+1); Rt=4.95 min.
Example 355
General Procedure (E)
7-(4-Formylphenyl)-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 293 (M+1); Rt=4.4 min.
Example 356
General Procedure (E)
6-Hydroxy-[1,2]binaphthalenyl-7-carboxylic acid




HPLC-MS (Method (A)): m/z: 315 (M+1); Rt=5.17 min.
Example 357
General Procedure (E)
7-(4-Carboxy-phenyl)-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=3.60 min.
Example 358
General Procedure (E)
7-Benzofuran-2-yl-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 305 (M+1); Rt=4.97 min.
Example 359
General Procedure (E)
3-Hydroxy-7-(4-methoxyphenyl)-naphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 295 (M+1); Rt=4.68 min.
Example 360
General Procedure (E)
7-(3-Ethoxyphenyl)-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=4.89 min.
Example 361
General Procedure (E)
7-Benzo[1,3]dioxol-5-yl-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 309 (M+1); Rt=5.61 min.
Example 362
General Procedure (E)
7-Biphenyl-3-yl-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method (A)): m/z: 341 (M+1); Rt=5.45 min.
General Procedure (F) for Preparation of Compounds of General Formula I5:




wherein R30 is hydrogen or C1-C6-alkyl and T is as defined above

This general procedure (F) is further illustrated in the following example:
Example 363
General Procedure (F)
3-Hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylic acid




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.
1H-NMR (DMSO-d6): δ 10.95 (1H, bs), 8.45 (1H, s), 7.96 (1H, s), 7.78 (1H, d), 7.62 (1H, d), 7.32 (1H, s), 7.13 (2H, bd), 6.98 (2H, bd), 4.48 (2H, s), 2.79 (1H, sept), 1.14 (6H, d); HPLC-MS (Method (A)): m/z: 336 (M+1); Rt=3.92 min.
The compounds in the following examples were made using this general procedure (F).
Example 364
General Procedure (F)
7-{[(4-Bromophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 372 (M+1); Rt=4.31 min.
Example 365
General Procedure (F)
7-{[(3,5-Dichlorophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 362 (M+1); Rt=4.75 min.
Example 366
General Procedure (F)
7-{[(Benzothiazol-6-yl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z 351 (M+1); Rt=3.43 min.
Example 367
General Procedure (F)
3-Hydroxy-7-{[(quinolin-6-yl)amino]methyl}naphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 345 (M+1); Rt=2.26 min.
Example 368
General Procedure (F)
3-Hydroxy-7-{[(4-methoxyphenyl)amino]methyl}naphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 324 (M+1); Rt=2.57 min.
Example 369
General Procedure (F)
7-{[(2,3-Dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 350 (M+1); Rt=2.22 min.
Example 370
General Procedure (F)
7-{[(4-Chlorobenzyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 342 (M+1); Rt=2.45 min.
Example 371
General Procedure (F)
3-Hydroxy-7-{[(naphthalen-1-ylmethyl)amino]methyl}naphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 357 (M+1); Rt=2.63 min.
Example 372
General Procedure (F)
7-{[(Biphenyl-2-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 384 (M+1); Rt=2.90 min.
Example 373
General Procedure (F)
3-Hydroxy-7-{[(4-phenoxybenzyl)amino]methyl}naphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 400 (M+1); Rt=3.15 min.
Example 374
General Procedure (F)
3-Hydroxy-7-{[(4-methoxybenzyl)amino]methyl}naphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 338 (M+1); Rt=2.32 min.
General Procedure (G) for Preparation of Compounds of General Formula I6:




wherein T is as defined above and the moiety (C1-C6-alkanoyl)2O is an anhydride.

The general procedure (G) is illustrated by the following example:
Example 375
General Procedure (G)
N-Acetyl-3-hydroxy-7-[(4-(2-propyl)phenylamino)methyl]naphthalene-2-carboxylic acid




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.
1H-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.
The compounds in the following examples were prepared in a similar fashion.
Example 376
General Procedure (G)
N-Acetyl-7-{[(4-bromophenyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 414 (M+1); Rt=3.76 min.
Example 377
General Procedure (G)
N-Acetyl-7-{[(2,3-dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 392 (M+1); Rt=3.26 min.
Example 378
General Procedure (G)
N-Acetyl-7-{[(4-chlorobenzyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic acid




HPLC-MS (Method C): m/z: 384 (M+1); Rt=3.67 min.
Example 379
5-(3-(Naphthalen-2-yloxymethyl)-phenyl)-1H-tetrazole




To a mixture of 2-naphthol (10 g, 0.07 mol) and potassium carbonate (10 g, 0.073 mol) in acetone (150 mL), alpha-bromo-m-tolunitril (13.6 g, 0.07 mol) was added in portions. The reaction mixture was stirred at reflux temperature for 2.5 hours. The cooled reaction mixture was filtered and evaporated in vacuo affording an oily residue (19 g) which was dissolved in diethyl ether (150 mL) and stirred with a mixture of active carbon and MgSO4 for 16 hours. The mixture was filtered and evaporated in vacuo affording crude 18.0 g (100%) of 3-(naphthalen-2-yloxymethyl)benzonitrile as a solid.
12 g of the above benzonitrile was recrystallised from ethanol (150 mL) affording 8.3 g (69%) of 3-(naphthalen-2-yloxymethyl)-benzonitrile as a solid.
M.p. 60-61° C.
Calculated for C18H13NO: C, 83.37%; H, 5.05%; N, 5.40%. Found C, 83.51%; H, 5.03%; N, 5.38%.
To a mixture of sodium azide (1.46 g, 22.5 mmol) and ammonium chloride (1.28 g, 24.0 mmol) in dry dimethylformamide (20 mL) under an atmosphere of nitrogen, 3-(naphthalen-2-yloxymethyl)-benzonitrile (3.9 g, 15 mmol) was added and the reaction mixture was stirred at 125° C. for 4 hours. The cooled reaction mixture was poured on to ice water (300 mL) and acidified to pH=1 with 1 N hydrochloric acid. The precipitate was filtered off and washed with water, dried at 100° C. for 4 hours affording 4.2 g (93%) of the title compound.
M.p. 200-202° C.
Calculated for C18H14N4O: C, 71.51%; H, 4.67%; N, 18.54%. Found C, 72.11%; H, 4.65%; N, 17.43%.
1H NMR (400 MHz, DMSO-d6) δH 5.36 (s, 2H), 7.29 (dd, 1H), 7.36 (dt, 1H), 7.47 (m, 2H), 7.66 (t, 1H), 7.74 (d, 1H), 7.84 (m, 3H), 8.02 (d, 1H), 8.22 (s, 1H).
Example 380
N-(3-(Tetrazol-5-yl)phenyl)-2-naphtoic acid amide




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.
M.p. 138-140° C.
The above naphthoic acid amide (10 g, 37 mmol) was dissolved in N,N-dimethylformamide (200 mL) and sodium azide (2.63 g, 40 mmol) and ammonium chloride (2.16 g, 40 mmol) were added and the mixture heated at 125° C. for 6 hours. Sodium azide (1.2 g) and ammonium chloride (0.98 g) were added and the mixture heated at 125° C. for 16 hours. After cooling, the mixture was poured into water (1.5 l) and stirred at room temperature for 30 minutes. The solid formed was filtered off, washed with water and dried in vacuo at 50° C. for 3 days affording 9.69 g (84%) of the title compound as a solid which could be further purified by treatment with ethanol at reflux temperature.
1H NMR (200 MHz, DMSO-d6): δH 7.58-7.70 (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
5-[3-(Biphenyl-4-yloxymethyl)phenyl]-1H-tetrazole




To a solution of 4-phenylphenol (10.0 g, 59 mmol) in dry N,N-dimethyl-formamide (45 mL) kept under an atmosphere of nitrogen, sodium hydride (2.82 g, 71 mmol, 60% dispersion in oil) was added in portions and the reaction mixture was stirred until gas evolution ceased. A solution of m-cyanobenzyl bromide (13 g, 65 mmol) in dry N,N-dimethylformamide (45 mL) was added dropwise and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was poured on to ice water (150 mL). The precipitate was filtered of and washed with 50% ethanol (3×50 mL), ethanol (2×50 mL), diethyl ether (80 mL), and dried in vacuo at 50° C. for 18 hours affording crude 17.39 g of 3-(biphenyl-4-yloxymethyl)benzonitrile as a solid.
1H NMR (200 MHz, CDCl3) δH 5.14 (s, 2H), 7.05 (m, 2H), 7.30-7.78 (m, 11H).
To a mixture of sodium azide (2.96 g, 45.6 mmol) and ammonium chloride (2.44 g, 45.6 mmol) in dry N,N-dimethylformamide (100 mL) under an atmosphere of nitrogen, 3-(biphenyl-4-yloxymethyl)-benzonitrile (10.0 g, 35.0 mmol) was added and the reaction mixture was stirred at 125° C. for 18 hours. The cooled reaction mixture was poured on to a mixture of 1N hydrochloric acid (60 mL) and ice water (500 mL). The precipitate was filtered off and washed with water (3×100 mL), 50% ethanol (3×100 mL), ethanol (50 mL), diethyl ether (50 mL), ethanol (80 mL), and dried in vacuo at 50° C. for 18 hours affording 8.02 g (70%) of the title compound.
1H NMR (200 MHz, DMSO-d6) δH 5.31 (s, 2H), 7.19 (m, 2H), 7.34 (m, 1H), 7.47 (m, 2H), 7.69 (m, 6H), 8.05 (dt, 1H), 8.24 (s, 1H).
Example 382
5-(3-Phenoxymethyl)-phenyl)-tetrazole




3-Bromomethylbenzonitrile (5.00 g, 25.5 mmol) was dissolved in N,N-dimethylformamide (50 mL), phenol (2.40 g, 25.5 mmol) and potassium carbonate (10.6 g, 77 mmol) were added. The mixture was stirred at room temperature for 16 hours. The mixture was poured into water (400 mL) and extracted with ethyl acetate (2×200 mL). The combined organic extracts were washed with water (2×100 mL), dried (MgSO4) and evaporated in vacuo to afford 5.19 g (97%) 3-(phenoxymethyl)benzonitrile as an oil.
TLC: Rf=0.38 (Ethyl acetate/heptane=1:4)
The above benzonitrile (5.19 g, 24.8 mmol) was dissolved in N,N-dimethylformamide (100 mL) and sodium azide (1.93 g, 30 mmol) and ammonium chloride (1.59 g, 30 mmol) were added and the mixture was heated at 140° C. for 16 hours. After cooling, the mixture was poured into water (800 mL). The aqueous mixture was washed with ethyl acetate (200 mL). The pH of the aqueous phase was adjusted to 1 with 5 N hydrochloric acid and stirred at room temperature for 30 minutes. Filtration, washing with water and drying in vacuo at 50° C. afforded 2.06 g (33%) of the title compound as a solid.
1H NMR (200 MHz, CDCl3+DMSO-d6) δH 5.05 (s, 2H), 6.88 (m, 3H), 7.21 (m, 2H), 7.51 (m, 2H), 7.96 (dt, 1H), 8.14 (s, 1H).
Example 383
5-[3-(Biphenyl-4-ylmethoxy)phenyl]-1H-tetrazole




To a solution of 3-cyanophenol (5.0 g, 40.72 mmol) in dry N,N-dimethylformamide (100 mL) kept under an atmosphere of nitrogen, sodium hydride (2 g, 48.86 mmol, 60% dispersion in oil) was added in portions and the reaction mixture was stirred until gas evolution ceased. p-Phenylbenzyl chloride (9.26 g, 44.79 mmol) and potassium iodide (0.2 g, 1.21 mmol) were added and the reaction mixture was stirred at room temperature for 60 hours. The reaction mixture was poured on to a mixture of saturated sodium carbonate (100 mL) and ice water (300 mL). The precipitate was filtered of and washed with water (3×100 mL), n-hexane (2×80 mL) and dried in vacuo at 50° C. for 18 hours affording 11.34 g (98%) of 3-(biphenyl-4-ylmethoxy)-benzonitrile as a solid.
To a mixture of sodium azide (2.37 g, 36.45 mmol) and ammonium chloride (1.95 g, 36.45 mmol) in dry N,N-dimethylformamide (100 mL) under an atmosphere of nitrogen, 3-(biphenyl-4-ylmethoxy)benzonitrile (8.0 g, 28.04 mmol) was added and the reaction mixture was stirred at 125° C. for 18 hours. To the cooled reaction mixture water (100 mL) was added and the reaction mixture stirred for 0.75 hour. The precipitate was filtered off and washed with water, 96% ethanol (2×50 mL), and dried in vacuo at 50° C. for 18 hours affording 5.13 g (56%) of the title compound.
1H NMR (200 MHz, DMSO-d6) δH 5.29 (s, 2H), 7.31 (dd, 1H), 7.37-7.77 (m, 12H).
Example 384
5-[4-(Biphenyl-4-ylmethoxy)-3-methoxyphenyl]-1H-tetrazol




This compound was made similarly as described in example 383.
Example 385




Example 386
5-(2-Naphtylmethyl)-1H-tetrazole




This compound was prepared similarly as described in example 379, step 2.
Example 387
5-(1-Naphtylmethyl)-1H-tetrazole




This compound was prepared similarly as described in example 379, step 2.
Example 388
5-[4-(Biphenyl-4-yloxymethyl)phenyl]-1H-tetrazole




A solution of alpha-bromo-p-tolunitrile (5.00 g, 25.5 mmol), 4-phenylphenol (4.56 g, 26.8 mmol), and potassium carbonate (10.6 g, 76.5 mmol) in N,N-dimethylformamide (75 mL) was stirred vigorously for 16 hours at room temperature. Water (75 mL) was added and the mixture was stirred at room temperature for 1 hour. The precipitate was filtered off and washed with thoroughly with water. Drying in vacuo over night at 50° C. afforded 7.09 g (97%) of 4-(biphenyl-4-yloxymethyl)benzonitrile as a solid.
The above benzonitrile (3.00 g, 10.5 mmol) was dissolved in N,N-dimethylformamide (50 mL), and sodium azide (1.03 g, 15.8 mmol) and ammonium chloride (0.84 g, 15.8 mmol) were added and the mixture was stirred 16 hours at 125° C. The mixture was cooled to room temperature and water (50 mL) was added. The suspension was stirred overnight, filtered, washed with water and dried in vacuo at 50° C. for 3 days to give crude 3.07 g (89%) of the title compound. From the mother liquor crystals were collected and washed with water, dried by suction to give 0.18 g (5%) of the title compound as a solid.
1H NMR (200 MHz, DMSO-d6): δH 5.21 (s, 2H), 7.12 (d, 2H), 7.30 (t, 1H), 7.42 (t, 2H), 7.56-7.63 (m, 6H), 8.03 (d, 2H).
Calculated for C20H16N4O, 2H2O: C, 65.92%; H, 5.53%; N, 15.37%. Found: C, 65.65%; H, 5.01%; N, 14.92%.
Example 389




This compound was prepared similarly as described in example 383.
Example 390




Example 391




Example 392




Example 393
5-(3-(Biphenyl-4-yloxymethyl)-benzyl)-1H-tetrazole




Example 394
5-(1-Naphthyl)-1H-tetrazole




This compound was prepared similarly as described in example 379, step 2.
Example 395
5-[3-Methoxy-4-(4-methylsulfonylbenzyloxy)phenyl]-1H-tetrazole




This compound was made similarly as described in example 383.
Example 396
5-(2-Naphthyl)-1H-tetrazole




This compound was prepared similarly as described in example 379, step 2.
Example 397
2-Amino-N-(1H-tetrazol-5-yl)-benzamide




Example 398
5-(4-Hydroxy-3-methoxyphenyl)-1H-tetrazole




This compound was prepared similarly as described in example 379, step 2.
Example 399
4-(2H-Tetrazol-5-ylmethoxy)benzoic acid




To a mixture of methyl 4-hydroxybenzoate (30.0 g, 0.20 mol), sodium iodide (30.0 g, 0.20 mol) and potassium carbonate (27.6 g, 0.20 mol) in acetone (2000 mL) was added chloroacetonitrile (14.9 g, 0.20 mol). The mixture was stirred at RT for 3 days. Water was added and the mixture was acidified with 1N hydrochloric acid and the mixture was extracted with diethyl ether. The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was dissolved in acetone and chloroacetonitrile (6.04 g, 0.08 mol), sodium iodide (12.0 g, 0.08 mol) and potassium carbonate (11.1 g, 0.08 mol) were added and the mixture was stirred for 16 hours at RT and at 60° C. More chloroacetonitrile was added until the conversion was 97%. Water was added and the mixture was acidified with 1N hydrochloric acid and the mixture was extracted with diethyl ether. The combined organic layers were dried over Na2SO4 and concentrated in vacuo to afford methyl 4-cyanomethyloxybenzoate in quantitative yield. This compound was used without further purification in the following step.
A mixture of methyl 4-cyanomethyloxybenzoate (53.5 g, 0.20 mol), sodium azide (16.9 g, 0.26 mol) and ammonium chloride (13.9 g, 0.26 mol) in DMF 1000 (mL) was refluxed overnight under N2. After cooling, the mixture was concentrated in vacuo. The residue was suspended in cold water and extracted with ethyl acetate. The combined organic phases were washed with brine, dried over Na2SO4 and concentrated in vacuo, to afford methyl 4-(2H-tetrazol-5-ylmethoxy)benzoate. This compound was used as such in the following step.
Methyl 4-(2H-Tetrazol-5-ylmethoxy)-benzoate was refluxed in 3N sodium hydroxide. The reaction was followed by TLC (DCM:MeOH=9:1). The reaction mixture was cooled, acidified and the product filtered off. The impure product was washed with DCM, dissolved in MeOH, filtered and purified by column chromatography on silica gel (DCM:MeOH=9:1). The resulting product was recrystallised from DCM:MeOH=95:5. This was repeated until the product was pure. This afforded 13.82 g (30%) of the title compound.
1H-NMR (DMSO-d6): 4.70 (2H, s), 7.48 (2H, d), 7.73 (2H, d), 13 (1H, bs).
Example 400
4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoic acid




To a solution of sodium hydroxide (10.4 g, 0.26 mol) in degassed water (600 mL) was added 4-mercaptobenzoic acid (20.0 g, 0.13 mol). This solution was stirred for 30 minutes. To a solution of potassium carbonate (9.0 g, 65 mmol) in degassed water (400 mL) was added chloroacetonitrile (9.8 g, (0.13 mol) portion-wise. These two solutions were mixed and stirred for 48 hours at RT under N2. The mixture was filtered and washed with heptane. The aqueous phase was acidified with 3N hydrochloric acid and the product was filtered off, washed with water and dried, affording 4-cyanomethylsulfanylbenzoic acid (27.2 g, 88%). This compound was used without further purification in the following step.
A mixture of 4-cyanomethylsulfanylbenzoic acid (27.2 g, 0.14 mol), sodium azide (11.8 g, 0.18 mol) and ammonium chloride (9.7 g, 0.18 mol) in DMF (1000 mL) was refluxed overnight under N2. The mixture was concentrated in vacuo. The residue was suspended in cold water and extracted with diethyl ether. The combined organic phases were washed with brine, dried over Na2SO4 and concentrated in vacuo. Water was added and the precipitate was filtered off. The aqueous layer was concentrated in vacuo, water was added and the precipitate filtered off. The combined impure products were purified by column chromatography using DCM:MeOH=9:1 as eluent, affording the title compound (5.2 g, 16%).
1H-NMR (DMSO-d6): 5.58 (2H, s), 7.15 (2H, d), 7.93 (2H, d), 12.7 (1H, bs).
Example 401
3-(2H-Tetrazol-5-yl)-9H-carbazole




3-Bromo-9H-carbazole was prepared as described by Smith et al. in Tetrahedron 1992, 48, 7479-7488.
A solution of 3-bromo-9H-carbazole (23.08 g, 0.094 mol) and cuprous cyanide (9.33 g, 0.103 mol) in N-methyl-pyrrolidone (300 ml) was heated at 200° C. for 5 h. The cooled reaction mixture was poured on to water (600 ml) and the precipitate was filtered off and washed with ethyl acetate (3×50 ml). The filtrate was extracted with ethyl acetate (3×250 ml) and the combined ethyl acetate extracts were washed with water (150 ml), brine (150 ml), dried (MgSO4) and concentrated in vacuo. The residue was crystallised from heptanes and recrystallised from acetonitrile (70 ml) affording 7.16 g (40%) of 3-cyano-9H-carbazole as a solid.
M.p. 180-181° C.
3-Cyano-9H-carbazole (5.77 g, 30 mmol) was dissolved in N,N-dimethylformamide (150 ml), and sodium azide (9.85 g, 152 mmol), ammonium chloride (8.04 g, 150 mmol) and lithium chloride (1.93 g, 46 mmol) were added and the mixture was stirred for 20 h at 125° C. To the reaction mixture was added an additional portion of sodium azide (9.85 g, 152 mmol) and ammonium chloride (8.04 g, 150 mmol) and the reaction mixture was stirred for an additional 24 h at 125° C. The cooled reaction mixture was poured on to water (500 ml). The suspension was stirred for 0.5 h, and the precipitate was filtered off and washed with water (3×200 ml) and dried in vacuo at 50° C. The dried crude product was suspended in diethyl ether (500 ml) and stirred for 2 h, filtered off and washed with diethyl ether (2×200 ml) and dried in vacuo at 50° C. affording 5.79 g (82%) of the title compound as a solid.
1H-NMR (DMSO-d6): 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.
The following commercially available tetrazoles do all bind to the His B10 Zn2+ site of the insulin hexamer:
Example 402
5-(3-Tolyl)-1H-tetrazole




Example 403
5-(2-Bromophenyl)tetrazole




Example 404
5-(4-Ethoxalylamino-3-nitrophenyl)tetrazole




Example 405




Example 406




Example 407




Example 408




Example 409
Tetrazole




Example 410
5-Methyltetrazole




Example 411
5-Benzyl-2H-tetrazole




Example 412
4-(2H-Tetrazol-5-yl)benzoic acid




Example 413
5-Phenyl-2H-tetrazole




Example 414
5-(4-Chlorophenylsulfanylmethyl)-2H-tetrazole




Example 415
5-(3-Benzyloxyphenyl)-2H-tetrazole




Example 416
2-Phenyl-6-(1H-tetrazol-5-yl)-chromen-4-one




Example 417




Example 418




Example 419




Example 420




Example 421




Example 422
5-(4-Bromo-phenyl)-1H-tetrazole




Example 423




Example 424




Example 425




Example 426




Example 427




Example 428




Example 429




Example 430




Example 431




Example 432




Example 433




Example 434




Example 435




Example 436




Example 437




Example 438




Example 439




Example 440




Example 441




Example 442




Example 443




Example 444




Example 445




Example 446




Example 447




Example 448




Example 449




Example 450




Example 451




Example 452




General Procedure (H) for Preparation of Compounds of General Formula I7:





wherein A1, AR1, and AR2 are as defined above.

The reaction is generally known as a reductive alkylation reaction and is generally performed by stirring an aldehyde with an amine at low pH (by addition of an acid, such as acetic acid or formic acid) in a solvent such as THF, DMF, NMP, methanol, ethanol, DMSO, dichloromethane, 1,2-dichloroethane, trimethyl orthoformate, triethyl orthoformate, or a mixture of two or more of these. As reducing agent sodium cyano borohydride or sodium triacetoxy borohydride may be used. The reaction is performed between 20° C. and 120° C., preferably at room temperature.
When the reductive alkylation is complete, the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art.
The general procedure (H) is further illustrated in the following example 453:
Example 453
General Procedure (H)
Biphenyl-4-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine




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.
HPLC-MS (Method C): m/z: 328 (M+1), 350 (M+23); Rt=4.09 min.
Example 454
General Procedure (H)
Benzyl-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 252 (M+1); Rt=3.74 min.
Example 455
General Procedure (H)
(4-Methoxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 282.2 (M+1); Rt=3.57 min.
Example 456
General Procedure (H)
4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenol




HPLC-MS (Method D): m/z: 268.4 (M+1); Rt=2.64 min.
Example 457
General Procedure (H)
(4-Nitrobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 297.4 (M+1); Rt=3.94 min.
Example 458
General Procedure (H)
(4-Chlorobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 287.2 (M+1); Rt=4.30 min.
Example 459
General Procedure (H)
(2-Chlorobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 286 (M+1); Rt=4.40 min.
Example 460
General Procedure (H)
(4-Bromobenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 332 (M+1); Rt=4.50 min.
Example 461
General Procedure (H)
(3-Benzyloxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.94 min.
Example 462
General Procedure (H)
Naphthalen-1-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.
Example 463
General Procedure (H)
Naphthalen-2-ylmethyl-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.60 min.
Example 464
General Procedure (H)
4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid




HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.24 min.
Example 465
General Procedure (H)
[3-(2H-Tetrazol-5-yl)-phenyl]-[3-(3-trifluoromethyl-phenoxy)benzyl]amine




HPLC-MS (Method D): m/z: 412 (M+1); Rt=5.54 min.
Example 466
General Procedure (H)
(3-Phenoxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.04 min.
Example 467
General Procedure (H)
(4-Phenoxy-benzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.00 min.
Example 468
General Procedure (H)
(4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenoxy)acetic acid




HPLC-MS (Method D): m/z: 326 (M+1); Rt=3.10 min.
Example 469
General Procedure (H)
(4-Benzyloxybenzyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.97 min.
Example 470
General Procedure (H)
3-(4-{[3-(2H-Tetrazol-5-yl)phenylamino]methyl}phenyl)acrylic acid




HPLC-MS (Method D): m/z: 322 (M+1); Rt=3.60 min.
Example 471
General Procedure (H)
Dimethyl-(4-{[3-(2H-tetrazol-5-yl)phenylamino]methyl}naphthalen-1-yl)amine




HPLC-MS (Method D): m/z: 345 (M+1); Rt=3.07 min.
Example 472
General Procedure (H)
(4′-Methoxybiphenyl-4-ylmethyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.97 min.
Example 473
General Procedure (H)
(2′-Chlorobiphenyl-4-ylmethyl)-[3-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 362 (M+1); Rt=5.27 min.
Example 474
General Procedure (H)
Benzyl-[4-(2H-tetrazol-5-yl)phenyl]amine




For preparation of starting material, see example 590.
HPLC-MS (Method D): m/z: 252 (M+1); Rt=3.97 min.
Example 475
General Procedure (H)
(4-Methoxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 282 (M+1); Rt=3.94 min.
Example 476
General Procedure (H)
4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenol




HPLC-MS (Method D): m/z: 268 (M+1); Rt=3.14 min.
Example 477
General Procedure (H)
(4-Nitrobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: (M+1); Rt=3.94 min.
Example 478
General Procedure (H)
(4-Chlorobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: (M+1); Rt=4.47 min.
Example 479
General Procedure (H)
(2-Chlorobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 286 (M+1); Rt=4.37 min.
Example 480
General Procedure (H)
(4-Bromobenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 331 (M+1); Rt=4.57 min.
Example 481
General Procedure (H)
(3-Benzyloxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.07 min.
Example 482
General Procedure (H)
Naphthalen-1-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.
Example 483
General Procedure (H)
Naphthalen-2-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 302 (M+1); Rt=4.70 min.
Example 484
General Procedure (H)
Biphenyl-4-ylmethyl-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 328 (M+1); Rt=5.07 min.
Example 485
General Procedure (H)
4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid




HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.34 min.
Example 486
General Procedure (H)
[4-(2H-Tetrazol-5-yl)phenyl]-[3-(3-trifluoromethylphenoxy)benzyl]amine




HPLC-MS (Method D): m/z: 412 (M+1); Rt=5.54 min.
Example 487
General Procedure (H)
(3-Phenoxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.07 min.
Example 488
General Procedure (H)
(4-Phenoxybenzyl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine




HPLC-MS (Method D): m/z: 344 (M+1); Rt=5.03 min.
Example 489
General Procedure (H)
3-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid




HPLC-MS (Method D): m/z: 286 (M+1); Rt=3.47 min.
Example 490
General Procedure (H)
(4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenoxy)acetic acid




HPLC-MS (Method D): m/z: 326 (M+1); Rt=3.40 min.
Example 491
General Procedure (H)
(4-Benzyloxybenzyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.14 min.
Example 492
General Procedure (H)
3-(4-{[4-(2H-Tetrazol-5-yl)phenylamino]methyl}phenyl)acrylic acid




HPLC-MS (Method D): m/z: 322 (M+1); Rt=3.66 min.
Example 493
General Procedure (H)
Dimethyl-(4-{[4-(2H-tetrazol-5-yl)phenylamino]methyl}naphthalen-1-yl)amine




HPLC-MS (Method D): m/z: 345 (M+1); Rt=3.10 min.
Example 494
General Procedure (H)
(4′-Methoxybiphenyl-4-ylmethyl)-[4-(2H-tetrazol-5-yl)phenyl]amine




HPLC-MS (Method D): m/z: 358 (M+1); Rt=5.04 min.
Example 495
General Procedure (H)
(2′-Chlorobiphenyl-4-ylmethyl)-[4-(2H-tetrazol-5-yl)-phenyl]-amine




HPLC-MS (Method D): m/z: 362 (M+1); Rt=5.30 min.
General Procedure (I) for Preparation of Compounds of General Formula I8:




wherein A1, AR1, and AR2 are as defined above.

This procedure is very similar to general procedure (A), the only difference being the carboxylic acid is containing a tetrazole moiety. When the acylation is complete, the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art.
The general procedure (I) is further illustrated in the following example 496:
Example 496
General Procedure (I)
4-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic acid




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 aliquot of this HOAt-ester solution (0.45 mL) was mixed with 0.25 mL of a solution of 4-aminobenzoic acid (1.2 mmol in 1 mL DMF). (Anilines as hydrochlorides can also be utilised, a slight excess of triethylamine was added to the hydrochloride suspension in DMF prior to mixing with the HOAt-ester.) The resulting mixture was shaken for 3 days at room temperature. 1N hydrochloric acid (2 mL) was added and the mixture was shaken for 16 hours at room temperature. The solid was isolated by centrifugation (alternatively by filtration or extraction) and was washed with water (3 mL). Drying in vacuo at 40° C. for 2 days afforded the title compound.
HPLC-MS (Method D): m/z: 310 (M+1); Rt=2.83 min.
Example 497
General Procedure (I)
3-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic acid




HPLC-MS (Method D): m/z: 310 (M+1); Rt=2.89 min.
Example 498
General Procedure (I)
3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}acrylic acid




HPLC-MS (Method D): m/z: 336 (M+1); Rt=3.10 min.
Example 499
General Procedure (I)
3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}propionic acid




HPLC-MS (Method D): m/z: 338 (M+1); Rt=2.97 min.
Example 500
General Procedure (I)
3-Methoxy-4-[4-(2H-tetrazol-5-yl)benzoylamino]benzoic acid




HPLC-MS (Method D): m/z: 340 (M+1); Rt=3.03 min.
Example 501
General Procedure (I)
N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-yl)benzamide




HPLC-MS (Method D): m/z: 372 (M+1); Rt=4.47 min.
Example 502
General Procedure (I)
N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-yl)benzamide




HPLC-MS (Method D): m/z: 358 (M+1); Rt=4.50 min.
Example 503
General Procedure (I)
N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-yl)benzamide




HPLC-MS (Method D): m/z: 354 (M+1); Rt=4.60 min.
Example 504
General Procedure (I)
N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-yl)benzamide




HPLC-MS (Method D): m/z: 383 (M+1); Rt=4.60 min.
Example 505
General Procedure (I)
N-Phenyl-4-(2H-tetrazol-5-yl)benzamide




HPLC-MS (Method D): m/z: 266 (M+1); Rt=3.23 min.
Example 506
General Procedure (I)
4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic acid




The starting material was prepared as described in example 399.
HPLC-MS (Method D): m/z: 340 (M+1); Rt=2.83 min.
Example 507
General Procedure (I)
3-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic acid




HPLC-MS (Method D): m/z: 340 (M+1); Rt=2.90 min.
Example 508
General Procedure (I)
3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}acrylic acid




HPLC-MS (Method D): m/z: 366 (M+1); Rt=3.07 min.
Example 509
General Procedure (I)
3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}propionic acid




HPLC-MS (Method D): m/z: 368 (M+1); Rt=2.97 min.
Example 510
General Procedure (I)
3-Methoxy-4-[4-(2H-tetrazol-5-ylmethoxy)benzoylamino]benzoic acid




HPLC-MS (Method D): m/z: 370 (M+1); Rt=3.07 min.
Example 511
General Procedure (I)
N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)benzamide




HPLC-MS (Method D): m/z: 402 (M+1); Rt=4.43 min.
Example 512
General Procedure (I)
N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)benzamide




HPLC-MS (Method D): m/z: 388 (M+1); Rt=4.50 min.
Example 513
General Procedure (I)
N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide




HPLC-MS (Method D): m/z: 384 (M+1); Rt=4.57 min.
Example 514
General Procedure (I)
N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide




HPLC-MS (Method D): m/z: 413 (M+1); Rt=4.57 min.
Example 515
General Procedure (I)
N-Phenyl-4-(2H-tetrazol-5-ylmethoxy)benzamide




HPLC-MS (Method D): m/z: 296 (M+1); Rt=3.23 min.
Example 516
General Procedure (I)
4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid




The starting material was prepared as described in example 400.
HPLC-MS (Method D): m/z: 356 (M+1); Rt=2.93 min.
Example 517
General Procedure (I)
3-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid




HPLC-MS (Method D): m/z: 356 (M+1); Rt=3.00 min.
Example 518
General Procedure (I)
3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}acrylic acid




HPLC-MS (Method D): m/z: 382 (M+1); Rt=3.26 min.
Example 519
General Procedure (I)
3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}propionic acid




HPLC-MS (Method D): m/z: 384 (M+1); Rt=3.10 min.
Example 520
General Procedure (I)
3-Methoxy-4-[4-(2H-tetrazol-5-ylmethylsulfanyl)benzoylamino]benzoic acid




HPLC-MS (Method D): m/z: 386 (M+1); Rt=3.20 min.
Example 521
General Procedure (I)
N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide




HPLC-MS (Method D): m/z: 418 (M+1); Rt=4.57 min.
Example 522
General Procedure (I)
N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide




HPLC-MS (Method D): m/z: 404 (M+1); Rt=4.60 min.
Example 523
General Procedure (I)
N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide




HPLC-MS (Method D): m/z: 400 (M+1); Rt=4.67 min.
Example 524
General Procedure (I)
N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide




HPLC-MS (Method D): m/z: 429 (M+1); Rt=4.67 min.
Example 525
General Procedure (I)
N-Phenyl-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide




HPLC-MS (Method D): m/z: 312 (M+1); Rt=3.40 min.
General Procedure (J) for Solution Phase Preparation of Amides of General Formula I9:




wherein AR2 is as defined above.

This general procedure (J) is further illustrated in the following example.
Example 526
General Procedure (J)
9-(3-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




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.
1H-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.
3-[2-(Triphenylmethyl)-2H-tetrazol-5-yl]-9H-carbazole (200 mg, 0.42 mmol) was dissolved in methyl sulfoxide (1.5 mL). Sodium hydride (34 mg, 60%, 0.85 mmol) was added, and the resulting suspension was stirred for 30 min at room temperature. 3-Chlorobenzyl chloride (85 μL, 108 mg, 0.67 mmol) was added, and the stirring was continued at 40° C. for 18 hours. The reaction mixture was cooled to ambient temperature and poured into 0.1 N hydrochloric acid (aq.) (15 mL) The precipitated solid was filtered off and washed with water (3×10 mL) to furnish 9-(3-chlorobenzyl)-3-[2-(triphenylmethyl)-2H-tetrazol-5-yl]-9H-carbazole, which was dissolved in a mixture of tetrahydrofuran and 6 N hydrochloric acid (aq.) (9:1) (10 mL) and stirred at room temperature for 18 hours. The reaction mixture was poured into water (100 mL). The solid was filtered off and rinsed with water (3×10 mL) and dichloromethane (3×10 mL) to yield the title compound (127 mg). No further purification was necessary.
1H-NMR (DMSO-d6): 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), 7.36-7.27 (4H, m), 7.08 (1H, bt), 5.78 (2H, s); HPLC-MS (Method B): m/z: 360 (M+1); Rt=5.07 min.
The compounds in the following examples were prepared in a similar fashion. Optionally, the compounds can be further purified by recrystallization from e.g. aqueous sodium hydroxide (1 N) or by chromatography.
Example 527
General Procedure (J)
9-(4-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 360 (M+1); Rt=4.31 min.
Example 528
General Procedure (J)
9-(4-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.26 min.
Example 529
General Procedure (J)
3-(2H-Tetrazol-5-yl)-9-(4-trifluoromethylbenzyl)-9H-carbazole




HPLC-MS (Method C): m/z: 394 (M+1); Rt=4.40 min.
Example 530
General Procedure (J)
9-(4-Benzyloxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 432 (M+1); Rt=4.70 min.
Example 531
General Procedure (J)
9-(3-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 340 (M+1); Rt=4.25 min.
Example 532
General Procedure (J)
9-Benzyl-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-NMR (DMSO-d6): δ 8.91 (1H, dd), 8.30 (1H, d), 8.13 (1H, dd), 7.90 (1H, d), 7.73 (1H, d), 7.53 (1H, t), 7.36-7.20 (6H, m), 5.77 (2H, s).
Example 533
General Procedure (J)
9-(4-Phenylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(3-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 356 (M+1); Rt=3.99 min.
Example 535
General Procedure (J)
9-(Naphthalen-2-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 376 (M+1); Rt=4.48 min.
Example 536
General Procedure (J)
9-(3-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 404 (M+1); Rt=4.33 min.
Example 537
General Procedure (J)
9-(Biphenyl-2-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 402 (M+1); Rt=480 min.
Example 538
General Procedure (J)
3-(2H-Tetrazol-5-yl)-9-[4-(1,2,3-thiadiazol-4-yl)benzyl]-9H-carbazole




Example 539
General Procedure (J)
9-(2′-Cyanobiphenyl-4-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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 (1H, d), 7.75 (1H, dt), 7.60-7.47 (5H, m), 7.38-7.28 (3H, m), 5.86 (2H, s); HPLC-MS (Method C): m/z: 427 (M+1); Rt=4.38 min.
Example 540
General Procedure (J)
9-(4-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 452 (M+1); Rt=4.37 min.
Example 541
General Procedure (J)
9-(3,5-Bis(trifluoromethyl)benzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 462 (M+1); Rt=4.70 min.
Example 542
General Procedure (J)
9-(4-Bromobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(Anthracen-9-ylmethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 426 (M+1); Rt=4.78 min.
Example 544
General Procedure (J)
9-(4-Carboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




3.6 fold excess sodium hydride was used.
1H-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)
9-(2-Chlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method B): m/z: 360 (M+1); Rt=5.30 min.
Example 546
General Procedure (J)
9-(4-Fluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(3-Fluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(2-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 452 (M+1); Rt=4.58 min.
Example 549
General Procedure (J)
9-(3-Carboxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




3.6 fold excess sodium hydride was used.
1H-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)
9-[4-(2-Propyl)benzyl]-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(3,5-Dimethoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 386 (M+1); Rt=4.03 min.
Example 552
General Procedure (J)
3-(2H-Tetrazol-5-yl)-9-(2,4,5-trifluorobenzyl)-9H-carbazole




HPLC-MS (Method B): m/z: 380 (M+1); Rt=5.00 min.
Example 553
General Procedure (J)
N-Methyl-N-phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide




HPLC-MS (Method B): m/z: 383 (M+1); Rt=4.30 min.
Example 554
General Procedure (J)
9-(4-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(2-Methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-NMR (DMSO-d6): 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)
9-(4-Cyanobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.74 min.
Example 557
General Procedure (J)
9-(3-Cyanobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 351 (M+1); Rt=3.73 min.
Example 558
General Procedure (J)
9-(5-Chloro-2-methoxybenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
N-Phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide




1H-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)
N-Butyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide




1H-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)
9-(2,4-Dichlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(2-Methylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(3-Nitrobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 371 (M+1); Rt=3.78 min.
Example 564
General Procedure (J)
9-(3,4-Dichlorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method B): m/z: 394 (M+1); Rt=5.62 min.
Example 565
General Procedure (J)
9-(2,4-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(3,5-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(3,4-Difluorobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




1H-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)
9-(3-Iodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method B): m/z: 452 (M+1); Rt=5.50 min.
Example 569
General Procedure (J)
3-(2H-Tetrazol-5-yl)-9-[3-(trifluoromethyl)benzyl]-9H-carbazole




1H-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)
N-(4-Carboxyphenyl)-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide




3.6 fold excess sodium hydride was used.
HPLC-MS (Method B): m/z: 413 (M+1); Rt=3.92 min.
Example 571
General Procedure (J)
N-(2-Propyl)-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide




HPLC-MS (Method B): m/z: 335 (M+1); Rt=3.70 min.
Example 572
General Procedure (J)
N-Benzyl-N-phenyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide




HPLC-MS (Method B): m/z: 459 (M+1); Rt=5.37 min.
Example 573
General Procedure (J)
N-[4-(2-Methyl-2-propyl)phenyl]-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide




HPLC-MS (Method B): m/z: 425 (M+1); Rt=5.35 min.
Example 574
General Procedure (J)
N-Phenethyl-2-[3-(2H-tetrazol-5-yl)carbazol-9-yl]acetamide




HPLC-MS (Method C): m/z: 397 (M+1); Rt=3.43 min.
Example 575
General Procedure (J)
3-(2H-Tetrazol-5-yl)-9-[2-(trifluoromethyl)benzyl]-9H-carbazole




HPLC-MS (Method C): m/z: 394 (M+1); Rt=4.44 min.
Example 576
General Procedure (J)
9-[2-Fluoro-6-(trifluoromethyl)benzyl]-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 412 (M+1); Rt=4.21 min.
Example 577
General Procedure (J)
9-[2,4-Bis(trifluoromethyl)benzyl)]-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 462 (M+1); Rt=4.82 min.
Example 578
General Procedure (J)
3-(2H-Tetrazol-5-yl)-9-(2,4,6-trimethylbenzyl)-9H-carbazole




HPLC-MS (Method C): m/z: 368 (M+1); Rt=4.59 min.
Example 579
General Procedure (J)
9-(2,3,5,6-Tetramethylbenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 382 (M+1); Rt=4.47 min.
Example 580
General Procedure (J)
9-[(Naphthalen-1-yl)methyl]-3-(2H-tetrazol-5-yl)-9H-carbazole




HPLC-MS (Method C): m/z: 376 (M+1); Rt=4.43 min.
Further preferred compounds of the invention that may be prepared according to general procedure (J) includes:







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).




General Procedure (K) for Preparation of Compounds of General Formula I10:





wherein AR2 is as defined above.

The general procedure (K) is further illustrated by the following example:
Example 581
General Procedure (K)
1-Benzyl-5-(2H-tetrazol-5-yl)-1H-indole




5-Cyanoindole (1.0 g, 7.0 mmol) was dissolved in N,N-dimethylformamide (14 mL) and cooled in an ice-water bath. Sodium hydride (0.31 g, 60%, 7.8 mmol) was added, and the resulting suspension was stirred for 30 min. Benzyl chloride (0.85 mL, 0.94 g, 7.4 mmol) was added, and the cooling was discontinued. The stirring was continued for 65 hours at room temperature. Water (150 mL) was added, and the mixture was extracted with ethyl acetate (3×25 mL). The combined organic phases were washed with brine (30 mL) and dried with sodium sulfate (1 hour). Filtration and concentration yielded the crude material. Purification by flash chromatography on silica gel eluting with ethyl acetate/heptanes=1:3 afforded 1.60 g 1-benzyl-1H-indole-5-carbonitrile.
HPLC-MS (Method C): m/z: 233 (M+1); Rt=4.17 min.
1-Benzyl-1H-indole-5-carbonitrile was transformed into 1-benzyl-5-(2H-tetrazol-5-yl)-1H-indole by the method described in general procedure (J) and in example 401. Purification was done by flash chromatography on silica gel eluting with dichloromethane/methanol=9:1.
HPLC-MS (Method C): m/z: 276 (M+1); Rt=3.35 min.
The compounds in the following examples were prepared by the same procedure.
Example 582
General Procedure (K)
1-(4-Bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole




HPLC-MS (Method C): m/z: 354 (M+1); Rt=3.80 min.
Example 583
General Procedure (K)
1-(4-Phenylbenzyl)-5-(2H-tetrazol-5-yl)-1H-indole




1H-NMR (200 MHz, DMSO-d6): δ=5.52 (2H, s), 6.70 (1H, d), 7.3-7.45 (6H, m), 7.6 (4H, m), 7.7-7.8 (2H, m), 7.85 (1H, dd), 8.35 (1H, d).
Calculated for C22H17N5, H2O: 73.32% C; 5.03% H; 19.43% N. Found: 73.81% C; 4.90% H; 19.31% N.
Example 584
General Procedure (K)
5-(2H-Tetrazol-5-yl)-1H-indole




5-(2H-Tetrazol-5-yl)-1H-indole was prepared from 5-cyanoindole according to the method described in example 401.
HPLC-MS (Method C): m/z: 186 (M+1); Rt=1.68 min.
Example 585
General Procedure (K)
1-Benzyl-4-(2H-tetrazol-5-yl)-1H-indole




1-Benzyl-1H-indole-4-carbonitrile was prepared from 4-cyanoindole according to the method described in example 581.
HPLC-MS (Method C): m/z: 233 (M+1); Rt=4.24 min.
1-Benzyl-4-(2H-tetrazol-5-yl)-1H-indole was prepared from 1-benzyl-1H-indole-4-carbonitrile according to the method described in example 401.
HPLC-MS (Method C): m/z: 276 (M+1); Rt=3.44 min.
Further preferred compounds of the invention that may be prepared according to general procedure (K) includes:










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).




General Procedure (L) for Preparation of Compounds of General Formula I11:





The general procedure (L) is further illustrated by the following example:
Example 586
General Procedure (L)
1-Benzoyl-5-(2H-tetrazol-5-yl)-1H-indole




To a solution of 5-cyanoindole (1.0 g, 7.0 mmol) in dichloromethane (8 mL) was added 4-(dimethylamino)pyridine (0.171 g, 1.4 mmol), triethylamine (1.96 mL, 1.42 g, 14 mmol) and benzoyl chloride (0.89 mL, 1.08 g, 7.7 mmol). The resulting mixture was stirred for 18 hours at room temperature. The mixture was diluted with dichloromethane (80 mL) and washed consecutively with a saturated solution of sodium hydrogencarbonate (40 mL) and brine (40 mL). The organic phase was dried with magnesium sulfate (1 hour). Filtration and concentration furnished the crude material which was purified by flash chromatography on silica gel, eluting with ethyl acetate/heptanes=2:3. 1-Benzoyl-1H-indole-5-carbonitrile was obtained as a solid.
HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.07 min.
1-Benzoyl-1H-indole-5-carbonitrile was transformed into 1-benzoyl-5-(2H-tetrazol-5-yl)-1H-indole by the method described in example 401.
HPLC (Method C): Rt=1.68 min.
The compound in the following example was prepared by the same procedure.
Example 587
General Procedure (L)
1-Benzoyl-4-(2H-tetrazol-5-yl)-1H-indole




1-Benzoyl-1H-indole-4-carbonitrile was prepared from 4-cyanoindole according to the method described in example 586.
HPLC-MS (Method C): m/z: 247 (M+1); Rt=4.24 min.
1-Benzoyl-4-(2H-tetrazol-5-yl)-1H-indole was prepared from 1-benzoyl-1H-indole-4-carbonitrile according to the method described in example 401.
HPLC (Method C); Rt=1.56 min.
The following known and commercially available compounds do all bind to the His B10 Zn2+ site of the insulin hexamer:
Example 588
1-(4-Fluorophenyl)-5-(2H-tetrazol-5-yl)-1H-indole




Example 589
1-Amino-3-(2H-tetrazol-5-yl)benzene




Example 590
1-Amino-4-(2H-tetrazol-5-yl)benzene




A mixture of 4-aminobenzonitrile (10 g, 84.6 mmol), sodium azide (16.5 g, 254 mmol) and ammonium chloride (13.6 g, 254 mmol) in DMF was heated at 125° C. for 16 hours. The cooled mixture was filtered and the filtrate was concentrated in vacuo. The residue was added water (200 mL) and diethyl ether (200 mL) which resulted in crystallisation. The mixture was filtered and the solid was dried in vacuo at 40° C. for 16 hours to afford 5-(4-aminophenyl)-2H-tetrazole.
1H NMR DMSO-d6): δ=5.7 (3H, bs), 6.69 (2H, d), 7.69 (2H, d). HPLC-MS (Method C): m/z: 162 (M+1); Rt=0.55 min.
Example 591
1-Nitro-4-(2H-tetrazol-5-yl)benzene




Example 592
1-Bromo-4-(2H-tetrazol-5-yl)benzene




General Procedure (M) for Solution Phase Preparation of Amides of General Formula I12:





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.

A-B1—B2—CO2H may be prepared eg by general procedure (D) or by other similar procedures described herein, or may be commercially available.
The procedure is further illustrated in the following example 593:
Example 593
General Procedure (M)
N-(4-Chlorobenzyl)-2-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)-1H-indol-1-yl]acetamide




[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.
HPLC-MS (Method C): m/z: 426 (M+1); Rt.=3.79 min.
Example 594
General Procedure (M)
1H-Benzotriazole-5-carboxylic acid 4-chlorobenzylamide




HPLC-MS (Method B): m/z: 287 (M+1); Rt=4.40 min.
Example 595
General Procedure (M)
N-(4-Chlorobenzyl)-4-[2-chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide




HPLC-MS (Method A): m/z: 465 (M+1); Rt=4.35 min.
Example 596
General Procedure (M)
N-(4-Chlorobenzyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide




HPLC-MS (Method A): m/z: 431 (M+1); Rt=3.68 min.
Example 597
General Procedure (M)
2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]-N-(4-chlorobenzyl)acetamide




HPLC-MS (Method A): m/z: 483 (M+1); Rt=4.06 min.
Example 598
General Procedure (M)
N-(4-Chlorobenzyl)-2-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetamide




HPLC-MS (Method A): m/z: 403 (M+1); Rt=4.03 min.
Example 599
General Procedure (M)
N-(4-Chlorobenzyl)-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]acrylamide




HPLC-MS (Method A): m/z: 399 (M+1); Rt=3.82.
Example 600
General Procedure (M)
N-(4-Chlorobenzyl)-4-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyramide




HPLC-MS (Method A): m/z: 431 (M+1); Rt=3.84 min.
Example 601
General Procedure (M)
4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]-N-(4-chlorobenzyl)butyramide




HPLC-MS (Method A): m/z: 511 (M+1)); Rt=4.05 min.
Example 602
General Procedure (M)
4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-phenoxy]-N-(4-chlorobenzyl)-butyramide




HPLC-MS (Method A): m/z: 527 (M+1); Rt=4.77 min.
Example 603
General Procedure (M)
N-(4-Chlorobenzyl)-2-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetamide




HPLC-MS (Method C): m/z: 431 (M+1); Rt.=4.03 min.
Example 604
General Procedure (M)
N-(4-Chlorobenzyl)-3-[3-(2,4-dioxothiazolidin-5-ylidenemethyl)-1H-indol-1-yl]propionamide




HPLC-MS (Method C): m/z: 440 (M+1); Rt.=3.57 min.
Example 605
General Procedure (M)
N-(4-Chlorobenzyl)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyramide




HPLC-MS (Method C): m/z: 481 (M+1); Rt=4.08 min.
Example 606
General Procedure (M)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-N-hexylbutyramide




HPLC-MS (Method C): m/z: 441 (M+1); Rt=4.31 min.
Example 607
General Procedure (M)
N-(4-Chlorobenzyl)-4-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzamide




HPLC-MS (Method C): m/z: 493 (M+1); Rt=4.19 min.
Example 608
General Procedure (M)
N-(4-Chlorobenzyl)-3-[3-(2H-tetrazol-5-yl)carbazol-9-ylmethyl]benzamide




HPLC-MS (Method C): m/z: 493 (M+1); Rt=4.20 min.
Example 609
4-({[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carbonyl]amino}methyl)benzoic acid Methyl Ester




HPLC-MS (Method C): m/z: 436 (M+1); Rt.=3.55 min.
The commercially available compounds in the following examples do all bind to the HisB10 Zn2+ site:
Example 610
1-(4-Bromo-3-methylphenyl)-1,4-dihydrotetrazole-5-thione




Example 611
1-(4-Iodophenyl)-1,4-dihydrotetrazole-5-thione




Example 612
1-(2,4,5-Trichlorophenyl)-1H-tetrazole-5-thiol




Example 613
1-(2,6-Dimethylphenyl)-1,4-dihydrotetrazole-5-thione




Example 614
1-(2,4,6-Trimethylphenyl)-1,4-dihydrotetrazole-5-thione




Example 615
1-(4-Dimethylaminophenyl)-1H-tetrazole-5-thiol




Example 616
1-(3,4-Dichlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione




Example 617
1-(4-Propylphenyl)-1,4-dihydro-1H-tetrazole-5-thione




Example 618
1-(3-Chlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione




Example 619
1-(2-Fluorophenyl)-1,4-dihydro-1H-tetrazole-5-thione




Example 620
1-(2,4-Dichlorophenyl)-1,4-dihydro-1H-tetrazole-5-thione




Example 621
1-(4-Trifluoromethoxyphenyl)-1,4-dihydro-1H-tetrazole-5-thione




Example 622
N-[4-(5-Mercaptotetrazol-1-yl)-phenyl]-acetamide




Example 623
1-(4-Chlorophenyl)-1,4-dihydrotetrazole-5-thione




Example 624
1-(4-Methoxyphenyl)-1,4-dihydrotetrazole-5-thione




Example 625
1-(3-Fluoro-4-pyrrolidin-1-ylphenyl)-1,4-dihydrotetrazole-5-thione




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
1-Aryl-1,4-dihydrotetrazole-5-thiones with a carboxylic acid tethered to the aryl group may be prepared as shown in the following scheme:




Step 1 is a phenol alkylation and is very similar to steps 1 and 2 of general procedure (D) and may also be prepared similarly as described in example 303.
Step 2 is a reduction of the nitro group. SnCl2, H2 over Pd/C and many other procedures known to those skilled in the art may be utilised.
Step 3 is formation of an arylisothiocyanate from the corresponding aniline. As reagents CS2, CSCl2, or other reagents known to those skilled in the art, may be utilised.
Step 4 is a conversion to mercaptotetrazole as described above.
Preferred compounds of the invention includes:




Example 626
4-(4-Hydroxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile




Phenylsulphonyl acetonitrile (2.0 g, 11.04 mmol) was mixed with 4-hydroxybenzaldehyde (1.35 g, 11.04 mmol) in DMF (10 mL) and toluene (20 mL). The mixture was refluxed for 3 hours and subsequently evaporated to dryness in vacuo. The residue was treated with diethyl ether and toluene. The solid formed was filtered to afford 2.08 g (66%) of 2-benzenesulfonyl-3-(4-hydroxyphenyl)acrylonitrile.
HPLC-MS (Method C): m/z: 286 (M+1); Rt.=3.56 min.
A mixture of 2-benzenesulfonyl-3-(4-hydroxyphenyl)acrylonitrile (2.08 g, 7.3 mmol) and sodium azide (0.47 g, 7.3 mmol) in DMF (50 mL) was heated at reflux temperature 2 hours. After cooling, the mixture was poured on ice. The mixture was evaporated in vacuo to almost dryness and toluene was added. After filtration, the organic phase was evaporated in vacuo. The residue was purified by silicagel chromatography eluting with a mixture of ethyl acetate and heptane (1:2). This afforded 1.2 g (76%) of the title compound.
1H NMR (DMSO-d6): 10.2 (broad, 1H); 7.74 (d, 2H); 6.99 (d, 2H); 3.6-3.2 (broad, 1H). HPLC-MS (Method C) m/z:=187 (M+1); Rt.=1.93 min
The compounds in the following examples are commercially available and may be prepared using a similar methodology:
Example 627
4-(4-Trifluoromethoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile




Example 628
4-Benzo[1,3]dioxol-5-yl-1H-[1,2,3]triazole-5-carbonitrile




Example 629
4-(3-Trifluoromethylphenyl)-1H-[1,2,3]triazole-5-carbonitrile




Example 630
4-Pyridin-3-yl-1H-[1,2,3]triazole-5-carbonitrile




Example 631
4-(2,6-Dichlorophenyl)-1H-[1,2,3]triazole-5-carbonitrile




Example 632
4-Thiophen-2-yl-1H-[1,2,3]triazole-5-carbonitrile




Example 633
3,5-Dimethylisoxazole-4-carboxylic acid 4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester




Example 634
3,3-Dimethyl-butyric acid 4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester




Example 635
4-Methyl-[1,2,3]thiadiazole-5-carboxylic acid 4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester




Example 636
4-Chlorobenzoic acid 4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl Ester




Example 637
4-(3-Phenoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile




Example 638
4-(5-Bromo-2-methoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile




Example 639
4-(2-Chloro-6-fluorophenyl)-1H-[1,2,3]triazole-5-carbonitrile




The following cyanotriazoles are also preferred compounds of the invention:

    4-(2-Chloro-6-fluorophenyl)-1H-[1,2,3]triazole-5-carbonitrile.
    Terephthalic acid mono[4-(5-cyano-1H-[1,2,3]triazol-4-yl)phenyl] ester.
    N-[4-(5-cyano-1H-[1,2,3]triazol-4-yl)-phenyl]terephthalamic acid
    4-(4-Octyloxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile
    4-(Styrylphenyl))-1H-[1,2,3]triazole-5-carbonitrile.
    4-(4′-Trifluoromethylbiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.
    4-(4′-Chlorobiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.
    4-(4′-Methoxybiphenyl-4-yl)-1H-[1,2,3]triazole-5-carbonitrile.
    4-(1-Naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.
    4-(9-Anthranyl)-1H-[1,2,3]triazole-5-carbonitrile.
    4-(4-Methoxy-1-naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.
    4-(4-Aminophenyl)-1H-[1,2,3]triazole-5-carbonitrile.
    4-(2-Naphthyl)-1H-[1,2,3]triazole-5-carbonitrile.

General Procedure (N) for Preparation of Compounds of General Formula I13:







wherein

    
    
        n is 1 or 3-20,
        AR1 is as defined above,
        R′′ is a standard carboxylic acid protecting group, such as C1-C6-alkyl or benzyl and Lea is a leaving group, such as chloro, bromo, iodo, methanesulfonyloxy, toluenesulfonyloxy or the like.
    
    


This procedure is very similar to general procedure (D), steps 1 and 2 are identical.
Steps 3 and 4 are described in the literature (eg Beck & Gûnther, Chem. Ber., 106, 2758-66 (1973))
Step 3 is a Knoevenagel condensation of the aldehyde obtained in step 2 with phenylsulfonylacetonitrile and step 4 is a reaction of the vinylsulfonyl compound obtained in step 3 with sodium azide. This reaction is usually performed in DMF at 90-110° C.
The following compounds may be prepared according to this general procedure (N):

    4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyric acid:






    2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetic acid:






    4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyric acid ethyl ester
    5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoic acid
    8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoic acid
    10-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)decanoic acid
    12-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)dodecanoic acid

General Procedure (O) for Preparation of Compounds of General Formula I7:







(SEQ ID NOS: 20-23 and 23, respectively, in order or appearance)
wherein PS is polymeric support, a Tentagel S RAM resin, n is 1-20, m is 0-5, and p is 0 or 1.
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:
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.
Step wise coupling of Fmoc-Arginine (Fmoc-Arg(Pmc)-OH), Fmoc-Glycine (Fmoc-Gly-OH) and Fmoc-4-aminobenzoic acid (Fmoc-4-Abz-OH):
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 Fmoc-4-aminobenzoic acid (Fluka and Neosystems) were coupled using the same procedure as described for Fmoc-Arg(Pmc)-OH.
Coupling of A-OH, e.g. 1H-benzotriazole-5-carboxylic acid on Gly.
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.
Coupling of A-OH, e.g. 1H-benzotriazole-5-carboxylic acid on Abz or 4-Apac:
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.
Introduction of Fragment 4-Apac Instead of 4-Abz:
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).
Cleavage of the Peptides from the Resin.
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.
RP-HPLC analysis and purification: The crude products were analysed on RP-HPLC C18 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)
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).












TABLE 1







Time (min.)
Flow (ml/min) (
% A
% B





















0
1.00
95.0
5.0



30.00
1.00
80.0
20.0



35.00
1.00
0.0
100.0



40.00
1.00
0.0
100.0



45.00
1.00
95.0
5.0






















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










The following examples were prepared using this general procedure (O).
Example 640
General Procedure (O)
Benzotriazol-5-ylcarbonyl-Gly2-Arg3-NH2 (BT-G2R3) (SEQ ID NO: 6)




MS (MALDI): m/z: 746.7 g/mol; calculated: 744.2 g/mol.
HPLC gradient:
















Time (min)
Flow (ml/min)
% A
% B





















0.00
6.00
90.0
10.0



120.00
6.00
90.0
10.0



121.00
0.10
90.0
10.0










Example 641
General Procedure (O)
Benzotriazol-5-ylcarbonyl-Gly2-Arg4-NH2 (BT-G2R4) (SEQ ID NO: 2)




MS (MALDI): m/z: 903.0 g/mol; calculated: 900.6 g/mol.
HPLC gradient:
















Time (min)
Flow (ml/min)
% A
% B





















0.00
6.00
95.0
5.0



30.00
6.00
80.0
20.0



35.00
6.00
0.0
100.0



40.00
6.00
0.0
100.0



45.00
6.00
95.0
5.0



64.00
6.00
95.0
5.0










Example 642
General Procedure (O)
Benzotriazol-5-ylcarbonyl-Gly2-Arg5-NH2 (BT-G2R5) (SEQ ID NO: 1)




MS (MALDI): m/z: 1060.8 g/mol; calculated: 1057 g/mol.
HPLC gradient
















Time (min)
Flow (ml/min)
% A
% B





















0.00
6.00
88.0
12.0



120.00
6.00
88.0
12.0



121.00
0.10
88.0
12.0










Example 643
General Procedure (O)
Benzotriazol-5-ylcarbonyl-Gly2-Arg6-NH2 (BT-G2R6) (SEQ ID NO: 5)




MS (MALDI): m/z: 1214.8 g/mol; calculated: 1213.4 g/mol.
HPLC gradient:
















Time (min)
Flow (ml/min)
% A
% B





















0.00
6.00
88.0
12.0



120.00
6.00
88.0
12.0



121.00
0.10
88.0
12.0










Example 644
General Procedure (O)
Benzotriazol-5-ylcarbonyl-4-Abz-Gly2-Arg5-NH2 (BT-4-Abz-G2R5) (SEQ ID NO: 10)




MS (MALDI): m/z: 1176.7 g/mol; calculated: 1177.9 g/mol.
HPLC gradient:
















Time (min)
Flow (ml/min)
% A
% B





















0.00
6.00
95.0
5.0



40.00
6.00
60.0
40.0



45.00
6.00
60.0
40.0



50.00
6.00
0.0
100.0



55.00
6.00
0.0
100.0



60.00
6.00
95.0
5.0










Example 645
General Procedure (O)
Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Arg5-NH2 (BT-4-Abz-GR5) (SEQ ID NO: 24)




MS (MALDI): m/z: 1122 g/mol; calculated: 1120.4 g/mol.
HPLC gradient:
















Time (min)
Flow (ml/min)
% A
% B





















0.00
6.00
95.0
5.0



40.00
6.00
60.0
40.0



45.00
6.00
60.0
40.0



50.00
6.00
0.0
100.0



55.00
6.00
0.0
100.0



60.00
6.00
95.0
5.0










Example 646
General Procedure (O)
Benzotriazol-5-ylcarbonyl-4-Abz-Arg5-NH2 (BT-4-Abz-R5) (SEQ ID NO: 13)




MS (MALDI): m/z: 1064.3 g/mol; calculated: 1063.2 g/mol.
HPLC gradient:
















Time (min)
Flow (ml/min)
% A
% B





















0.00
6.00
95.0
5.0



40.00
6.00
60.0
40.0



45.00
6.00
60.0
40.0



50.00
6.00
0.0
100.0



55.00
6.00
0.0
100.0



60.00
6.00
95.0
5.0










General Procedure (P) for Preparation of Compounds of General Formula I8:





(SEQ ID NOS: 20-22, 22 and 22, respectively, in order or appearance)
wherein X, Y, R10, E, B1, B2 are as defined above,
p is 0 or 1,
m is 0-5 and
n is 1-20.
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):




Example 647
General Procedure (P)
4-{2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetylamino}benzoyl-Gly2-Arg5-NH2 (SEQ ID NO: 25)




Example 648
General Procedure (P)
3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg5-NH2 (SEQ ID NO: 26)




MS (MALDI): m/z: 1057.3 g/mol; calculated: 1055.3 g/mol.
Example 649
General Procedure (P)
3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg4-NH2 (SEQ ID NO: 27)




MS (MALDI): m/z: 899.1 g/mol; calculated: 901.6 g/mol.
Example 650
General Procedure (P)
3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg3-NH2




MS (MALDI): m/z: 746.2 g/mol; calculated: 742.9 g/mol.
Example 651
General Procedure (P)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH2 (SEQ ID NO: 26)




MS (MALDI): m/z: 1088.7 g/mol; calculated: 1087 g/mol.
Example 652
General Procedure (P)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH2 (SEQ ID NO: 27)




MS (MALDI): m/z: 933.0 g/mol; calculated: 931 g/mol.
Example 653
General Procedure (P)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2




MS (MALDI): m/z: 776.9 g/mol; calculated: 774.0 g/mol.
Example 654
General Procedure (P)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethypnaphthalen-1-yloxy]butyryl-Arg12-NH2 (SEQ ID NO: 28)




MS (MALDI): m/z: 2232.9.4 g/mol; calculated: 2230.3 g/mol.
Example 655
General Procedure (P)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg8-NH2 (SEQ ID NO: 29)




MS (MALDI): m/z: 1607.4 g/mol; calculated: 1605.5 g/mol.
Example 656
General Procedure (P)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg5-NH2 (SEQ ID NO: 26)




MS (MALDI): m/z: 1141.9 g/mol; calculated: 1137.4 g/mol.
Example 657
General Procedure (P)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyDnaphthalen-1-yloxy]butyryl-Arg4-NH2 (SEQ ID NO: 27)




MS (MALDI): m/z: 985.4 g/mol; calculated: 981.2 g/mol.
Example 658
General Procedure (P)
4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg3-NH2




MS (MALDI): m/z: 828.5 g/mol; calculated: 825.0 g/mol.
The following compounds were prepared according to the methodology described in general procedure (O) and (P):
Example 659
4-(2H-Tetrazol-5-yl)benzoyl-4-Abz-Gly2-Arg5-NH2 (SEQ ID NO: 19)




MS (MALDI): m/z: 1203.8 g/mol; calculated: 1203.8 g/mol.
Example 660
4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg5-NH2 (SEQ ID NO: 26)




MS (MALDI): m/z: 1152.5 g/mol; calculated: 1149.3 g/mol.
Example 661
4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg8-NH2 (SEQ ID NO: 29)




MS (MALDI): m/z: 1621.0 g/mol; calculated: 1617.5 g/mol.
Example 662
4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg12-NH2 (SEQ ID NO: 28)




MS (MALDI): m/z: 2247.9 g/mol; calculated: 2242.3 g/mol.
Other preferred compounds of the invention that may be prepared according to general procedure (O) and/or general procedure (P) includes:












Building block from example 291:


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg10-NH2


(SEQ ID NO: 30)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg9-NH2


(SEQ ID NO: 31)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg8-NH2


(SEQ ID NO: 29)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg7-NH2


(SEQ ID NO: 32)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg11-NH2


(SEQ ID NO: 33)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg12-NH2


(SEQ ID NO: 28)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg13-NH2


(SEQ ID NO: 34)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg14-NH2


(SEQ ID NO: 35)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg15-NH2


(SEQ ID NO: 36)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg16-NH2


(SEQ ID NO: 37)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg17-NH2


(SEQ ID NO: 38)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg18-NH2


(SEQ ID NO: 39)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg19-NH2


(SEQ ID NO: 40)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg20-NH2


(SEQ ID NO: 41)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys6-NH2


(SEQ ID NO: 42)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys5-NH2


(SEQ ID NO: 43)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys4-NH2


(SEQ ID NO: 44)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys3-NH2


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys7-NH2


(SEQ ID NO: 45)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys8-NH2


(SEQ ID NO: 46)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys9-NH2


(SEQ ID NO: 47)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys10-NH2


(SEQ ID NO: 48)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys11-NH2


(SEQ ID NO: 49)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys12-NH2


(SEQ ID NO: 50)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys13-NH2


(SEQ ID NO: 51)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys14-NH2


(SEQ ID NO: 52)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys15-NH2


(SEQ ID NO: 53)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys16-NH2


(SEQ ID NO: 54)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys17-NH2


(SEQ ID NO: 55)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys18-NH2


(SEQ ID NO: 56)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys19-NH2


(SEQ ID NO: 57)


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Lys20-NH2


(SEQ ID NO: 58)


Building block from example 292:


5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg6-NH2


(SEQ ID NO: 59)


5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg5-NH2


(SEQ ID NO: 26)


5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg4-NH2


(SEQ ID NO: 27)


5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg3-NH2


Building block from page 164:


6-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg3-NH2


6-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg4-NH2


(SEQ ID NO: 27)


6-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]hexanoyl-Arg5-NH2


(SEQ ID NO: 26)


Building block from page 164:


7-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg3-NH2


7-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg4-NH2


(SEQ ID NO: 27)


7-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]heptanoyl-Arg5-NH2


(SEQ ID NO: 26)


Building block from page 164:


8-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg3-NH2


8-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg4-NH2


(SEQ ID NO: 27)


8-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]octanoyl-Arg5-NH2


(SEQ ID NO: 26)


Building block from page 164:


10-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg3-NH2


10-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg4-NH2


(SEQ ID NO: 27)


10-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]decanoyl-Arg5-NH2


(SEQ ID NO: 26)


Building block from page 164:


11-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg3-NH2


11-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg4-NH2


(SEQ ID NO: 27)


11-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]undecanoyl-Arg5-NH2


(SEQ ID NO: 26)


Building block from page 164:


12-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg3-NH2


12-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg4-NH2


(SEQ ID NO: 27)


12-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]dodecanoyl-Arg5-NH2


(SEQ ID NO: 26)


Building block from page 164:


15-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg3-NH2


15-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg4-NH2


(SEQ ID NO: 27)


15-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentadecanoyl-Arg5-NH2


(SEQ ID NO: 26)


Building block from example 298:


2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg6-NH2


(SEQ ID NO: 59)


2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg5-NH2


(SEQ ID NO: 26)


2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg4-NH2


(SEQ ID NO: 27)


2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]acetyl-Arg3-NH2


Building block from example 302:


2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-


yl}acetyl-Arg6-NH2


(SEQ ID NO: 59)


2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-


yl}acetyl-Arg5-NH2


(SEQ ID NO: 26)


2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-


yl}acetyl-Arg4-NH2


(SEQ ID NO: 27)


2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-


3-yl}acetyl-Arg3-NH2


4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]butyryl-Arg3-NH2


15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg6-NH2


(SEQ ID NO: 59)


15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg5-NH2


(SEQ ID NO: 26)


15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg4-NH2


(SEQ ID NO: 27)


15-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]pentadecanoyl-Arg3-NH2


5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg6-NH2


(SEQ ID NO: 59)


5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg5-NH2


(SEQ ID NO: 26)


5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg4-NH2


(SEQ ID NO: 27)


5-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]pentanoyl-Arg3-NH2


Building block from example 284:


3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyl]acryloyl-Arg6-NH2


(SEQ ID NO: 59)


Building block from example 295:


2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg6-NH2


(SEQ ID NO: 59)


2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg5-NH2


(SEQ ID NO: 26)


2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg4-NH2


(SEQ ID NO: 27)


2-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg3-NH2


8-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg6-NH2


(SEQ ID NO: 59)


8-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg5-NH2


(SEQ ID NO: 26)


8-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg4-NH2


(SEQ ID NO: 27)


8-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]octanoyl-Arg3-NH2


6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg6-NH2


(SEQ ID NO: 59)


6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg5-NH2


(SEQ ID NO: 26)


6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg4-NH2


(SEQ ID NO: 27)


6-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]hexanoyl-Arg3-NH2


Building block from example 288:


4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2


Building block from example 282:


4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


Building block from example 289:


4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2


11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg6-NH2


(SEQ ID NO: 59)


11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg5-NH2


(SEQ ID NO: 26)


11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg4-NH2


(SEQ ID NO: 27)


11-[6-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxy]undecanoyl-Arg3-NH2


4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg3-NH2


Building block from example 286:


4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg6-NH2


(SEQ ID NO: 59)


4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg5-NH2


(SEQ ID NO: 26)


4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg4-NH2


(SEQ ID NO: 27)


4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoyl-Arg3-NH2


Building block from example 285:


2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg6-NH2


(SEQ ID NO: 59)


2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg5-NH2


(SEQ ID NO: 26)


2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg4-NH2


(SEQ ID NO: 27)


2-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg3-NH2


Building block from example 283:


2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg6-NH2


(SEQ ID NO: 59)


2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg5-NH2


(SEQ ID NO: 26)


2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg4-NH2


(SEQ ID NO: 27)


2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]acetyl-Arg3-NH2


Building block from example 296:


4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2


Building block from example 290:


4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-[2-Bromo-4-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)phenoxy]butyryl-Arg3-NH2


Building block from example 544:


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg6-NH2


(SEQ ID NO: 59)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg4-NH2


(SEQ ID NO: 27)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg3-NH2


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg7-NH2


(SEQ ID NO: 32)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg8-NH2


(SEQ ID NO: 29)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg9-NH2


(SEQ ID NO: 31)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg10-NH2


(SEQ ID NO: 30)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg11-NH2


(SEQ ID NO: 33)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg12-NH2


(SEQ ID NO: 28)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg13-NH2


(SEQ ID NO: 34)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg14-NH2


(SEQ ID NO: 35)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg15-NH2


(SEQ ID NO: 36)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg16-NH2


(SEQ ID NO: 37)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg17-NH2


(SEQ ID NO: 38)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg18-NH2


(SEQ ID NO: 39)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg19-NH2


(SEQ ID NO: 40)


4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg20-NH2


(SEQ ID NO: 41)


Building block from page 251:


4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg6-NH2


(SEQ ID NO: 59)


4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg5-NH2


(SEQ ID NO: 26)


4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg4-NH2


(SEQ ID NO: 27)


4′-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]biphenyl-4-carbonyl-Arg3-NH2


Building block from example 549:


3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg6-NH2


(SEQ ID NO: 59)


3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg5-NH2


(SEQ ID NO: 26)


3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg4-NH2


(SEQ ID NO: 27)


3-[3-(2H-Tetrazol-5-yl)carbazol-9-ylmethyl]benzoyl-Arg3-NH2


Building block from page 252:


4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg6-NH2


(SEQ ID NO: 59)


4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg5-NH2


(SEQ ID NO: 26)


4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg4-NH2


(SEQ ID NO: 27)


4′-[5-(2H-Tetrazol-5-yl)indol-1-ylmethyl]biphenyl-4-carbonyl-Arg3-NH2


Building block from example 412:


4-(2H-Tetrazol-5-yl)benzoyl-Gly2-Arg6-NH2


(SEQ ID NO: 59)


4-(2H-Tetrazol-5-yl)benzoyl-Gly2-Arg5-NH2


(SEQ ID NO: 26)


4-(2H-Tetrazol-5-yl)benzoyl-Gly2-Arg4-NH2


(SEQ ID NO: 27)


4-(2H-Tetrazol-5-yl)benzoyl-Gly2-Arg3-NH2


Building block from example 355:


[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg6-NH2


(SEQ ID NO: 59)


[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg5-NH2


(SEQ ID NO: 26)


[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg4-NH2


(SEQ ID NO: 27)


[4-(7-Carboxy-6-hydroxynaphthalen-2-yl)phenyl]methyl-Arg3-NH2


Building block from example 342:


(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg6-NH2


(SEQ ID NO: 59)


(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg5-NH2


(SEQ ID NO: 26)


(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg4-NH2


(SEQ ID NO: 27)


(7-Carboxy-6-hydroxynaphthalen-2-yl)methyl-Arg3-NH2


Building block from example 342:


4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg6-NH2


(SEQ ID NO: 59)


4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg5-NH2


(SEQ ID NO: 26)


4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg4-NH2


(SEQ ID NO: 27)


4-[(7-Carboxy-6-hydroxynaphthalen-2-ylmethyl)amino]benzoyl-Arg3-NH2


4-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg6-NH2


(SEQ ID NO: 59)


4-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg5-NH2


(SEQ ID NO: 26)


4-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg4-NH2


(SEQ ID NO: 27)


4-[4-(5-Mercaptotetrazol-1-yl)benzoylamino]benzoyl-Arg3-NH2


4-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


4-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-[4-(5-Mercaptotetrazol-1-yl)phenoxy]butyryl-Arg3-NH2


4-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg6-NH2


(SEQ ID NO: 59)


4-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-[4-(5-Mercaptotetrazol-1-yl)naphthalen-1-yloxy]butyryl-Arg3-NH2


Benzotriazol-5-ylcarbonyl-4-Abz-Gly2-Arg6-NH2


(SEQ ID NO: 60)


Benzotriazol-5-ylcarbonyl-4-Abz-Gly2-Arg4-NH2


(SEQ ID NO: 61)


Benzotriazol-5-ylcarbonyl-4-Abz-Gly2-Arg3-NH2


(SEQ ID NO: 62)


4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg3-


NH2


4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg4-


NH2


(SEQ ID NO: 27)


4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyl]benzoyl-Arg5-


NH2


(SEQ ID NO: 26)


3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg3-NH2


3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg4-NH2


(SEQ ID NO: 27)


3′,5′-Dichloro-4′-(2,4-dioxothiazolidin-5-ylidenemethyl)biphenyl-4-oyl-Arg5-NH2


(SEQ ID NO: 26)


2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg3-NH2


2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg4-NH2


(SEQ ID NO: 27)


2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetyl-Arg5-NH2


(SEQ ID NO: 26)


4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg5-NH2


(SEQ ID NO: 26)


4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg4-NH2


(SEQ ID NO: 27)


4-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)butyryl-Arg3-NH2


5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg5-NH2


(SEQ ID NO: 26)


5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg4-NH2


(SEQ ID NO: 27)


5-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)pentanoyl-Arg3-NH2


8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg5-NH2


(SEQ ID NO: 26)


8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg4-NH2


(SEQ ID NO: 27)


8-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)octanoyl-Arg3-NH2


4-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg6-NH2


(SEQ ID NO: 59)


4-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg5-NH2


(SEQ ID NO: 26)


4-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg4-NH2


(SEQ ID NO: 27)


4-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)benzoylamino]-benzoyl-Arg3-NH2


N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg5-NH2


(SEQ ID NO: 26)


N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg4-NH2


(SEQ ID NO: 27)


N-[4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenyl]terephthalamoyl-Arg3-NH2









Example 663
Equilibrium Solubility. For pH-solubility profiles, a 0.6 mM human insulin stock solution containing 0.2 mM Zn2+, 30 mM phenol, 0.2 M 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 (SEQ ID NO: 1) on the pH-dependence of insulin solubility is illustrated in FIG. 1.
Example 664
The effect of increasing concentrations of the ligand BTG2R4 (SEQ ID NO: 2) on the pH-dependence of insulin solubility is illustrated in FIG. 2. The solubility was determined as in example 24. 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
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 γ-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.
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 HisB10Zn2+-site ligand. This is illustrated below for AspB28 human insulin employing two different levels of the ligand TZD-Abz-G2R5 (SEQ ID NO: 4) (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.


















125I-Prep. 1
125I-Prep. 2
125I-Prep. 3
125I-Prep. 4
125I-Prep. 5
125I-Prep. 6






















Insulin (mM)
0.6 human
0.6 human
0.6 human
0.6 human
0.6 AspB28
0.6 AspB28



insulin
insulin
insulin
insulin
insulin
insulin


Zn2+ (mM)
0.3
0.3
0.3
0.3
0.3
0.3


Phenolic
30 mM
30 mM
30 mM
30 mM 7-
30 mM
30 mM


ligand
phenol
phenol
phenol
hydroxyindole
phenol
phenol


Zn2+ ligand
6 mM BTG2R4
6 mM BTG2R6
2 mM BT-
2 mM BT-
0.4 mM TZD-
0.15 mM TZD-



(Ex. 641)
(Ex. 643)
AbzG2R5
AbzG2R5
AbzG2R5
AbzG2R5



(SEQ ID
(SEQ ID
(Ex. 644)
(Ex. 644)
(Ex. 647)
(Ex. 647)



NO: 2)
NO: 5)
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID





NO: 3)
NO: 3)
NO: 4)
NO: 4)


Mannitol (mM)
112
112
150
150
154
176


Phosphate
2
2
2
2
2
2


buffer (mM)


pH
7.4
7.4
7.4
7.4
7.4
7.4


T50% (hrs)
10.2
10.3
>22
20.2
14.8
biphasic









ANALYTICAL METHODS
Assays to quantify the binding affinity of ligands to the metal site of the insulin R6 hexamers:
4H3N-Assay:
The binding affinity of ligands to the metal site of insulin R6 hexamers are measured in a UV/vis based displacement assay. The UV/vis spectrum of 3-hydroxy-4-nitro benzoic acid (4H3N) which is a known ligand for the metal site of insulin R6 shows a shift in absorption maximum upon displacement from the metal site to the solution (Huang et al., 1997, 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.
A stock solution with the following composition 0.2 mM human insulin, 0.067 mM Zn-acetate, 40 mM phenol, 0.101 mM 4H3N is prepared in a 10 mL quantum as described below. Buffer is always 50 mM tris buffer adjusted to pH=8.0 with NaOH/ClO4−.
1000 μL of 2.1 mM human insulin in buffer
66.7 μL of 10 mM Zn-acetate in buffer
800 μL of 500 mM phenol in H2O
201 μL of 4H3N in H2O
7.93 ml buffer
The ligand is dissolved in DMSO to a concentration of 20 mM.
The ligand solution is titrated to a cuvette containing 2 mL stock solution and after each addition the UV/vis spectrum is measured. The titration points are listed in Table 3 below.









TABLE 3





ligand addition
ligand conc.
dilution


(μl)
(mM)
factor

















1
0.010
1.0005


1
0.020
1.0010


1
0.030
1.0015


2
0.050
1.0025


5
0.100
1.0050


10
0.198
1.0100


20
0.392
1.0200


20
0.583
1.0300


20
0.769
1.0400


20
0.952
1.0500









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.
The following equation is fitted to these datapoints to determine the two parameters KD (obs), the observed dissociation constant, and absmax the absorbance at maximal ligand concentration.

abs([ligand]free)=(absmax*[ligand]free)/(KD(obs)+[ligand]free)

The observed dissociation constant is recalculated to obtain the apparent dissociation constant

KD(app)=KD(obs)/(1+[4H3N]/K4H3N)

The value of K4H3N=50 μM is taken from Huang et al., 1997, Biochemistry 36, 9878-9888.
TZD-Assay:
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.
Preparation
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 mM.
Measurements
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.
Data Analysis
This equation is fitted to the datapoints

ΔF(455 nm))=ΔFmax*[ligand]free/(KD(app)*(1+[TZD]/KTZD)+[ligand]free))

KD(app) is the apparent dissociation constant and Fmax is the fluorescence at maximal ligand concentration. The value of KTZD is measured separately to 230 nM

Two different fitting-procedures can be used. One in which both parameters, KD(app) and Fmax, are adjusted to best fit the data and a second in which the value of Fmax is fixed (Fmax=1) and only KD(app) is adjusted. The given data are from the second fitting procedure. The Solver module of Microsoft Excel can be used to generate the fits from the datapoints.

## Claims

1. A zinc-binding ligand of the following formula (III)


A-B-C-D-X  (III)

wherein:
A is a chemical group of the following formula which reversibly binds to a HisB10 Zn2+ site of an insulin hexamer;




wherein A1 is a valence bond;
AR1 is the group





where C1 is attached to the nitrogen;
C1 is CH2;
AR2 is phenyl;
B is a linker selected from the group consisting of
A valence bond and
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—,
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)—;
wherein the alkylene, alkenylene, and alkynylene moieties are optionally substituted with —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene and heteroarylene moieties are optionally substituted with halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl;
R6 and R7 are independently H, C1-C4-alkyl;

C is a fragment consisting of 0 to 5 neutral amino acids, wherein the individual neutral amino acids are the same or different
D is a fragment consisting of 1 to 20 basic amino acids independently selected from the group consisting of Lys and Arg or D-isomers thereof; and
X is —OH, —NH2 or a diamino group,

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

2. A zinc-binding ligand according to claim 1 wherein 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.

3. A zinc-binding ligand according to claim 2 wherein 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.

4. A zinc-binding ligand according to claim 3 wherein A1 is a valence bond, C1-C6-alkylene, or —NH—C(═O)-A2, wherein any C1-C6-alkyl moiety is optionally substituted by R1A.

5. A zinc-binding ligand according to claim 4 wherein A1 is a valence bond or C1-C6-alkylene, wherein any C1-C6-alkyl moiety is optionally substituted by R1A.

6. A zinc-binding ligand according to claim 5 wherein A1 is a valence bond.

7. A zinc-binding ligand according to claim 1 wherein A2 is a valence bond or —C1-C6-alkyl-O—.

8. A zinc-binding ligand according to claim 7 wherein A2 is a valence bond.

9. A zinc-binding ligand according to claim 1 wherein AR1 is arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted by one or more R1B independently.

10. A zinc-binding ligand according to claim 9 wherein AR1 is selected from the group of compounds consisting of phenylene, biphenylylene, naphthylene, anthra-cenylene, 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, and acridinylene, optionally substituted by one or more R1B independently.

11. A zinc-binding ligand according to claim 10 wherein AR1 is selected from the group of compounds consisting of phenylene, biphenylylene, naphthylene, pyridinylene, fyrylene, indolylene and carbazolylene, optionally substituted by one or more R1B independently.

12. A zinc-binding ligand according to claim 11 wherein AR1 is selected from the group of compounds consisting of phenylene, indolylene and carbazolylene, optionally substituted by one or more R1B independently.

13. A zinc-binding ligand according to claim 12 wherein AR1 is phenylene optionally substituted by one or more R1B independently.

14. A zinc-binding ligand according to claim 12 wherein AR1 is indolylene optionally substituted by one or more R1B independently.

15. A zinc-binding ligand according to claim 14 wherein AR1 is

16. A zinc-binding ligand according to claim 12 wherein AR1 is carbazolylene optionally substituted by one or more R1B independently.

17. A zinc-binding ligand according to claim 16 wherein AR1 is

18. A zinc-binding ligand according to claim 1 wherein R1B is selected from the group consisting of
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
C1-C6-alkyl or C2-C6-alkenyl
which is optionally substituted with one or more substituents selected from the group consisting of halogen, —CN, —CF3, —OCF3, —OR1C, and —NR1CR1D and
aryl, aryloxy, aryl-C1-C6-alkoxy, aryl-C1-C6-alkyl, aryl-C2-C6-alkenyl, heteroaryl, heteroaryl-C1-C6-alkyl, or heteroaryl-C2-C6-alkenyl

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

19. A zinc-binding ligand according to claim 18 wherein R1B is selected from the group consisting of
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 and
C1-C6-alkyl.

20. A zinc-binding ligand according to claim 1 wherein R1C and R1D independently are hydrogen, C1-C6-alkyl, or aryl, wherein the aryl moieties may optionally be substituted by halogen or —COOH.

21. A zinc-binding ligand according to claim 20 wherein R1C and R1D independently are hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties may optionally be substituted by halogen or —COOH.

22. A zinc-binding ligand according to claim 1 wherein C1 is a valence bond, C1-C6-alkylene, —NH—C(═O)—, —C(═O)—NH—, —C(═O)—, or —C1-C6-alkyl-C(═O)—N(R1E)— wherein the alkyl moieties are optionally substituted by one or more R1F independently.

23. A zinc-binding ligand according to claim 22 wherein 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)—.

24. A zinc-binding ligand according to claim 1 wherein R1E and R1F are independently selected from C1-C6-alkyl.

25. A zinc-binding ligand according to claim 1 wherein AR2 is
a valence bond
C1-C6-alkylene, wherein the alkyl is optionally substituted by one or more R2A independently
arylene, aryl-C1-C6-alkyl, heteroarylene, wherein the arylene and heteroarylene moieties are optionally substituted by one or more R2A independently.

26. A zinc-binding ligand according to claim 25 wherein AR2 is
a valence bond
C1-C6-alkylene, wherein the alkyl is optionally substituted by one or more R2A independently
phenyl, phenyl-C1-C6-alkyl, wherein the phenyl moieties are optionally substituted by one or more R2A independently.

27. A zinc-binding ligand according to claim 1 wherein 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).

28. A zinc-binding ligand according to claim 27 wherein 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).

29. A zinc-binding ligand according to claim 28 wherein 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.

30. A zinc-binding ligand according to claim 29 wherein R2A is C1-C6-alkyl, C1-C6-alkoxy, phenyl, halogen, —CF3, wherein the phenyl is optionally substituted by one or more C1-C6-alkyl, halogen, —COOH, —CF3, or —CN.

31. A zinc-binding ligand according to claim 1, wherein GB is of the formula B1—B2—C(O)—, B1—B2—SO2— or —B1—B2—CH2—.

32. A zinc-binding ligand according to claim 1, wherein GB is of the formula B1—B2—C(O)—, B1—B2—SO2— or B1—B2—NH—.

33. A zinc-binding ligand according to claim 1, wherein GB is of the formula B1—B2—C(O)—, —B1—B2—CH2— or B1—B2—NH.

34. A zinc-binding ligand according to claim 31, wherein GB is of the formula B1—B2—C(O)— or B1—B2—SO2.

35. A zinc-binding ligand according to claim 31, wherein GB is of the formula B1—B2—C(O)— or —B1—B2—CH2—.

36. A zinc-binding ligand according to claim 32 wherein GB is of the formula B1—B2—C(O)— or B1—B2—NH—.

37. A zinc-binding ligand according to claim 34, wherein GB is of the formula B1—B2—C(O)—.

38. A zinc-binding ligand according to claim 1 wherein B1 is a valence bond, —O—, or —S—.

39. A zinc-binding ligand according to claim 1 wherein B1 is a valence bond, —O—, or —N(R6)—.

40. A zinc-binding ligand according to claim 1 wherein B1 is a valence bond, —S—, or —N(R6)—.

41. A zinc-binding ligand according to claim 1 wherein B1 is —O—, —S— or —N(R6)—.

42. A zinc-binding ligand according to claim 38 wherein B1 is a valence bond or —O—.

43. A zinc-binding ligand according to claim 38 wherein B1 is —O— or —S—.

44. A zinc-binding ligand according to claim 39 wherein B1 is —O— or —N(R6)—.

45. A zinc-binding ligand according to claim 42, wherein B1 is —O—.

46. A zinc-binding ligand according to claim 1, wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-NR6—C1-C18-alkyl-C(═O)—; and the alkylene moieties are optionally substituted with —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene moieties are optionally substituted with halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.

47. A zinc-binding ligand according to claim 46, wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, —C(═O)—C1-C18-alkyl-O—C1-C18-alkyl-C(═O)—, and the alkylene moieties are optionally substituted with —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene moieties are optionally substituted with halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.

48. A zinc-binding ligand according to claim 47, wherein B2 is a valence bond, C1-C18-alkylene, C2-C18-alkenylene, C2-C18-alkynylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, and the alkylene moieties are optionally substituted with —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene moieties are optionally substituted with halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.

49. A zinc-binding ligand according to claim 48, wherein B2 is a valence bond, C1-C18-alkylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, —C(═O)—C1-C18-alkyl-C(═O)—, and the alkylene moieties are optionally substituted with —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene moieties are optionally substituted with halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.

50. A zinc-binding ligand according to claim 49 wherein B2 is a valence bond, C1-C18-alkylene, arylene, heteroarylene, —C1-C18-alkyl-aryl-, and the alkylene moieties are optionally substituted with —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene moieties are optionally substituted with halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.

51. A zinc-binding ligand according to claim 50, wherein B2 is a valence bond, C1-C18-alkylene, arylene, —C1-C18-alkyl-aryl-, and the alkylene moieties are optionally substituted with —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene moieties are optionally substituted with halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.

52. A zinc-binding ligand according to claim 51, wherein B2 is a valence bond or —C1-C18-alkylene, and the alkylene moieties are optionally substituted moieties are optionally substituted with —CN, —CF3, —OCF3, —OR6, or —NR6R7 and the arylene moieties are optionally substituted with halogen, —C(O)OR6, —C(O)H, OCOR6, —SO2, —CN, —CF3, —OCF3, —NO2, —OR6, —NR6R7, C1-C18-alkyl, or C1-C18-alkanoyl.

53. A zinc-binding ligand according to claim 1, wherein C consists of 0 to 5 neutral amino acids independently selected from the group consisting of Abz, Gly, Ala, Thr, and Ser.

54. A zinc-binding ligand according to claim 53, wherein C consists of 0 to 5 Gly.

55. A zinc-binding ligand according to claim 54, wherein C consists of 1 Gly.

56. A zinc-binding ligand according to claim 54, wherein C consists of 2 Gly.

57. A zinc-binding ligand according to claim 54, wherein C consists of 3 Gly.

58. A zinc-binding ligand according to claim 54 wherein C consists of 4 Gly.

59. A zinc-binding ligand according to claim 54, wherein C consists of 5 Gly.

60. A zinc-binding ligand according to claim 1, wherein the positively charged groups of D number from 1 to 16.

61. A zinc-binding ligand according to claim 60, wherein the positively charged groups of D number from 1 to 12.

62. A zinc-binding ligand according to claim 61, wherein the positively charged groups of D number from 1 to 10.

63. A zinc-binding ligand according to claim 1, wherein the basic amino acid is Arg.

64. A zinc-binding ligand according to claim 1, wherein X is —OH or —NH2.

65. A zinc-binding ligand according to claim 64, wherein X is —NH2.

66. Method of prolonging the action of an insulin preparation which comprises adding a zinc-binding ligand according to claim 1 to the insulin preparation.

67. A method of preparing a zinc-binding ligand according to claim 1 comprising the steps of
Identifying starter compounds that are able to displace a ligand from the R-state HisB10-Zn2+ site
optionally attaching a fragment consisting of 0 to 5 neutral α- or β-amino acids attaching a fragment comprising 1 to 20 positively charged groups independently selected from amino or guanidino groups.