ZA200401839B

ZA200401839B - Novel ligands for the HisB10 Zn2+ sites of the R-state insulin hexamer.

Info

Publication number: ZA200401839B
Application number: ZA200401839A
Authority: ZA
Inventors: Lle Birk Olsen; Niels C Karsholm; Peter Madsen; Soren Ostergaard; Svend Ludvigsen; Palle Jakobsen; Anders Klarskov Petersen; Dorte Bjerre Steensgaard
Original assignee: Novo Nordisk As
Priority date: 2001-09-14
Filing date: 2004-03-05
Publication date: 2005-09-16

Description

NOVEL LIGANDS FOR THE HisB10 Zn? SITES OF THE R-STATE INSULIN HEXAMER.

FIELD OF THE INVENTION

The present invention discloses novel ligands for the HisB10 Zn?" sites of the R-state insulin hexamer, R-state insulin hexamers comprising such ligands, and aqueous insulin prepara- tions 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 behav- jour consists of two interrelated allosteric transitions designated L"; and L%, three inter- converting allosteric conformation states (eq. 1),

L% L%

Teg T:Ry Rs (1) designated Te, T3R3, and Rg and two classes of allosteric ligand binding sites designated as the phenolic pockets and the His®™ 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 a-helical conformation in the R-state. This coil-to-helix transition causes the N-terminal residue, Phe®", to undergo an ~ 30 A change in position.

This conformational change creates hydrophobic pockets (the phenolic pockets) at the sub- unit interfaces (three in T3R3, and six in Rg), and the new B-chain helices form 3-helix bun- dies (one in T3R3 and two in Rg) with the bundle axis aligned along the hexamer three-fold symmetry axis. The His?" Zn?" in each Rs unit is forced to change coordination geometry from octahedral to either tetrahedral (monodentate ligands) or pentahedral (bidentate ligands). Formation of the helix bundle creates a narrow hydrophobic tunnel in each R; unit that extends from the surface ~12 A down to the His?" metal ion. This tunnel and the His?"

Zn?" 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 Zn* at the His®" sites to give Ts, and the subsequent ligand-mediated transition of T¢ to TsR; and to Rs is known to greatly enhance the physical and chemical stability of the resulting formula- tions. .

Ligand Binding and Long Acting Insulin Formulations. Although the conversion of Tg to TaRs and Rg improves the stability of the preparation, the rate of absorption following subcutane- “ ous injection of a soluble hexameric preparation is not much affected by the addition of phe- nol and cloride.

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 Zn for e.g. albumin and the large ef- fective space available for diffusion of the lipophilic phenol will tend to speed up the separa- tion. In about 10-15 minutes after injection, the distribution of insulin species in the subcuta- neous tissue will roughly correspond to that of a zinc-free insulin preparation at the same di- 16 lution. 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 Asp®® human insulin) and about 4 hours (Co*"-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 modifica- tions to obtain the desired profraction. 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.

Gly*2'Arg®*'Arg®3? human insulin belongs to this category of insulin analogues.

Most recently, a series of soluble insulin derivatives with a hydrophobic moiety covalently at- } tached to the side chain of Lys®® have been synthesized. These derivatives may show pro- longed action profile due to various mechanisms including albumin binding (e.g. B29-Ns- myristoyl-des(B30) human insulin), extensive protein self-association and/or stickiness (e.g.

B29-N°-(N-lithocholyl-y-glutamyl)-des(B30) human insulin) induced by the attached hydro- phobic group.

SUMMARY OF THE INVENTION

The present invention provides novel ligands for the His®'® Zn* sites of the R-state insulin : 5 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 re- sulting 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, Brand |.

The term “C,-Ce-alkyl” as used herein represents a saturated, branched or straight hydrocar- bon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methy!, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl and the like.

The term “C,-Cs-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 “C,-Cg-alkenyl” as used herein represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, iso-propenyl, 1,3-buta- dienyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 1-pentenyl, 2-pentenyl, 3- pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 2,4-hexadienyl, 5- hexenyl and the like.

The term “C»Ce-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-hexadiynyl and the like.

The term “C,-Cg-alkoxy” as used herein refers to the radical -O-C;-Cg-alkyl, wherein C;-Cg-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 “C,-Cs-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 “C, g-cycloalkenyl” as used herein represents a non-aromatic, carbocyclic group hav- ing 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-cyclooctadieny! and the like.

The term “heterocyclyl” as used herein represents a non-aromatic 3 to 10 membered ring con- taining one or more heteroatoms selected from nitrogen, oxygen and sulphur and optionally containing one or two double bonds. Representative examples are pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, aziridinyl, tetrahydrofuranyl and the like.

The term “aryl” as used herein is intended to include carbocyclic, aromatic ring systems such as 6 membered monocyclic and 9 to 14 membered bi- and tricyclic, carbocyclic, aromatic ring systems. Representative examples are phenyl, biphenylyl, naphthyl, anthracenyl, phe- nanthrenyl, fluorenyl, indenyl, azulenyl and the like. Aryl is also intended to include the par- tially 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 deriva- tives are 1,2,3,4-tetrahydronaphthyiene, 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 sys- tems 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, heterocyc- lic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sul- phur. Representative examples are furyl, thienyl, pyrrolyl, pyrazolyl, 3-oxopyrazolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, pyranyl, pyridyl, 5 pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5- triazinyl, 1,2,3- : oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4- thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, ben- zofuryl, benzothienyl, indazolyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl, thiazolidinyl, 2- thiooxothiazolidinyl and the like. Heteroaryl is also intended to include the partially hydrogen- ated derivatives of the ring systems enumerated above. Non-limiting examples of such par- tially hydrogenated derivatives are 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.

The term “heteroarylene” as used herein is intended to include divalent, aromatic, heterocyc- lic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sul- phur 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, oxy- gen and sulphur. Representative examples are furylene, thienylene, pyrrolylene, oxa- zolylene, 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, ben- zisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene, quinazolinylene, quinoliz- inylene, quinolinylene, isoquinolinylene, quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene, azepinylene, diazepinylene, acridinylene and the like. Heteroaryl is also in- tended to include the partially hydrogenated derivatives of the ring systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydro- benzofuranylene, pyrrolinylene, pyrazolinylene, indolinylene, oxazolidinylene, oxazolinylene, oxazepinylene and the like. “Aryl-C4-Cg-alkyl”, “heteroaryl-C4-Cg-alkyl”, “aryl-C-Ce-alkenyl” etc. is intended to mean C,-C,- alkyl or C,-Cg-alkenyl as defined above, substituted by an aryl or heteroaryl as defined above, for example: : :

OY Cv

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

S 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 condi- tion. 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 a- or B-aminocarboxylic acids, including D-isomers of these (when applicable) without charges at physiologically relevant pH in the side chain, such as glycine, alanine, B-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 pharmaceuti- . 25 cally acceptable group that contains a positive charge at physiologically relevant pH, such as amino (primary, secondary and tertiary), ammonium and guanidino groups.

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

The term “f amino acid” as used herein is intended to mean any B-aminocarboxylic acid, ’ such as B-alanine, isoserine or the like.

When in the specification or claims mention is made of groups of compounds such as car- boxylates, dithiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imi-

dazoles, triazoles, 4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thia- zolidinediones, tetrazoles, S-mercaptotetrazoles, rhodanines, N-hydroxyazoles, hydantoines, thiohydantoines, naphthoic acids and salicylic acids, these groups of compounds are in- : tended 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 ac- tivity.

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 pro- longed 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 Diisopropyicarbodiimide

EDAC 1-ethyl-3~(3'-dimethylamino-propyl)carbodiimide, hydrochloride .

Fmoc 9H-Fluorene-9-yimethoxycarbonyl

G, Gly Glycine -

HOAt 1-hydroxy-7-azabenzotriazole

HOBT 1-Hydroxybenzotriazole

L, 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: 4-Abz O 4-Apac 0 BT jon oJ -N Oo

Oo a

NN ~N N

H H

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1: Effect of BTG,Rs-NH; on pH-solubility profile of an insulin preparation.

Fig. 2: Effect of BTG,R.-NH; 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-AbzG,Rs-NH, with phenol and 7-hydroxy indole (a-b); and BT -G2Rs-NH, and BT-G,R, with phenol (c-d) . The bottom panels (e-f) show slow- and dual release pro- files, respectively, obtained from Asp®® human insulin formulated with variable concentration of TZD-Abz-G;Rs

Fig. 4: 4H3N-assay. UV/vis spectra resulting from a titration of hexameric insulin with the compound 3-hydroxy-2-naphthoic acid in the presence of 4-hydroxy-3-nitrobenzoic acid

(4H3N). Inserted in the upper right corner is the absorbance at 444nm vs. the concentration of ligand

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

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 mole- cules 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 His?” Zn?" 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 ba- sis, 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 de- livery of human insulin (or analogues or derivatives thereof) as the active species.

The anions curently used in insulin formulations as allosteric ligands for the R-state hexam- ers (notably chloride ion) bind only weakly to the His®'® anion site. The present invention, which is based on the discovery of suitable higher affinity ligands for these anion sites, pro- vides ligands which are extended to modify timing via changes in hexamer solubility as out- lined above.

Most ligand binding sites in proteins are highly asymmetric. Because the His®'® Zn* 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 His? Zn? site consists of a tunnel or cavity with a triangular-shaped cross-section that extends ~12 A from the surface of the hexamer down to the His?" Zn? 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 Asn®® and Phe®' side chains. The walls of the 30 . tunnel are made up of the side chains of the amino acid residues along one face each of the three a-helices. The side chains from each helix that make up the lining of the tunnel are

Phe®', Asn®?, and Leu®®. Therefore, except for the zinc ion, which is coordinated to three

His? residues and is positioned at the bottom of the tunnel, the site is principally hydropho-

bic. Depending on the ligand structure, it may be possible for substituents on the ligand to make H-bonding interactions with Asn? and with the peptide linkage to Cys®’.

The present invention originates from a search for compounds with suitable binding proper- ) ties 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 His®'-Zn* site by the incoming ligand in question. These compounds will be referred to as “starter com- - pounds” in the following. These assays are easily transformed into a high-throughput format capable of handling libraries constructed around hits from the initial search of compound da- tabases.

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 as- say(s) it will be apparent for those skilled in the art to modify the starter compounds in ques- tion 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 posi- tion 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 inthe 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 Zn**-binding functionality

The zinc-binding ligands of the present invention are characterised by the following formula a () ’

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

A is a functionality capable of reversibly coordinating to a His®'® Zn?" site of an insulin hexamer;

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

Cis a valence bond or a fragment consisting of 1 to 5 neutral a- or p-amino acids;

E 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 in- dependently selected from the group consisting of Lys and Arg and D-isomers of these; and

X is OH, NH; or a diamino group.

The length of the zinc-binding ligand should be such that it extends from the His?" zn* site : to beyond the hexamer surface.

A is preferably a chemical structure selected from the group consisting of carboxylates, di- thiocarboxylates, phenolates, thiophenolates, alkylthiolates, sulfonamides, imidazoles, tria- zoles, benzimidazoles, benzotriazoles, purines, thiazolidinediones, naphthoic acids and sali- "15 cylic acids.

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

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

0]

N i i ?

N N N

H EAS .

HN HN Nes

INE N= HN

N N N=N N=N . lo) (CH,),

CH.)

N Nay ( 0

HN © HN, ° UX JT

N=N N=N N=N : lo) 3 SN G4

Ss Ss

N ~ ~

BE JOO

N=N HO HO

R' R' oO rR o 0 lo] “0 SARE JJ oJ 0 HN Ne : é TM mn - oO R N=N (0) 1

R o 0

Ms sa eae o HN ; REG 1 0] R wherein

R'is hydrogen, fluoro, chloro, bromo or iodo, misOori.

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

[0]

[0] oe 1 y oY « (IY

H ~N “n lo} Mu Son J )

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.

SNTTN

X is preferably OH, NH; or " Chom

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

Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-Gly-Gly-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-Gly-Arg-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-Gly-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH, Benzotriazol-5-ylcarbonyi-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-yltarbonyl-4-Abz-Gly-Gly-Arg-Arg-Arg-NH

Benzotriazol-5-ylcarbonyl-4-Abz-Arg-Arg-Arg-Arg-Arg-NH;

Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH

Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-4-Apac-Gly-Gly-Arg-Arg-Arg-NH,

Benzotriazol-5-yicarbonyi-4-Apac-Arg-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-Arg-NH,

Benzotriazol-5-ylcarbonyl-4-Apac-Arg-Arg-Arg-NH, [4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxylacetyl-4-Abz-Gly-Gly-Arg-Arg-Arg-

Arg-Arg-NH [3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxylacetyl-4-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-

NH . 4-(2H-Tetrazol-5-yl)benzoyl-Abz-Gly-Gly-Arg-Arg-Arg-Arg-Arg-NH.

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

A-B-C-D-X (Il) wherein:

A is a chemical group which reversibly binds to a His®'® Zn®* site of an insulin hexamer;

B is a linker selected from + A valence bond - « A chemical group G® of the formula -B'-B*-C(O)-, -B'-B%-S0,-, -B'-B*CH_-, or -B'-BZNH-; wherein B’ is a valence bond, -O-, -S-, or -NR®-; -

B?is a valence bond, C-Cys-alkylene, C-Cig-alkenylene, C,-Cys-alkynylene, arylene, heteroarylene, -C;-Cqg-alkyl-aryl-, -C,-Cyg-alkenyl-aryl-, -C,-C,g-alkynyl-aryl-, -C(=0)-

C,-Cie-alkyl-C(=0Y)-, -C(=0)-C,-C,g-alkenyl-C(=0)-, -C(=0)-C1-Cz-alkyl-O-C4-Cyq- alkyl-C(=O)-, -C(=0)- C1-C1g-alkyl-S-C-Cyg-alkyl-C(=0)-, -C(=0)-C,-C1g-alkyl-NR®-Cs-

C,s-alkyl-C(=0)-, -C(=0)-aryl-C(=0)-, -C(=0)-heteroaryl-C(=0)-; wherein the alkylene, alkenylene, and alkynyl enemoieties are optionally substituted by —CN, -CF3, -OCF;, -OR?, or -NR°R’ and the arylene and heteroarylene moieties are optionally substituted by halogen, -C(O)OR®, -C(O)H, OCORS, -SO,, -CN, -CF3, -

OCF, -NO,, -OR®, -NR®R’, C,-Cys-alkyl, or C,-C,s-alkanoyl;

Rfand R’ are independently H, C;-C,-alkyt;

C is a fragment consisting of 1 to 5 neutral a- or B-amino acids

Dis a fragment comprising 1 to 20 positively charged groups independently selected from : amino or guanidine groups; and

Xis -OH, -NH; 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 car- boxylates, dithiocarboxylates, phenolates, thiophenolates, akylthiolates, sulfonamides, imi- dazoles, triazoles, 4-cyano-1,2,3-triazoles, benzimidazoles, benzotriazoles, purines, thia- =zolidinediones, 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 ben- zotriazoles, 3-hydroxy 2-napthoic acids, salicylic acids, tetrazoles or thiazolidinediones

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

X X

Ng = or wd) Gc . rire yh 0] IN 0 R RY : wherein

Xis =0, =S or =NH

Yis-S-, -O-or-NH-

R® and R'! are independently hydrogen or C;-Cg-alkyl,

R? is hydrogen or C,-Cs-alky! or aryl, R® and R® may optionally be combined to form a double bond,

Rand R" are independently hydrogen, aryl, C;-Ce-alkyl, or -C(O)NR"R"

E and G are independently C-Cg-alkylene, arylene, -aryl-C4-Cgq-alkyl, -aryl-C-Ce-alkenyl- or heteroarylene, wherein the alkylene or alkenylene is optionally substituted with one or more substituents independently selected from halogen, -CN, -CF3, -OCF;, aryl, -COOH and —NH_, and the arylene or heteroarylene is optionally substituted with up to three substituents R",

Rand R",

E and R'™ may be connected through one or two valence bonds, G and R'? may be con- nected through one or two valence bonds;

R"™,R™ and R" are independently selected from e hydrogen, halogen, -CN, -CH.CN, -CHF,, -CF3, -OCF;, -OCHF,, -OCH,CF3, -OCF,CHF,, -S(0),CF3, -OS(0),CFs, -SCF3, -NO,, -OR?, -NR'"R"’, -SR'®, -NR"S(0),R", -S(0);NR™R", -S(O)NR'R", -S(O)R'®, -S(0).R"®, -0S(0), R", -C{O)NR"RY, -OC(O)NR'®R", -NR™C(O)R"7, -CH,C(O)NR*R", -OC-Cs- alkyl-C(O)NR'®R"", -CH,OR', -CH,0C(O)R", -CH,NR'"R", -OC(O)R*, -OC;-C;- alkyl-C(O)OR', -OC,-Ce-alkyl-OR™, -SC-Cg-alkyl-C(O)OR'®, ~C,-Cg-alkenyl-

C(=0)OR'®, -NR'"-C(=0)-C,-Cs-alkyl-C(=0)OR", -NR'®-C(=0)-C;-Cs- alkenyl-C(=0)OR , -C(O)OR', or —C,-Cs-alkenyl-C(=O)R", e C,;-Ce-alkyl, C,-Cs-alkenyl or C,-Cg-alkynyl,

which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF,, -OCF3, -OR'®, and -NR'®R" e aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, .

S aryl-C,-Ce-alkenyl, aroyl-C,-Ce-alkenyl, aryl-C,-Cg-alkynyl, heteroaryl, heteroaryl-C+-

Cs-alkyl, heteroaryl-C,-Cg-alkenyl or heteroaryl-C,-Cg-alkynyl, ‘ of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR’®, -CH,C(0)OR'®, -CH,OR'®, -CN, -CF3, -OCFs, -NO,, -OR'®, -NR'*R"" and C,-C¢-alkyl,

R' and R" independently are hydrogen, OH, C,-Ce-alkyl, aryi-C,-Ce-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from - halogen, -CN, -CF3, -OCF3;, -OC;-Cg-alkyl, -C(O)OC,-Cs-alkyl, -COOH and —NH,, and the aryl groups may optionally be substituted by halogen, -C(O)OC,-Cg-alkyl, -COOH, -CN, -CF3, -

OCF3, -NO,, -OH, -OC;-Cg-alkyl, -NH,, C(=0) or C,-Ce-alkyt; R'® and R'” 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 is -O- . In another embodiment Y is -S-

In another embodiment E is arylene optionally substituted with up to three substituents R'™,

R™ and R™. in another embodiment E is phenylene or naphtylene optionally substituted with up to three substituents R*, R™ and R". in another embodiment E is heteroarylene optionally substituted with up to three substituents

R™, R" and R".

In another embodiment E is indolylene optionally substituted with up to three substituents

R™ R'™ and R'S. : ’

In another embodiment R® is hydrogen. :

In another embodiment R® is hydrogen.

In another embodiment R® and R® are combined to form a double bond.

In another embodiment R" is C;Cs-alkyl.

In another embodiment R is methyl.

In another embodiment G is phenylene optionally substituted with up to three substituents

R™, R" and R". . In another embodiment R'" is hydrogen.

In another embodiment R'is hydrogen. in another embodiment R"™, R™ and R' are independently selected from «hydrogen, halogen, -NO,, -OR?, -NR™R", -SR', -NR'"S(0),R"", -S(0),NR"*R", -S(O)NR'™R"7, -S(O)R’®, -8(0),R"®, -0S(0), R™®, -NR™C(O)R", -CH,OR'™, -

CH,OC(O)R', -CH,NR"R'"’, -OC(0)R®, -OC,-Cg-alkyl-C(O)OR®, -OC;-Ce- alkyl-C(O)NR™R'"", -OC,-Cg-alkyl-OR®, -SC4-Ce-alky}l-C(O)OR™, —~C,-Ce-alkenyl-

C(=0)OR'®, -C(O)OR', or —~C,-Ce-alkenyl-C(=O)R™, » C;-Cg-alkyl, C»-Ce-alkenyl or C,-Cs-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF,, -OR'®, and -NR'*R"’ « aryl, aryloxy, aroyl, arylsulfanyl, aryl-C,-Ce-alkoxy, aryl-C;-Ces-alkyl, aryl-C,-

Ce-alkenyl, aroyl-C,-Ce-alkenyl, aryl-C>-Ce-alkynyl, heteroaryl, heteroaryl-C,-Ce-alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR®, -CH,C(O)OR™, -CH,0R", -CN, -CF3, -OCF,, -NO,, -OR'®, -NR"R"" and C,-Cg-alkyl.

In another embodiment R'™®, R* and R' are independently selected from « hydrogen, halogen, -NO,, -OR®, -NR™R", -SR', -S(O),R", -0S(0), R", -

CH,OC(O)R'®, -OC(O)R™, -OC;-Cs-alkyl-C(O)OR', -OC,-Cg-alkyl-OR", -SC1-Cy- alkyl-C(O)OR'®, -C(O)OR'®, or —C,-C-alkenyl-C(=O)R"®,

» C,-Cs-alkyl or C4-Ce-alkenyl which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF;3, OR, and -NR"R" e aryl, aryloxy, aroyl, aryl-C,-Cs-alkoxy, aryl-C;-Cg-alkyl, heteroaryl, . of which the cyclic moieties optionally may be substituted with one or more substitu- : ents selected from halogen, -C(O)OR, -CH,C(O)OR, -CH,OR', -CN, -CF3, -OCF;, -NO,, -OR™, -NR'"R*'" and C,-Cs-alkyl.

In another embodiment R*®, R* and R*® are independently selected from « hydrogen, halogen, -NO,, -OR®, -NR'"R"", -SR'®, -S(0),R"®, -0S(0), R", -

CH,OC(O)R', -OC(O)R?', -OC,-Cg-alkyl-C(O)OR', -OC,-Cs-alkyl-OR'®, -SC4-Ce- alkyl-C(O)OR', -C(O)OR', or —C,-Cs-alkenyl-C(=O)R, » C;-Cs-alkyl or C,4-Ces-alkenyl which may optionally be substituted with one or more substituents selected from halogen, -CFs, -OR™, and -NR'"R"’ « aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, C(O)OR™, -CN, -NO,, -OR', -NR"R" and C,-C¢-alkyl.

In another embodiment R'?, R™ and R* are independently selected from « hydrogen, halogen, -OR®, -OC,-C¢-alkyl-C(O)OR'®, or -C(O)OR', « C;-Cg-alkyl which may optionally be substituted with one or more substituents se- lected from halogen, -OR'®, and -NR"™R" s aryl, aryloxy, aryl-C,-Cg-alkoxy, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, C(O)OR", OR’, and C,-Cs-alkyl.

In another embodiment R'® and RY independently are hydrogen, C-Ce-alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CF3, -OCF3, -OC;-Ce-alkyl, -COOH and —NH,, and the aryl groups may optionally be substituted by halogen, -COOH, -CN, -CF3, -OCF3, -NO,, -OH, -OC4-Ce-alkyl, -NH,, C(=O) or C,-Ce-alkyl; R'® and RY when attached to the same nitrogen atom may forma 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally con- taining one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and op- tionally containing one or two double bonds

In another embodiment R™ and R" independently are hydrogen, C4-Ce-alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CF3, -OC;-Cgs-alkyl, -COOH and ~NHj, and the aryl groups may optionally be sub- stituted by halogen, -COOH, -CN, -CF3;, -OCF;, -OH, -NH,,or C;-Cs-alkyl.

In another embodiment A is ong of the following structures

R'® : N N o U N RV

CTD WELYA US EVAN

N wo N N I or Ty L—

H R H RX H lo) wherein

R? is hydrogen or C,-Cg-alkyl,

R?% is hydrogen or C;-Ce-alkyl,

U and V are a valence bond or C;-Cg-alkylene optionally substituted with one or more hy- droxy, C4-Ce-alkyl, or aryl independently,

Jis Ci-Ce-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is option- ally substituted with up to three substituents R*, R* and R%,

L is C,-Ce-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is option- ally substituted with up to three substituents R®, R® and R”,

RB, RY RZ RZ R* R% R® and R? are independently selected from

« hydrogen, halogen, -CN, -CH,CN, -CHF,, -CF3, -OCF3, -OCHF2, -OCH,CF3, -OCF,CHF,, -S(0),CF3, -SCF3, -NO,, -OR%, -NR*R?, -SR%, -NR*S(0),R”, -S(O),NRZRZ, -S(0)NRZRZ, -S(O)R?, -S(0),R%, -C(O)NR®R®, -OC(O)NR*R?, . -NR®C(0)RZ, -NREC(O)OR?, -CH,C(O)NRZR?, -OCH,C(O)NRZR?, -CH,OR?, : -CH,NR®R?, -OC(O)R%, -OC,-C¢-alkyl-C(O)OR?, -SC;-Cs-alkyl-C(O)OR?, —C,-Ce- alkenyl-C(=O)OR?%, -NR?-C(=0)-C,-Cg-alkyl-C(=0)OR?, -NR®-C(=0})-C;-Cq- . alkenyl-C(=0)OR?, -C(=0)NR?-C,-Cq-alkyl-C(=0)ORZ, -C;-Ce-alkyl-C(=0)OR? or -C(O)OR?, » C;-Cs-alkyl, Co-Ce-alkenyl or C-Cg-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR?, and -NR*R? « aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, aryl-C

Ce-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C;-Ce-alkyl, heteroaryl-C,-Ce- alkenyl or heteroaryl-C,-Cs-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, -CF;, -OCF3, -NO,, -OR%, -NR?R” and

C,-Cs-alkyl,

R2 and R? independently are hydrogen, C,-Cs-alkyl, aryl-C,-C¢-alkyl or aryl, or R®® and R*® when attached to the same nitrogen atom together with the said nitrogen atom may forma 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms se- lected 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 C;-Ce-alkylene optionally substituted with one or more hydroxy,

C;-Cg-alkyl, or aryl

In another embodiment J is arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R%, R? and R*

In another embodiment J is arylene optionally substituted with up to three substituents R2,

R? and R*

In another embodiment J is phenylene optionally substituted with up to three substituents

R# R® and R*

In another embodiment R%, RZ and R? are independently selected from «hydrogen, halogen, -CHF,, -CF, -OCF;, -OCHF2, -OCH,CF;, -OCF,CHF, -SCF3, -

NO,, -OR?, -NR?R?, -SR?, -C(O)NR®R?, -OC(O)NR®R?, -NR®C(O)R, ~ -NRZ®C(O)ORZ, -CH,C(O)NRZRZ, -OCH,C(O)NR®R?, -CH,0R?, -CH,NR?*R?, -OC(O)R%, -OC,-Cs-alkyl-C(OYOR?, -SC,-Ce-alkyl-C(O)OR?, —C,-Ce-alkenyl-

C(=0)OR?, -NR®-C(=0)-C,-C¢-alkyl-C(=0)OR?, -NR?®-C(=0)-C;-Cq- alkenyl-C(=0)OR%-, -C(=O)NR%-C,-Ce-alkyl-C(=0)OR?, -C4-Ce-alkyl-C(=0)OR?, or -C(O)OR?, ¢ C,;-Ce-alkyl, C-Ce-alkenyl or C,-Cs-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR®, and -NR*R* «aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C,-Cg-alkoxy, aryl-C,-Ce-alkyl, aryl-C,-

Cg-alkenyl, aryl-C,-Cs-alkynyl, heteroaryl, heteroaryl-C,-Ce-alkyl, heteroaryl-C»-Ce- alkenyl or heteroary!l-C,-Ce-alkynyi, : of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(OJOR?, -CN, -CF,, -OCFs3, -NO,, -OR?, -NR”R? and

C,-Ce-alkyl

In another embodiment RZ, RZ and R* are independently selected from «hydrogen, halogen, -OCF;, -OR?, -NR®R?, -SR%, -NR®C(O)R?, -NR*C(O)OR?, -OC(0)R?, -OC;-Ce-alkyl-C(OYOR?, -SC,-Cs-alkyl-C(OYOR?, ~C,-Cs-alkenyl-

C(=0)OR?, -C(=0)NR®-C,-Ce-alkyl-C(=0)OR?, -C,-Cs-alkyl-C(=0)OR?, or -C(O)OR?, « C,-C-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CF,, -OCF3, -OR?, and -NR®R*

e aryl, aryloxy, aroyl, aryl-C,-C¢-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-Ci-Ce- alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- . ents selected from halogen, -C(O)OR%, -CN, -CF3, -OCF3, -NO,, -OR?, -NR®R” and

C1-Ce-alkyl ’

In another embodiment R%, RZ and R** are independently selected from «hydrogen, halogen, -OCFa, -OR%, -NR®R?, -SR?, -NRZC(0)R%, -NR®C(0)OR?, -OC(O)R?, -OC;-Ce-alkyl-C(O)OR?, -SC,-Ce-alkyl-C(OYOR?, —C,-Ce-alkenyl-

C(=0)OR?, -C(=0)NR?-C,-C-alkyl-C(=0)OR?, -C,-Cs-alkyl-C(=0)OR?, or -C(O)OR?, » C,-C¢-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, or -CF; - e aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Cs-alkyl, heteroaryl, heteroary!l-C,-Ce- alkyl, ) of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OH, -CN, -CF3, -NO,, or C,-Cs-alkyl

In another embodiment R% is hydrogen or methyl

In another embodiment R% is hydrogen in another embodiment R? is hydrogen, C,-Cs-alkyl or aryl

In another embodiment R? is hydrogen or C,-Cs-alky!

In another embodiment RZ is hydrogen or C;-Ce-alkyl

In another embodiment V is a valence bond

In another embodiment V is Cy-Cg-alkylene optionally substituted with one or more hydroxy,

C,-Cs-alkyl, or aryl

In another embodiment L is C,-Cs-alkylene or arylene, wherein the arylene is optionally sub- stituted with up to three substituents R?, R%® and R?

In another embodiment L is Cy-Cg-alkyl

In another embodiment L is phenylene optionally substituted with up to three substituents

R%, R® and R%

In another embodiment R®, R® and RZ are independently selected from «hydrogen, halogen, -CHF,, -CF3, -OCF;, -OCHF2, -OCH,CF3, -OCF,CHF,, -SCF3, -

NO,, -OR%, -NR®R?, -SR?, -C(O)NR®R?, -OC(O)NR®R?, -NR®C(O)R?, -NRZC(0)ORZ, -CH,C(O)NRZRZ, -OCH,C(0)NR?R?, -CH,0R?, -CH,NRZR, -OC(O)R?%, -OC,-Cs-alkyl-C(O)OR?, -SC;-Cs-alkyl-C(O)OR?, —C,-Cs-alkenyl-

C(=0)OR?%, -NR?-C(=0)-C,-Cs-alkyl-C(=0)OR?, -NR®-C(=0)-C;-C- alkenyl-C(=O)ORZ-, -C(=O)NR?-C,-C-alkyl-C(=O)OR?, -C,-C;-alkyl-C(=0)OR?, or -C(O)OR?®, ¢ C-Cg-alkyl, C,-Cs-alkenyl or C,-Cg-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCFs, -OR?, and -NR*R? « aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C,-Cs-alkoxy, aryl-C,-Ce-alkyl, aryl-Co-

Ce-alkenyl, aryl-C,-Cs-alkynyl, heteroaryl, heteroaryl-C4-Ce-alkyl, heteroaryl-C»-Ce- alkenyl! or heteroaryl-C,-Ce-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(OYOR?, -CN, -CF3, -OCFs, -NO,, -OR%, -NR*R* and

C,-Cs-alkyl

In another embodiment R®, R* and R? are independently selected from « hydrogen, halogen, -OCF3, -OR®, -NR*R?, -SR?, -NR®C(O)R%, -NR*C(O)ORZ, -OC(0O)R?, -OC,-Cs-alkyl-C(O)OR?, -SC,-Ce-alkyl-C(O)OR?, —C,-Ce-alkenyl-

C(=0)ORZ, -C(=0)NR?-C,-Ce-alkyl-C(=O)OR®, -C,-C¢-alkyl-C(=0)OR?, or -C(O)OR?, « C,-Ce-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CFs, -OCF3, -OR%, and -NR¥*R?

«aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Cs-alkyl, heteroaryl, heteroaryl-C4-Ce- alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- : ents selected from halogen, -C(O)OR?, -CN, -CF3, -OCF3, -NO,, -OR®, -NR*R* and

C,-Cs-alkyl .

In another embodiment R®, R® and R” are independently selected from « hydrogen, halogen, -OCF3, -OR%, -NRZR?, -SR%, -NR®C(O)R%, -NR*C(O)OR?, -OC(O)R®, -OC,-Cs-alkyl-C(O)OR?, -SC,-Ce-alkyl-C(O)OR?, —C,-Cq-alkenyl-

C(=0)OR?%, -C(=O)NR?-C,-Cg-alkyl-C(=0)OR?, -C,-C¢-alkyl-C(=0)OR?, or -C(O)OR®, « C,-Cg-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, or -CF; e aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-C,-Ce- alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OH, -CN, -CF3, -NO,, or C,-Ce-alkyl in another embodiment R?' is hydrogen or methyl

In another embodiment R* is hydrogen

In another embodiment R? is Hydrogen, C;-Cg-alkyl or aryl

In another embodiment R? is Hydrogen or C,-Cg-alky!

In another embodiment R® is Hydrogen or C,-Ce-alkyl

In another embodiment R'® and R'® are independently selected from + hydrogen, halogen, -CN, -CFs, -OCF3, -NO,, -OR%, -NR®R?, -SR?, -S(O)R”, -S(0),R%, -C(O)NR®R?, -CH,OR?, -OC(O)R?®, -OC;-Ce-alkyl-C(O)OR?, -SC-Cq- alkyl-C(O)OR?, or -C(O)OR?, « C,-Ce-alkyl, C,-Cs-alkenyl or Co-Ce-alkynyl,

which may optionally be substituted with one or more substituents selected from “halogen, -CN, -CF3, -OCF;, -OR®, and -NR?**R*® ‘earyl, aryloxy, aryl-C;-Ce-alkoxy, aryl-C:-Ce-alkyl, heteroaryl, heteroaryi-C,-Ce-alky! - of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)ORZ, -CN, -CFa, OCF, -NO,, -OR?, -NR*R? and

C4-Ce-alkyl

In another embodiment R'® and R*® are independently selected from «hydrogen, halogen, -CN, -CF;, -NO,, -OR%, -NR®R?, or -C(O)OR?, C,-Cs-alky! optionally substituted with one or more substituents selected from halo- gen, -CN, -CFj, -OCF3, -OR?, and -NR#R? » aryl, aryloxy, aryl-C,-Ce-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR%, -CN, -CF3, -OCF3, -NO,, -OR%, -NR®R? and

C4-Cs-alkyl in another embodiment A is a compound of the form M-Q-T- wherein M is one of the following structures

HO, o - en 3 2 oo HO HO or Re or w' w? we [>

N wherein W', W2, and W? are independently OH, SH or NH, and the phenyl, naphthalene or benzocarbazole rings are optionally substituted by one or more R* independently

Q is selected from the following: s a valence bond o —CH,N(R*)- or —-SO,N(R*)-

A compound of the formula wherein Z' is S(O), or CH,, Z%is N,-O- or-S-,and nis 1 or 2; :

Tis ¢ A valence bond » C,-Cs-alkylene, C,-Cs-alkenylene or C,-Cg-alkynylene, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF3, -OR*, and -NR*R* « Arylene, -aryloxy-, -aryloxycarbonyl-, -aroyl-, -aryl-C4-C¢-alkoxy-, -aryl-C4-Cs-alky!-, -aryl-C-Ce-alkenyl-, -aryl-C,-Cg-alkynyl-, heteroarylene, -heteroaryi-C,-Cs-alkyl-, -heteroaryl-C,-Ce-alkenyl- or -heteroaryl-C,-Ce-alkynyi-, wherein the cyclic moieties are optionally substituted by one or more substituents selected from halogen, - -

C(O)OR™, -C(O)H, -CN, -CF3, -OCF3, -NO,, -OR%, -NR*R%®, C,-Cs-alkyl or C-Cs- alkanoyl,

R*2 and R™ independently are hydrogen, C;-Cs-alkyl, aryl-C,-Cs-alkyl or aryl, or R* and R* when attached to the same nitrogen atom together with the said nitrogen atom may forma 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms se- lected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,

R¥ and R* are independently hydrogen, C,-Cg-alkyl or C,-Cs-alkanoyl.

R* is hydrogen, halogen, -CN, -CH,CN, -CHF;, -CF3, -OCF;, -OCHF3, -OCH,CF,, -OCF,CHF,, -S(0),CF3, -SCF3, -NO,, -OR¥, -C(O)R*, -NR¥R¥®, -SR*, -NR¥S(0).R*®, -S(0);NR¥R¥®, -S(0)NR¥R*, -S(0)R¥, -5(0),R%, -C(O)NR¥R%*, -OC(O)NR¥*R*, -NR¥®C(0)R®, -CH,C(O)NR¥R*, -OCH,C(O)NR¥R®, -CH,0R%®, -CH,NR¥R*, -OC(O)R*, -

OC;-Cg-alkyl-C(O)OR¥, -SC,-Cg-alkyl-C(O)OR?* —C,-C¢-alkenyl-C(=0)OR*, -NR¥-C(=0)-C,-C¢-alkyl-C(=0)OR*, -NR*?-C(=0)-C,-Cs-alkenyl-C(=0)OR¥-, C,-Cs-alkyl,

C+-Ce-alkanoyl or -C(O)OR®,

In another embodiment M is one of the following structures @] e] wy “3 : w' w?

SUBSTITUTE SHEET (RULE 26)

In another embodiment M is 0 oN w'

In another embodiment Mis 0

AES

WwW?

In another embodiment the salicylic acid moiety is of the formula

HO 0] :

HO. :

In another embodiment the napthoic acid moiety is of the formula 0 00

HO in another embodiment Q is a valence bond, —=CHN(R¥)-, or —=SON(R*)-

In another embodiment Q is a valence bond

In another embodiment T is e A valence bond « C,-Cg-alkylene, C,-Cg-alkenylene or C,-Ce-alkynylene, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR*, and -NR*R* « 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)OR%, : -CN, -CF,, -OR®, -NR¥*R™®, C,-Cs-alkyl or C,-Cs-alkanoyl

In another embodiment the cyclic moiety in T is optionally substituted by halogen, -C(OJOR™, : -OR%®, -NR¥R®, C,-C¢-alkyl or C-Ce-alkanoyl

In another embodiment the cyclic moiety in T is optionally substituted by halogen, -C(OJOR™ or -OR*

In another embodiment T is a valence bond

In another embodiment R® and R*! are independently hydrogen or C4-Cg-alkyl

In another embodiment R* is hydrogen, halogen, -CN, -CF3, -OCF3, -SCF3, -NO,, -OR®, -C(O)R®, -NR¥ZR®, -SR%, -C(O)NRZR®, -OC(0)NR¥R¥®, -NR?C(O)R*, -OC(O)R¥, -OC1-

Ce-alkyl-C(O)OR, -SC;-Cg-alkyl-C(O)OR® or -C(O)OR™

In another embodiment R* is hydrogen, halogen, -CFs, -NO,, -OR®, -NR¥R¥, -SR%, -NR¥C(O)R™, or -C(O)OR*

In another embodiment R* is hydrogen, halogen, -CFa, -NO,, -OR¥, -NR¥R%, or -NR*C(O)R*

In another embodiment R* is hydrogen, halogen, or -OR*

In another embodiment R* and R* independently are hydrogen, C;-Cg-alkyt, or aryl

In another embodiment R* and R* independently are hydrogen or C4-Ce-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 |n another embodiment GE is of the formula -B'-B%-C(O)-, -B'-B*-SO,- or -B'-B*CHj-, wherein B! and B? are as defined above

In another embodiment G8 is of the formula -B'-B%-C(O)-, -B'-B%SO- or -B'-B*NH-, wherein B' and B? are as defined above

In another embodiment GB is of the formula -B'-B%-C(O)-, -B'-B%“CH,- or -B'-B%-NH-, wherein B' and B? are as defined above

In another embodiment G® is of the formula -B'-B%-CH,-, -B'-B>-SO,- or -B'-B*NH-, wherein

B' and B? are as defined above

In another embodiment G8 is of the formula -B'-B2-C(O)- or -B'-B*-S0,-, wherein B' and B? are as defined above

In another embodiment G® is of the formula -B'-B%-C(O)- or -B'-B?-CH,-, wherein B' and B are as defined above

In another embodiment G is of the formula -B'-B2-C(O)- or -B'-B2-NH-, wherein B' and B? are as defined above - In another embodiment G8 is of the formula -B'-B%-CH;- or -B'-B%-SO,- , wherein B' and B "areas defined above

In another embodiment G® is of the formula -B'-B?NH- or -B'-B%-SO,- , wherein B' and B are as defined above

In another embodiment G2 is of the formula -B'-B%-CH,- or -B'-B>NH- , wherein B' and B? are as defined above in another embodiment G® is of the formula -B'-B%-C(O)-

In another embodiment G® is of the formula -B*-B>-CH,-

In another embodiment G® is of the formula -B'-B%SO,-

In another embodiment GB is of the formula -B'-B%-NH-

In another embodiment B' is a valence bond, -O-, or -S-

In another embodiment B' is a valence bond, -O-, or -N(R®)-

In another embodiment B' is a valence bond, -S-, or -N(R®)-

In another embodiment B' is -O-, -S- or -N(R®)-

In another embodiment B' is a valence bond or —-O- in another embodiment B' is a valence bond or -S-

In another embodiment B' is a valence bond or —-N(R®)- in another embodiment B' is -O-or -S-

In another embodiment B' is -O-or -N(R°®)-

In another embodiment B' is -S-or -N(R®)-

In another embodiment B' is a valence bond

In another embodiment B' is -O-

In another embodiment B' is -S-

In another embodiment B' is -N(R®)-

In another embodiment B? is a valence bond, C;-Cis-alkylene, C,-Cg-alkenylene, C-Cig- alkynylene, arylene, heteroarylene, -C;-C,g-alkyl-aryl-, -C(=0)-C;-C,g-alkyl-C(=0)-, -C(=0)-

C4-Ci-alkyl-O-C-Cig-alkyl-C(=0)-, -C(=0)-C,-Cyg-alkyl-S-C,-Cyg-alkyl-C(=0)-, -C(=0)-C;-

C,s-alkyl-NR®-C,-Cys-alkyl-C(=0)-; and the alkylene and arylene moieties are optionally } substituted as defined above

In another embodiment B? is a valence bond, C,-Cis-alkylene, C,-Cys-alkenylene, C2-Cig- alkynylene, arylene, heteroarylene, -C4-C,g-alkyl-aryl-, -C(=0)-C;-Cys-alkyl-C(=0)-, -C(=0)- :

C1-C,g-alkyl-O-C,-C5-alkyl-C(=0)-, and the alkyl and aryl moieties are optionally substituted as defined above

In another embodiment BZ is a valence bond, C,-Cis-alkylene, C-Cie-alkenylene, C-Cie- alkynylene, arylene, heteroarylene, -C,-C,g-alkyl-aryl-, -C(=0)-C;-Cg-alkyl-C(=O})-, and the . alkylene and arylene moieties are optionally substituted as defined above

In another embodiment Bis a valence bond, C,-C,s-alkylene, arylene, heteroarylene, -C4- :

Cig-alkyl-aryl-, -C(=0)-C,-C,s-alkyl-C(=0)-, and the alkylene and arylene moieties are option- ally substituted as defined above

In another embodiment B? is a valence bond, C,-Cys-alkylene, arylene, heteroarylene, -Ci- Cye-alkyl-aryl-, and the alkylene and arylene moieties are optionally substituted as defined above

In another embodiment B? is a valence bond, C;-Cis-alkylene, arylene, -C-Cyg-alkyl-aryl-, and the alkylene and arylene moieties are optionally substituted as defined above

In another embodiment B2is a valence bond or C;-Cis-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 —NH,

In another embodiment X is —-NH;

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 829 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 lipo- philic substituents.

In another embodiment the insulin derivative is selected from the group consisting of B29-N"- myristoyl-des(B30) human insulin, B29-N°-paimitoyl-des(B30) human insulin, B29-N°- myristoyl human insulin, B29-N®-palmitoyl human insulin, B28-N°-myristoyl Lys®? Pro®® hu- man insulin, B28-N=-palmitoyl Lys®? Pro®2® human insulin, B30-N°*-myristoyl-Thr®2*Lys®* hu- man insulin, B30-N°-palmitoyl-Thr®®Lys®* human insulin, B29-N°-(N-palmitoyl-y-glutamyl)- des(B30) human insulin, B29-N*-(N-lithocholyl-y-glutamyl)-des(B30) human insulin, B29-N*- (w-carboxyheptadecanoyl)-des(B30) human insulin and B29-N*-(w-carboxyheptadecanoyl) human insulin. in another embodiment the insulin derivative is B29-N°*-myristoyl-des(B30) human insulin. in another embodiment the insulin hexamer of the invention further comprises at least 3 phe- nolic molecules.

In another embodiment the invention provides an insulin preparation comprising R-state insu- lin 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 (li)

A-B-C-D-X (I) : wherein: ‘

Ais a chemical group which reversibly binds to a His®'® Zn?" site of an insulin hexamer;

Bis a linker selected from + A valence bond « A chemical group G® of the formula -B'-B-C(O)-, -B'-B%-S0,-, -B'-B%CH.-, or -B'-

B%-NH-; wherein B' is a valence bond, -O-, -S-, or -NR®-,

Bis a valence bond, Ci-Cis-alkylene, C,-Cys-alkenylene, C,-Cyg-alkynylene, arylene, heteroarylene, -C,-C,s-alkyl-aryl-, -C>-C,g-alkenyl-aryl-, -C,-Cyg-alkynyl-aryl-, -C(=0)-

C4-Cyg-alkyl-C(=0)-, -C(=0)-C4-Cys-alkenyl-C(=0)-, -C(=0)-C;-C1s-alkyl-O-C1-C1s- : alkyl-C(=0)-, -C(=0)- C;-Cys-alkyl-S-C;-Cys-alkyl-C(=0)-, -C(=0)-C,-Cs-alkyl-NR®-C;-

Cys-alkyl-C(=0)-, -C(=0)-aryl-C(=0)-, -C(=0)-heteroaryl-C(=0}-; wherein the alkylene, alkenylene, and alkynylene moieties are optionally substituted by ~CN, -CF,, -OCF,, -OR®, or -NR°R’ and the arylene and heteroarylene moieties are optionally substituted by halogen, -C(O)OR®, -C(O)H, OCOR®, -SO;, -CN, -CF,, -

OCF, -NO,, -OR®, -NR®R’, C,-C,s-alkyl, or C-C,s-alkanoyl;

R®and R are independently H, C,-C,-alkyl;

Cis 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 ~ 30 different; and

X is -OH, -NH, 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, sul- fonamides, 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, thia- zolidinediones, 5-mercaptotetrazoles, or 4-cyano-1,2,3-triazoles.

In another embodiment of the invention A is § X

Y — vy

HN ET or HN Gc

R® RY UN 0 re o R R" wherein

Xis =0, =S or =NH

Yis-S-, -O-or -NH-

R® and R!' are independently hydrogen or C,-Ce-alkyl, :

R® is hydrogen or C,-Cg-alky! or aryl, R® and R® may optionally be combined to form a double bond,

R™ and R' are independently hydrogen, aryl, C;-Cs-alkyl, or -C(O)NR™R"’ £ and G are independently C,-Cs-alkylene, arylene, -aryl-C,-Cg-alkyl-, -aryl-C-Cs-alkenyl- or heteroarylene, wherein the alkylene or alkenylene is optionally substituted with one or more substituents independently selected from halogen, -CN, -CF;, -OCF3, aryl, -COOH and —NH,, and the arylene or heteroarylene is optionally substituted with up to four substituents R'3, R'4,

RS, and R™

E and R'® may be connected through one or two valence bonds, G and R'? may be con- nected through one or two valence bonds;

R™ R“ Rand R"™ are independently selected from

«hydrogen, halogen, -CN, -CH,CN, -CHF,, -CF,, -OCF3, -OCHF, -OCH,CF;, -OCF CHF, -S(0),CFa, -OS(0),CFs, -SCFs, -NO2, -OR™, -NR*R", -SR™, -NR™S(0),R", -S(0),NR*RY, -S(O)NR"R", -S(O)R™, -S(O):R"*, -0S(0), R*, -C(O)NR™R", -OC(O)NR'RY, -NR'®C(O)R", -CH,C(O)NR"R", : -OC,-Ce-alkyl-C(O)NR"R", -CH,OR'®, -CH,0C(O)R'®, -CH.NR"R"", -OC(O)R", -OC,-Cs-alkyl-C(O)OR'®, -OC,-Ce-alkyl-OR'®, -SC,-Ce-alkyl-C(O)OR™, : -C,-Ce-alkenyl-C(=0)OR™, -NR'-C(=0)-C,-C-alkyl-C(=O)OR'®, -NR*.C(=0)-C,-Ce-alkenyl-C(=O)OR"® , -C(O)OR™, or -C-Ce-alkenyl-C(=O)R™®, =O, or -C,-Ce-alkenyl-C(=0)-NR"R", ¢ C,-Cg-alkyl, C,-Ce-alkenyl or C-Cs-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, OR", and -NR"™R" « aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, . aryl-C-Ce-alkenyl, aroyl-C,-Ce-alkenyl, aryl-C,-Cg-alkynyl, heteroaryl, heteroaryt-Ci-

Cs-alkyl, heteroaryl-C,-Cg-alkenyl or heteroaryl-C,-Ce-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR'®, -CH,C(O)OR', -CH,0R'®, -CN, -CF3, -OCF, -NO,, -OR, -NR"R", S(O),R'®, aryl and C;-Cs-alkyl,

R'® and R'7 independently are hydrogen, OH, C,-Cx-alkyl, aryl-C-Ce-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CN, -CF;, -OCF3, -OC,-Ce-alkyl, -C(O)OC,-Cs-alkyl, -COOH and —NH, and the aryl groups may optionally be substituted by halogen, -C(O)OC,-Cs-alkyl, -COOH, -CN, -CF;, -

OCF, -NO,, -OH, -OC;-Cg-alkyl, -NH,, C(=0) or C,-Ce-alkyl; R'® and R'” 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, R", R"™, R', and R™A, } In another embodiment of the invention E is phenylene or naphtylene optionally substituted with up to four substituents, R'3, R", R™, and R"*. in another embodiment of the invention E is 15 ’

R RS

; ) or 3 o rR" 14 R rR" R 15

R R' ra : 14 rR" R

R™ R'*

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, R'3, R™, R'®, and R'™*

In another embodiment of the invention E is benzofuranylidene optionally substituted with up to four substituents R'>, R", R'®, and R™"

In another embodiment of the invention E is _ rR" o rR oO

VT / rR" le) rR"

RrR®

In another embodiment of the invention E is carbazolylidene optionally substituted with up to four substituents R™>, R™ R', and R"™*,

In another embodiment of the invention E is

R' rR" /

Ia rR"? ’

In another embodiment of the invention E is quinolylidene optionally substituted with up to } four substituents R', R™, R", and R"%A,

In another embodiment of the invention E is 15

R R'S = ) N or RY RY 3 rR"

R

In another embodiment of the invention E is indolylene optionally substituted with up to four substituents R™?, R™, R', and R"™, :

In another embodiment of the invention E is

RY RY RS RM RY R™ RY RY y ) — y " NH : N—

PAE R'

In another embodiment of the invention R® is Hydrogen.

In another embodiment of the invention R® and R® are combined to form a double bond.

In another embodiment of the invention R' is C,Cs-alkyl.

In another embodiment of the invention R'® is methyl.

In another embodiment of the invention G is phenylene optionally substituted with up to four substituents, R™>, R™, R'®, and R", in another embodiment of the invention R" is Hydrogen.

In another embodiment of the invention R'? is Hydrogen.

In another embodiment of the invention R'?, R*, R'® and R'>* are independently selected from : « hydrogen, halogen, -NO,, -OR®, -NR'R", -SR', -NR"®S(0),R", -S(0).NR*R", -S(O)NR™R', -S(O)R®, -S(O),R®, -0S(0), R™, -NR™C(0)RY, -CH,0OR", - :

CH,OC(O)R, -CHNR™R", -OC(O)R*®, -OC,-Cs-alkyl-C(O)OR', -OC;-Cs- alkyl-C(O)NR'R", -OC;-Ce-alkyl-OR®, -SC,-Cs-alkyl-C(O)OR™, ~C,-Cs-alkenyl-

C(=O)OR’, -C(O)OR'®, or —C,-Cs-alkenyl-C(=0)R"®, ¢ C,-Ce-alkyl, Co-Cs-alkenyl or C,-Cg-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF;, -OCF,, -OR™, and -NR"™R" e aryl, aryloxy, aroyl, arylsulfanyl, aryl-C,;-Ce-alkoxy, aryl-C;-Cs-alkyl, aryl-C,-

Ce-alkenyl, aroyl-C,-Cg-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C,-Ce-alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR", -CH,C(O)OR'®, -CH,OR'®, -CN, -CF3, -OCF3, -NO,, -OR™, -NR™R"” and C,-Cs-alkyl.

In another embodiment of the invention R*, R™, R'® and R'** are independently selected from « hydrogen, halogen, -NO., -OR?, -NR'*R"’, -SR'®, -S(0O),R"®, -0S(C), R"®, - : CH,OC(O)R'®, -OC(O)R’®, -OC,-Cs-alkyl-C(O)OR'®, -OC,-C¢-alkyl-OR'®, -SC,-C¢- alkyl-C(O)OR'®, -C(O)OR", or —C,-C¢-alkenyl-C(=0)R™, ¢ C,-Ce-alkyl or C4-Cs-alkenyl which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF;, -OR', and -NR'RY e aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Cg-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR'®, -CH,C(O)OR', -CH,OR", -CN, -CF3, -OCF,, -NO,, OR’, -NR"R"” and C,-C¢-alkyl.

In another embodiment of the invention R*, R™, R'® and R*** are independently selected from

« hydrogen, halogen, -NO,, -OR?, -NR"R", -SR', -S(0),R"®, -0S(0). R™, -

CH,OC(O)R'®, -OC(O)R®, -OC,-Cs-alkyl-C(O)OR", -OC;-Ce-alkyl-OR'™, -SC;-Ce- alkyl-C(O)OR'®, -C(OYOR, or ~C,-Ce-alkenyl-C(=0)R'®, « C,-C¢-alkyl or C,-C¢-alkenyl which may optionally be substituted with one or more substituents selected from halogen, -CF3, -OR’®, and -NR"™R" ’ e aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, C(O)OR', -CN, -NO,, OR, -NR'"R"” and C;-Ce-alkyl. in another embodiment of the invention R'®, R*, R' and R*** are independently selected from « hydrogen, halogen, -OR®, -OC,-C-alkyl-C(O)OR', or -C(O)OR, « C,-Cs-alkyl which may optionally be substituted with one or more substituents se- lected from halogen, -OR'®, and -NR'"R"” « aryi, aryloxy, aryl-C,-Cg-alkoxy, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, C(O)OR™, OR, and C;-Ce-alkyl.

In another embodiment of the invention R'® and R" independently are hydrogen, C1-Cx- alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more sub- stituents selected from halogen, -CF3, -OCF3, -OC,-Cg-alkyl, -COOH and —NH,, and the aryl groups may optionally be substituted by halogen, -COOH, -CN, -CF3, -OCF;, -NO;, -OH, -

OC,-Ce-alkyl, -NH,, C(=0) or C4-Ce-alkyl; R' and R'” when attached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom, the hetero- cyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxy- gen and sulphur, and optionally containing one or two double bonds

In another embodiment of the invention R'® and R" independently are hydrogen, Cy-Cax- alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more sub-

stituents selected from halogen, -CF,, -OC,-C;-alkyl, -COOH and —NH,, and the aryl groups may optionally be substituted by halogen, -COOH, -CN, -CF3, -OCF3, -OH, -NH,,0r C4-

Ce-alkyl.

In another embodiment of the invention Ais

R" © N N o Mu N RM

N, N, /\ N, N \

N w ON N ds OF Nn L—

H R H rR H 0) wherein

RZ is hydrogen or C,-Cg-alkyl, R* is hydrogen or C;-Cg-alkyl,

UandV are a valence bond or C,-Ce-alkylene optionally substituted with one or more hy- droxy, C+-Ce-alkyl, or aryl independently, Jis Ci-Cs-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is option- ally substituted with up to three substituents R%, R* and R%,

L is C,-Ce-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is option- ally substituted with up to three substituents R*, R® and R%,

R', R™, RZ R®, R*, R%, R® and R” are independently selected from «hydrogen, halogen, -CN, -CH.CN, -CHF,, -CF3;, -OCF;, -OCHF;, -OCH,CF;, -OCF,CHF,, -S(0),CF3, -SCF3, -NO,, -OR®, -NR*R?, -SR?, -NR?®S(0),R?, -S(0),NR?R?, -S(O)NR®R?, -S(0)R*, -S(0),R®, -C(O)NR®R?, -OC(O)NR*R*,

NRZC(0)RZ, -NRZC(0)OR?, -CH,C(O)NR®R?, -OCH,C(O)NR?®R?, -CH,0R?, -CH,NR?®R?, -OC(0)R?, -OC;-Cg-alkyl-C(O)OR?, -SC4-Cs-alkyl-C(O)OR?, ~C2-C¢- alkenyl-C(=0)OR?, -NR?-C(=0)-C;-Cg-alkyl-C(=0)OR?, -NR?-C(=0)-C1-Ce- alkenyl-C(=0)OR?, -C(=0)NR?®-C,-Cg-alkyl-C(=0)OR?, -C,-Ce-alkyl-C(=O)OR or -C(O)OR?, ¢ C,-Cs-alkyl, C>-Ce-alkenyl or C,-Cg-alkynyl, :

which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR?, and -NR®R* e aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C,-Cg-alkoxy, aryl-C+-Cs-alkyl, aryl-C,-

Ce-alkenyl, aryl-C,-Cs-alkynyl, heteroaryl, heteroaryl-C4-Cs-alkyl, heteroaryl-C»-Ce- : alkenyl or heteroaryl-C,-Cg-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)ORZ, -CN, -CF3, -OCF;, -NO,, -OR%, -NR*R? and

C;-Cs-alkyl,

R? and R? independently are hydrogen, C-Ce-alkyl, aryl-C;-Ce-alkyl or aryl, or R?® and R* when attached to the same nitrogen atom together with the said nitrogen atom may forma 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms se- lected 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 C;-Cg-alkylene optionally substituted with one or more hydroxy, Ci-Ce-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 R*, R® and R*

In another embodiment of the invention J is arylene optionally substituted with up to three substituents R%, R? and R*

In another embodiment of the invention J is phenylene optionally substituted with up to three substituents RZ, R* and R*

In another embodiment of the invention J is

RZ lo) Jag

SY R*

H in another embodiment of the invention R?, R? and R* are independently selected from :

» hydrogen, halogen, -CHF,, -CF,, -OCF3;, -OCHF, -OCH,CF;, -OCF,CHF,, -SCF3, -

NO, -OR?, -NRZR?, -SR%, -C(O)NR®R?, -OC(O)NR”R”, -NR*C(O)R”,

NRZC(O)OR?, -CH,C(OINRZR?, -OCH,C(O)NR®R?, -CH,OR?, -CH,NR*R”, -OC(O)R?, -0C,-Cs-alkyl-C(O)OR?, -SC,-Ce-alkyl-C(O)OR?, ~C,-Cs-alkenyl-

C(=0)ORZ, -NR?-C(=0)-C,-Ce-alkyl-C(=0)OR?, -NR?*-C(=0)-C:-Cs- : alkenyl-C(=O)OR?-, -C(=O)NRZ-C,-Cs-alkyl-C(=O)OR?, -C;-C-alkyl-C(=O)OR?, or -C(O)OR?, » C;-Cg-alkyl, C,-Cs-alkenyl or C,-Cs-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR?, and -NR*?R* = aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C,-Cs-alkoxy, aryl-C-Ce-alkyl, aryl-C2-

Ce-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C;-Ce-alkyl, heteroaryl-C,-Ce- alkenyl or heteroaryl-C,-Ces-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(OYOR?, -CN, -CF;, -OCFa, -NO,, -OR?, -NR*R* and

C,-Ce-alkyi

In another embodiment of the invention R%2, R® and R* are independently selected from « hydrogen, halogen, -OCF,, -OR%, -NR?R?, -SR?, -NR®C(0)R”, -NR?*C(O)OR?, -OC(O)R?, -OC;-Cy-alkyl-C(O)OR?, -SC,-Ce-alkyl-C(O)OR?, —C-C¢-alkenyl-

C(=0)OR?, -C(=0)NR?-C,-Ce-alkyl-C(=O)OR?, -C;-Cg-alkyl-C(=0)OR*, or -C(O)OR?, » C,-Ce-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CF;, -OCF;, -OR?, and -NR*R? «aryl, aryloxy, aroyl, aryl-C,-Cg-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-C.-C- alkyl,

of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)ORZ, -CN, -CFs, -OCFs, -NO,, -OR%, -NR*?R* and

C,-Ce-alkyl

In another embodiment of the invention R%, R? and R* are independently selected from « hydrogen, halogen, -OCF,, -OR%, -NR®R?, -SR?, -NR®C(O)R”, -NR®C(0)OR”, -OC(O)R?®, -OC,-Cs-alkyl-C(0)OR?, -SC,-Cq-alkyl-C(O)OR?, —C,-Ce-alkenyl-

C(=0)ORZ, -C(=0)NR?-C,-Ce-alkyl-C(=0)OR?, -C,-Cq-alkyl-C(=0)OR?, or -C(O)OR?%, « C,-Cs-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, or -CF, 16 «aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-C-Ce- alkyl, : of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C{O)OH, -CN, -CF3, -NO,, or C;-C¢-alkyl in another embodiment of the invention R® is hydrogen or methyl

In another embodiment of the invention R% is hydrogen

In another embodiment of the invention R? is Hydrogen, C4-C¢-alkyl or aryl

In another embodiment of the invention R? is Hydrogen or C4-Ce-alkyl

In another embodiment of the invention R% is Hydrogen or C-Ce-alkyl

In another embodiment of the invention V is a valence bond

In another embodiment of the invention V is C,-Cs-alkylene optionally substituted with one or more hydroxy, C;-Ce-alkyl, or aryl

In another embodiment of the invention L is C4-Cg-alkylene or arylene, wherein the arylene is optionally substituted with up to three substituents R*, R* and R”

In another embodiment of the invention L is C-Ce-alkylene

In another embodiment of the invention L is phenylene optionally substituted with up to three substituents R%, R® and R%’ in another embodiment of the invention R?°, R® and R? are independently selected from

« hydrogen, halogen, -CHF,, -CF,, -OCF5, -OCHF,, -OCH,CF3, -OCF,CHF2, -SCFs, -

NO,, -OR?, -NR®R?, -SR%, -C(O)NR®R?, -OC(0)NR¥R?, -NR®C(O)R?, -NR%G(O)ORZ, -CH,C(OINR®R?, -OCH,C(O)NRZR?, -CH,0R®, -CH,NRZR, -OC(O)R?, -0C,-Ce-alkyl-C(O)OR?, -SC,-Cg-alkyl-C(O)OR?, —C,-Ce-alkenyl-

C(=0)OR?, -NR*-C(=0)-C;-Ce-alkyl-C(=0)OR?, -NR?-C(=0)-C:-Ce- alkenyl-C(=0)OR?-, -C(=0)NR?-C,-Cg-alkyl-C(=O)OR?, -C,-Ce-alkyl-C(=O)OR?, or -C(O)ORZ, ¢ C,-C¢-alkyl, C,-Cs-alkenyl or C-Ce-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR%®, and -NR*®R%® e aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, aryl-Co-

Ce-alkenyl, aryl-C,-Cg-alkynyl, heteroaryl, heteroaryl-C,-Cs-alkyl, heteroaryl-C-Cs- alkenyl or heteroaryl-C,-Cg-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, -CF3, -OCF3, -NO,, -OR?, -NR?R? and

C,-Ce-alkyl

In another embodiment of the invention R**, R* and R¥ are independently selected from « hydrogen, halogen, -OCF;, -OR?, -NR?R?, -SR?, -NR?®C(0)R%, -NR®*C(O)OR?, -OC(0O)R®, -OC;-Cs-alkyl-C(O)ORZ, -SC,-Cq-alkyl-C(O)OR?, —C,-C¢-alkenyl-

C(=0)OR?®, -C(=O)NR%-C,-C¢-alkyl-C(=0)OR?, -C,-C¢-alkyl-C(=O)OR?, or ~ -C(O)OR?, » C,-Ce-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CF, -OCF3, -OR?, and -NR*R?% « aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-C,-Ce- alkyl,

of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, -CF3, -OCF3, -NO,, -OR%, -NR*R% and

C,-Cs-alkyl

In another embodiment of the invention R%®, R* and R¥ are independently selected from «hydrogen, halogen, -OCF;, -OR®, -NR®R?, -SR?, -NR*C(O)R*, NRZC(O)ORZ, -OC(O)RZ, -OC;-Ce-alkyl-C(O)OR?, -SC,-Ce-alkyl-C(O)OR?, ~C,-Cq-alkenyl-

C(=0)OR?, -C(=0)NR?-C,-C;-alkyl-C(=0)OR?, -C,-Ce-alkyl-C(=O)OR?, or -C(O)OR?, » C;-Ce-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, or -CF, e aryl, aryloxy, aroyl, aryl-C,-Cg-alkoxy, aryl-C;-Ce-alkyl, heteroaryl, heteroaryl-C-Ce- alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OH, -CN, -CF;, -NO,, or C,-Cs-alkyl

In another embodiment of the invention R?! is hydrogen or methyl

In another embodiment of the invention R*' is hydrogen

In another embodiment of the invention R? is Hydrogen, C,-Ce-alkyl or aryl

In another embodiment of the invention R? is Hydrogen or C,-Cg-alkyl

In another embodiment of the invention R? is Hydrogen or C,-Ce-alkyl

In another embodiment of the invention R' and R'® are independently selected from « hydrogen, halogen, -CN, -CFs, -OCF,, -NO,, -OR?, -NR®R%, -SR%, -S(O)R%, -S(0),R?, -C(O)NR®R?, -CH,0R?, -OC(0)R?, -OC,-C¢-alkyl-C(O)OR?, -SC;-C,- alkyl-C(O)OR?, or -C(O)OR?, + C1-Ce-alkyl, Co-Ce-alkenyl or C,-Ce-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF3, -OR%, ‘and -NR**R*

« aryl, aryloxy, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-C,-Ce-alkyl of which the cyclic moieties optionally may be substituted with one or mare substitu- ents selected from halogen, -C(O)ORZ%, -CN, -CFs, -OCF3, -NO,, -OR?, -NR?®R* and

C,-Cs-alkyl

In another embodiment of the invention R'® and R'® are independently selected from « hydrogen, halogen, -CN, -CF3, -NO,, -OR%, -NR¥R?, or -C(O)OR?, « C,-Ce-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CF3, -OCF3, -OR%, and -NR®R? « aryl, aryloxy, aryl-C,-Ce-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, -CFs, OCF, -NO,, -OR?, -NR®R* and

C;-Ce-alkyl in another embodiment of the invention A is .N EN

N Tr

N _

H

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

HO 25 0 0 0 <3 (2 I or or w' w? w? (=

H wherein W*, W2, and W? are independently OH, SH or NH, and the phenyl, naphthalene or benzocarbazole rings are optionally substituted by one or more R* independently

Q is selected from the following: + a valence bond o —CHN(R*®)- or ~SO,N(R*')- 1 + A compound of the formula wherein Z' is S(O), or CH,, Z%is N,-O-or

S -S-,and nis 1 or 2;

Tis » C,-Cg-alkylene, C,-Cs-alkenylene or C,-Cg-alkynylene, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF,, -OR*, and -NR¥R* e Arylene, arylene-oxy, -aryl-oxycarbonyl-, -aroyl-, -aryl-C;-Ce-alkoxy-, -aryl-C4-Ce¢- alkyl-, -aryl-C,-Ce-alkenyl-, -aryl-C,-Ce-alkynyl-, heteroarylene, -heteroaryl-C;-Ce- . alkyl-, -heteroaryl-C,-Ce-alkenyl- or -heteroaryl-C,-Cs-alkynyl-, wherein the cyclic moieties are optionally substituted by one or more substituents selected from halo- “gen, -C(O)OR®, -C(O)H, -CN, -CF3, -OCF3, -NO,, -OR¥, -NR¥R®, C,-Ce-alkyl or Cy-

Cs-alkanoyl, * A valence bond

Rand R® independently are hydrogen, Ci-Ce-alkyl, aryl-C;-Ce-alky! or aryl, or R* and R® 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 se- lected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds,

R¥ and R* are independently hydrogen, C-Ce-alkyl or C,-Ce-alkanoyl.

R3 is hydrogen, halogen, -CN, -CH.CN, -CHF;, -CF3, -OCF3, -OCHF;, -OCH.CF3, -OCF,CHF,, -S(0).CFs, -SCF3, -NO,, -OR®, -C(O)R*%, -NR¥R®, -SR¥, -NR¥S(0).R*, -S(0),NRZR®, -S(0O)NR*R®, -5(0)R®, -S(0),R*, -C(O)NR¥R™®, -OC(O)NR¥R®, : -NRS2C(O)R®, -CH,C(O)NR¥R®, -OCH,C(0)NR¥R®, -CH,OR%, -CH,NR*R®, -OC(O)R¥, -

OC,-Ce-alkyl-C(O)OR™, -SC,-Ce-alkyl-C(O)OR ~C,-Cg-alkenyl-C(=O)OR, :

-NR¥-C(=0)-C1-Cs-alkyl-C(=0)OR™, -NR*-C(=0)-C:-Ce-alkenyl-C(=0)OR™-, C1-Ce-alkyl

C1-Ce-alkanoyl or -C(O)OR™,

In another embodiment of the invention Mis 10) 0) or w! Ww?

In another embodiment of the invention M is (] ast

Ww!

In another embodiment of the invention M is

O anes

WwW? in another embodiment of the invention Mis

HO Oo in another embodiment of the invention Mis (0)

YS

HO

In another embodiment of the invention Q is a valence bond, =CH,N(R*)-, or ~SO,N(R*)-

In another embodiment of the invention Q is a valence bond

In another embodiment of the invention T is ¢ A valence bond

» C,-Cs-alkylene, C,-Ce-alkenylene or C,-Cg-alkynylene, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF;, -OR®, and -NR¥R* « Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as : defined in claim 70

In another embodiment of the invention T is : e 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)OR¥, -CN, -CF3, -OR%, -NR*R*, C,-Cs-alky! or C;-Ce-alkanoyl

In another embodiment of the invention the cyclic moiety in T is optionally substituted by halogen, -C(O)OR®, -OR%, -NR®2R®, C,-Cs-alkyl or C-Cs-alkanoyl

In another embodiment of the invention the cyclic moiety in T is optionally substituted by ‘halogen, -C(OYOR™ or -OR*

In another embodiment of the invention T is a valence bond

In another embodiment of the invention R* and R*' are independently hydrogen or C;-C¢- alkyl in another embodiment of the invention R* is hydrogen, halogen, -CN, -CF3, -OCF3, -SCF3, -NO,, -OR%, -C(O)R¥, -NR*?2R®, -SR®, -C(O)NR¥R¥, -OC(O)NR*R¥, -NR*C(O)R®, -OC(0)R%, -OC;-Cs-alkyl-C(O)OR?, -SC,-Ce-alkyl-C(O)OR* or -C(O)OR™

In another embodiment of the invention R* is hydrogen, halogen, -CF3, -NO,, -OR*, -NR¥R®, -SR%, -NR*’C(0)R*, or -C(0)OR*

In another embodiment of the invention R* is hydrogen, halogen, -CF3, -NO,, -OR*, -NR¥R™®, or -NR*C(O)R®

In another embodiment of the invention R* is hydrogen, halogen, or -OR>

In another embodiment of the invention R* and R* independently are hydrogen, C-Cs-alkyl, or aryl - In another embodiment of the invention R* and R® independently are hydrogen or C;-Cq- alkyl

In another embodiment of the invention A is

A Al 1 : \ rr Sar TON AR wherein A’ is a valence bond, C;-Ce-alkylene, -NH-C(=0)-A%, -C,-C¢-alkyl-S-, -Cs-

Ce-alkyl-O-, -C(=0)-, or -C(=0)-NH-, wherein any C,-C¢-alkyl moiety is optionally substituted by RA

A?is a valence bond, C,-Cg-alkylene, C4-Cs-alkenylene, or -C4-Ces-alkyl-O-;

R™ is C,-Ce-alkyl, aryl, wherein the alkyl or aryl moieties are optionally substituted by one or more halogen, cyano, nitro, amino;

AR! is a valence bond, arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted by one or more R'® independently

R™ is selected from « hydrogen, halogen, -CN, -CH,CN, -CHF,, -CF;, -OCF;, -OCHF,, -OCH,CF3, -OCF,CHF,, -S(0),CF3, -0S(0),CF3, -SCF3, -NO,, -OR, -NR'R'®, -SR'C,

NR'®S(0),R™, -S(0),NR'°R®, -S(O)NR'°R™, -S(0)R'C, -S(0),RC, -0S(0), R'°, -C(O)NR'CR'®, -OC(O)NR'R'®, -NR'°C(O)R™, -CH,C(ONR'°R®, -OC;-C¢- alkyl-C(O)NR'®R®, -CH,OR'®, -CH,OC(O)R'®, -CH,NR'*R®, -OC(O)R'®, -OC;-Cs- alky-C{O)OR'C, -OC,-Cs-2ikyl-OR'C, -S-C,-Cg-alky!-C(Q)OR'®, ~C,-Ce-alkenyl-

C(=0)OR®, -NR'C-C(=0)-C,-Ce-alkyl-C(=O)OR’®, -NR'®-C(=0)-C;-Cs- alkenyl-C(=0)OR'C , -C(O)OR'®, —C,-C¢-alkenyl-C(=0)R'®, =O, -NH-C(=0)-0-C;-

Ce-alkyl, or -NH-C(=0)-C(=0)-0-C,-C¢-alkyl » C,-Cg-alkyl, C,-Ce-alkenyl or C-Cg-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, “OCF, -OR'C, and -NR'°R™ « aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C-Ce-alkoxy, aryl-C4-Ce-alkyl, aryl-C,-Ce-alkenyl, aroyl-C-Cs-alkenyl, aryl-C,-Cs-alkynyl, heteroaryl, heteroaryl-C,-

Ce-alkyl, heteroaryl-C,-Ce-alkenyl or heteroaryl-C,-Cg-alkynyl,

of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR'C, -CH,C(O)OR'®, -CH,OR'®, -CN, -CF;, -

OCF, -NO,, -OR'®, -NR"°R'® and C,-Cq-alkyl,

R' and R"™ independently are hydrogen, -OH, C;-Cg-alkyl, Ci-Ce-alkenyl, aryl-C;-Ce-alkyl or aryl, wherein the alkyl moieties may optionally be substituted with one or more substituents ‘ selected from halogen, -CN, -CF3, -OCF3, -O-C,-Ce-alkyl, -C{0)-0-C,-Ce-alkyl, -COOH and -

NH,, and the aryl moieties may optionally be substituted by halogen, -C(O)OC;-C¢-alkyl, -

COOH, -CN, -CF3, -OCF3, -NO;, -OH, -OC;-Cs-alkyl, -NH,, C(=0) or C,-Ce-alkyl; R'® and R'® 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 heteroa- toms selected from nitrogen, oxygen and sulphur, and optionally containing one or two dou- ble bonds,

C'is a valence bond, C;-Cs-alkkylene, -C-Cg-alkyl-O-, -C4-Cs-alkyl-NH-, -NH-C,-Ce-alkyl, -NH-C(=0}-, -C(=0O)-NH-, -O-C;-C¢-alkvl, -C(=0)-, or -C4-Cg-alkyl-C(=0)-N(R)- wherein the alkyl moieties are optionally substituted by one or more R'¥ independently

R'E and RF are independently selected from C,-Ce-alkyl, aryl optionally substituted by one or more halogen, -COOH,;

AR? is ¢ a valence bond « C;-Cs-alkylene, C,-Cq-alkenylene , C,-Ce-alkynylene wherein the alkyl, alkenyl and alkynyl moieties are optionally substituted by one or more R? independently; « arylene, -aryloxy-, -aryloxy-carbonyl-, aryl-C,-Cs-alkyl, -aroyl-, aryl-C;-Ce-alkoxy-, aryl-C,-Ce-alkenyl-, aryl-C,-Ce-alkynyl-, heteroarylene, -heteroaryl-C,-Ce-alkyl-, -heteroaryl-C,-Ce-alkenyl-, -heteroaryl-C,-Ce-alkynyl- wherein the aryl and heteroaryl moieties are optionally substituted by one or more R? independently;

R? is C,-Ce-alkyl, C1-Ce-alkoxy, aryl, aryloxy, aryl-C,-Ce-alkoxy, -C(=0)-NH-C,-Ce-alkyl-aryl, heteroaryl, heteroaryl-C-Cg-alkoxy, -C4-Ce-alkyl-COOH, -O-C4-Ce-alkyl-COOH, -S(0),R%, -C,-Ce-alkenyl-COOH, -OR?, -NO,, halogen, -COOH, -CF;, -CN, -N(R®R?"), wherein the aryl or heteroaryl moieties are optionally substituted by one or more C,-Ces-alkyl, Ci

Ce-alkoxy, -C,-Ce-alkyl-COOH, -C,-Cg-alkenyl-COOH, -OR?®, -NQ,, halogen, -COOH, -CFs. -CN, or -N(R®R*)

R? and R* are independently selected from hydrogen and C,-Ce-alkyl

In another embodiment of the invention A’ is a valence bond, C,-Ce-alkylene, -NH-C(=O)-A%, ~C;-Cg-alkyl-S-, -C4-Ce-alkyl-O-, or -C(=0)-, wherein any C,-Ce-alkyl moiety is optionally sub- stituted by R™

In another embodiment of the invention A' is a valence bond, C4-Ce-alkylene, NH-C(=0)-A%, -C,-Ce-alkyl-S-, or -C,-Ce-alkyl-O, wherein any C,-Ce-alkyl moiety is optionally substituted by

RA

In another embodiment of the invention A' is a valence bond, C,-Cs-alkylene, or -NH-C(=0)-A?, wherein any C;-C¢-alkyl moiety is optionally substituted by R'™

In another embodiment of the invention A' is a valence bond or C,-Ce-alkylene, wherein any C4-Ce-alkyl moiety is optionally substituted by R'A in another embodiment of the invention A’ is a valence bond

In another embodiment of the invention A? is a valence bond or -C,-Ce-alkyl-O-

In another embodiment of the invention A? is a valence bond

In another embodiment of the invention AR! is arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted by one or more R'® independently

In another embodiment of the invention AR' is selected from the group of compounds con- sisting of phenylene, biphenylylene, naphthylene, anthracenylene, phenanthrenylene, fluo- renylene, indenylene, azulenylene, furylene, thienylene, pyrrolylene, oxazolylene, thia- zolylene, 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-oxa- diazolylene, 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, ben- zisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene, quinazolinylene, quinoliz- inylene, quinolinylene, isoquinolinylene, quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene, azepinylene, diazepinylene, or acridinylene, optionally substituted by one or more R'® independently

In another embodiment of the 4nvention AR" is selected from phenylene, biphenylylene, naphthylene, pyridinylene, fyrylene, indolylene, or carbazolylene, optionally substituted by one or more R'® independently

In another embodiment of the invention AR" is selected from the group of compounds con- sisting of phenylene, indolylene, or carbazolylene, optionally substituted by one or more R* independently :

In another embodiment of the invention AR’ is phenylene optionally substituted by one or more R'® independently

In another embodiment of the invention AR' is indolylene

In another embodiment of the invention AR" is carbazolylene

In another embodiment of the invention AR' is

R'® oo \

In another embodiment of the invention AR' is

RrR® oe ’ 15 In another embodiment of the invention R'® is selected from « hydrogen, halogen, -CN, -CF3, -OCF;, -NO,, -OR'C, -NR'°R™®, -SR, -S(0),R", -NR'°C(O)R™®, -OC,-Ce-alkyl-C(O)NR'’R'®, —C,-C¢-alkenyl-C(=0)OR’®, -C(O)OR'®, =0, -NH-C(=0)-0-C;-Cg-alkyl, or -NH-C(=0)-C(=0)-O-C,-Cs-alkyl » C-Cs-alkyl or C,-Cs-alkenyl which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF3, -OR'C, and -NR'R'° e aryl, aryloxy, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, aryl-C,-Ce-alkenyl, heteroaryl, het- eroaryl-C,-Ce-alkyl, or heteroaryl-C,-Ce-alkenyl of which the cyclic moieties optionally may be substituted with one or more substituents se- lected from halogen, -C(O)OR'S, -CN, -CF3, -OCF,, -NO;, -OR'®, -NR'“R'" and C;-Cs-alkyl

In another embodiment of the invention R'® is selected from

«hydrogen, halogen, -CF3, -NO,, -OR'®, -NR'°R', -C(O)OR'¢, =0, -NH-C(=0)-0-C+-

Cs-alkyl, or -NH-C(=0)-C(=0)-0-C;-Ce-alkyl ¢ C,-Cg-alkyl

In another embodiment of the invention R'® and R'® independently are hydrogen, C4-Ce- alkyl, or aryl, wherein the aryl moieties may optionally be substituted by halogen or -COOH

In another embodiment of the invention R'® and R'° 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 C' is a valence bond, C;-Cs-alkylene, -C;-Ce-alkyl-O-, -Cy-Cg-alkyl-NH-, -NH-C;-Cg-alkyl, -NH-C(=0)-, -C(=0)-NH-, -O-C4-Ce-alkyl, -C(=O}-, or -C1-

Ce-alkyl-C(=0)-N(RF)- wherein the alkyl moieties are optionally substituted by one or more

RF independently

In another embodiment of the invention C' is a valence bond, -CH-, -CHx-CH-, -CH-O-, -CHy-CH,-O-, -CH,-NH-, -CH,-CH-NH-, -NH-CH-, -NH-CH,-CH.-, -NH-C(=0})-, -C(=O}-NH-, -0O-CHz-, -O-CH-CHg-, or -C(=0)-

In another embodiment of the invention R'® and R'F are independently selected from C,-

Ce-alkyl "In another embodiment of the invention AR? is « a valence bond : « C,-Ce-alkylene, wherein the alkyl is optionally substituted by one or more R* inde- pendently « arylene, aryl-C,-Ce-alkyl, heteroarylene, wherein the aryl and heteroaryl moieties are optionally substituted by one or more R* independently

In another embodiment of the invention AR? is « a valence bond « C,-Ce-alkylene, wherein the alkyl is optionally substituted by one or more R* inde- pendently oo « phenyl, phenyl-C,-Ce-alkyl, wherein the phenyl moieties are optionally substituted by one or more R* independently

In another embodiment of the invention R# is C,-Ce-alkyl, C,-Ce-alkoxy, aryl, aryloxy, het- eroaryl, -C4-Ce-alkyl-COOH, -0-C,-Ce-alkyl-COOH, -S{0).R%, -C,-Ce-alkenyl-COOH, -OR®, -NO,, halogen, -COOH, CF, -CN, -N(R?®R*"), wherein the aryl or heteroaryl moieties are optionally substituted by one or more C,-Ce-alkyl, C1-Ce-alkoxy, -C,-Cs-alkyl-COOH, -C,-

Cgalkenyl-COOH, -OR?, -NO,, halogen, -COOH, -CF;, -CN, or -N(R®R?®)

In another embodiment of the invention R? is C,-Ce-alkyl, C4-Cg-alkoxy, aryl, -OR?, -NOz, halogen, -COOH, -CFs, -CN, -N(R?®R?%), wherein the aryl is optionally substituted by one or more C;-Cs-alkyl, C1-Ce-alkoxy, -OR?, -NO,, halogen, -COOH, -CFs, -CN, or -N(R*R*)

In another embodiment of the invention R? is C;-Cg-alkyl, C1-Cs-alkoxy, aryl, halogen, -CF3, . wherein the aryl is optionally substituted by one or more C+-Ce-alkyl, halogen, -COOH, -CF3, or -CN :

In another embodiment of the invention R* is C;-Cs-alkyl, C,-Cs-alkoxy, phenyl, halogen, -CF3, wherein the phenyl is optionally substituted by one or more C,-Cs-alkyl, halogen, -COOH, -CF3, or -CN

In another embodiment of the invention A is

N=N "NN ar es

S wherein AR? is C,-Cs-alkylene, arylene, heteroarylene, -aryl-Ci.¢-alky}- or -aryl-C,.¢-alkenyl-, wherein the alkylene or alkenylene is optionally substituted with one or more substituents in- dependently selected from halogen, -CN, -CFs, OCF, aryl, -COOH and ~NH,, and the ary- lene or heteroarylene is optionally substituted with one or more R* independently

R* is independently selected from « hydrogen, halogen, -CN, -CH.CN, -CHF, -CF3, -OCF3, -OCHF,, -OCH.CF3;, -OCF,CHF, -S(0),CF3, -OS(0),CF3, -SCF3, -NO,, -OR*®, -NR*R*, -SR™, -NR®S(0),R*, -S(0).NR®R*, -S(O)NR*®R*, -S(0)R®, -S(0).R*®, -0S(0), R®, -C(O)NR®R*, -OC(O)NR*®R*, -NR®C(0)R*¢, -CH,C(O)NR®R*’, -OC;-C- alkyl-C(O)NR®R®, -CH,0R®, -CH,0C(0)R?, -CH,NR*R*", -OC(0)R®, -0C,-Ce- alkyl-C(O)OR?®, -OC,-Ce-alkyl-OR*®, -SC,-C¢-alkyl-C(O)OR*®, —~C,-Cs-alkenyl-

C(=O)OR™®, -NR*-C(=0)-C,-Cs-alkyl-C(=0)OR®, -NR*-C(=0)-C;-Cs- alkenyl-C(=0)OR® , -C(O)OR™®, or —C,-Ce-alkenyl-C(=0)R™, ¢ C,;-Cg-alkyl, C,-Ce-alkenyl or C,-Ce-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, OCF, -OR®®, and -NR*®R*

« aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C,-Ce-alkoxy, aryl-C4-Ce-alkyl, -aryl-C,-Cg-alkenyl, aroyl-C,-Ce-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C1-

Ce-alkyl, heteroaryl-C,-Cg-alkenyl or heteroaryl-C»-Cs-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?®, -CH,C(O)OR®, -CH,0R®, -CN, -CF3, -OCFs, -NO,, -OR®, -NR*®R*¢ and C,-Cs-alkyl,

R® and R* are independently hydrogen, OH, CF,, C-Ciz-alkyl, aryl-C,-Ce-alkyl, -C(=0)-C,-Ce-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, OCF, -OC4-Cs-alkyl, -C(O)OC4-Ce- alkyl, -C(=0)-R*®, -COOH and —NH,, and the aryl groups may optionally be substituted by halogen, -C(0)OC;-Ce-alkyl, -COOH, -CN, -CF3, -OCF3, -NO,, -OH, -OC4-Cs-alkyl, -NH,

C(=0) or C,-Cs-alkyl; R*® and R3® when attached to the same nitrogen atom may forma 3 to 8 membered heterocyclic ring with the said nitrogen atom, the heterocyclic ring optionally containing one or two further heteroatoms selected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds

R® is C,-Ce-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl option- ally substituted with one or more C,-Ce-alkyl.

In another embodiment of the invention AR? is arylene, heteroarylene, or aryl-C,s-alkyl, wherein the alkyl! is optionally substituted with one or more substituents independently se- lected from halogen, -CN, -CF, -OCF;, aryl, -COOH and —NH,, and the arylene or heteroary- lene is optionally substituted with one or more R* independently

In another embodiment of the invention AR? is arylene optionally substituted with one or more R* independently

In another embodiment of the invention AR? is phenylene, naphthalene or anthranylene op- tionally substituted with one or more R3 independently

In another embodiment of the invention AR? is phenylene optionally substituted with one or more R* independently in another embodiment of the invention R* is independently selected from «halogen, -CN, -CF3, -NO,, -OR%®, -NR®R?, -SR*®, -0C,-Ce-alkyl-C(0)OR™ or -C(O)OR®

« C,-Cq-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CF3, -OCF;, -OR*, and -NR%®R*° e aryl, aryl-C,-Ce-alkyl, heteroaryl, or heteroaryl-C,-Ce-alkyl E of which the cyclic moieties optionally may be substituted with one or more substitu- : ents selected from halogen, -C(O)OR®, -CN, -CFs, -OCF3, -NO,, -OR®, -NR®R* and C,-Cg-alkyl )

In another embodiment of the invention R* is independently selected from halogen, -OR®, -NR¥R*, .C(O)OR®, -OC;-C,-alkyl-C(O)OR®, or C4-Ce-alkyl

In another embodiment of the invention R* and R*® are independently hydrogen, CFs, Ci-Cy-alkyl, or -C(=0)-C,-Ce-alkyl; R®® and R* 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

NR y - yr 74 “NAR N wherein AR! is C;-Cg-alkylene, arylene, heteroarylene, -aryl-C,.s-alkyl- or -aryl-C,¢-alkenyl-, wherein the alkylene or alkenylene is optionally substituted with one or more substituents in- dependently selected from halogen, -CN, -CF;, -OCF,, aryl, -COOH and —NH,, and the ary- lene or heteroarylene is optionally substituted with one or more R* independently

R* is independently selected from « hydrogen, halogen, -CN, -CH,CN, -CHF,, -CF3;, -OCF3, -OCHF,, -OCH.CF;3, -OCF,CHF,, -S(0),CF3, -OS(0),CF3, -SCF3, -NO,, -OR*®, -NR*R*°, -SR*, -NR*S(0);R*, -S(0).NR*®R*’, -S(O)NR*®R®, -S(O)R®, -S(0),R*®, -0S(0), R®, -C(O)NR*®R*C, -OC(O)NR*RC, -NR*®C(O)R*°, -CH,C(O)NR**R*®, -OC-Cs- alkyl-C(O)NR®R*°, -CH,OR*®, -CH,0C(O)R*®, -CH,NR*R*’, -OC(0)R*, -OC;-C;- alkyl-C(O)OR®, -OC,-C¢-alkyl-OR*, -SC,-Cs-alkyl-C(O)OR*®, —C,-Cs-alkenyl-

C(=0)OR*®, -NR*8-C(=0)-C;-C¢-alkyl-C(=O)OR*®, -NR**-C(=0)-C4-Cq- alkenyl-C(=0)OR*® , -C(O)OR*, or ~C,-Ce-alkenyl-C(=O)R*®,

¢ C,-Cs-alkyl, Co-Ce-alkenyl or C,-Cs-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, “OCF, -OR®, and -NR*®R* « aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C,-Cs-alkoxy, aryl-C,-Ce-alkyl, aryl-C,-Ce-alkenyl, aroyl-C,-Ce-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C+-

C.-alkyl, heteroaryl-C,-Ce-alkenyl or heteroaryl-C,-Ce-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR*®, -CH,C(O)OR®*, -CH,OR*, -CN, -CF;, -OCFs, -NO,, -OR*®, -NR*R*® and C,-C¢-alky!,

R*® and R* are independently hydrogen, OH, CFs, C;-Ci2-alkyl, aryl-Cy-Ce-alkyl, -C(=0)-R®, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCFs, -OC4-Cg-alkyl, -C(O)OC-Ce-alkyl, -COOH and —

NH,, and the aryl groups may optionally be substituted by halogen, -C(0)OC-Ce-alkyl, -

COOH, -CN, -CF3, OCF, -NO,, -OH, -OC,-Ce-alkyl, -NH,, C(=0) or C+-Ce-alkyl; R*® and R* 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 heteroa- toms selected from nitrogen, oxygen and sulphur, and optionally containing one or two dou- ble bonds

R%¥ is C,-Ce-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl option- ally substituted with one or more C;-Cg-alkyl.

In another embodiment of the invention AR! is arylene, heteroarylene or aryl-Cs¢-alkyl-, wherein the alkyl is optionally substituted with one or more substituents independently se- lected from halogen, -CN, -CF3, -OCF;, aryl, -COOH and —NH_, and the arylene or heteroaryl is optionally substituted with one or more R* independently

In another embodiment of the invention AR? is arylene or heteroarylene optionally substituted with one or more R** independently

In another embodiment of the invention AR* is phenylene, naphtylene, anthrylene, thienylene, pyridylene, or benzodioxylene optionally substituted with one or more R** inde- pendently

In another embodiment of the invention AR* is phenylene optionally substituted with one or more R* independently

In another embodiment of the invention R** is independently selected from hydrogen, halo- gen, -CF, -OR*®, -NR*®R*, C,-Ce-alkyl, aryl-C,-Cs-alkenyl or aryl optionally substituted with : one or more substituents selected from halogen, -CF;, or -OR®

In another embodiment of the invention R*® and R*° are independently hydrogen, CF,

C4-Cz-alkyl, -C(=0)-R*®, or ary!

In another embodiment of the invention R* is C,-Cs-alkyl, phenyl optionally substituted with one or more halogen, or a heteroaryl selected from isoxazole and thiadiazole optionally sub- stituted with one or more C;-Cs-alkyl

In another embodiment of the invention C consists of 0 to 5 neutral amino acids independ- ently 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 G® is of the formula -B'-B%-C(0)-, -B'-B%SO,- or -8'-

B?-CH,-, wherein B' and B? are as defined in claim 1 in another embodiment of the invention G® is of the formula -B'-B2-C(O)-, -B'-B%-S0O,- or -B'-

B2-NH-, wherein B' and B? are as defined in claim 1

In another embodiment of the invention G® is of the formula -B'-B%-C(0)-, -B'-B*-CH,- or -B'-

B%NH-, wherein B' and B? are as defined in claim 1

In another embodiment of the invention G® is of the formula -B*-B%-CH,-, -B'-B%-SO,- or -B'-

B2-NH-, wherein B' and B? are as defined in claim 1 in another embodiment of the invention G? is of the formula -8'-B2-C(O)- or -B'-B?-SO_-, wherein B' and B? are as defined in claim 1

In another embodiment of the invention G® is of the formula -B'-B2-C(O)- or -B'-B*-CH_-, wherein B' and B? are as defined in claim 1

In another embodiment of the invention G® is of the formula -B'-B2-C(O)- or -B'-B%NH-, wherein B' and B? are as defined in claim 1

In another embodiment of the invention G® is of the formula -B'-B2-CH;- or -B'-B*SO-, wherein B' and B? are as defined in claim 1 :

In another embodiment of the invention G8 is of the formula -B'-B%-NH- or -B'-B*-SO~, wherein B' and B? are as defined in claim 1

In another embodiment of the invention GB is of the formula -B'-B%-CH,- or -B'-B*NH- , wherein B' and B? are as defined in claim 1

In another embodiment of the invention G® is of the formula -B'-B?-C(O)-

In another embodiment of the invention G® is of the formula -B'-B?-CH,-

In another embodiment of the invention G® is of the formula -B'-B?-SO,-

In another embodiment of the invention GP? is of the formula -B'-B?-NH-

In another embodiment of the invention B' is a valence bond, -O-, or -S- - 10 In another embodiment of the invention B' is a valence bond, -O-, or -N(R°®)-

In another embodiment of the invention B' is a valence bond, -S-, or -N(R°)-

In another embodiment of the invention B' is -O-, -S- or -N(R®)-

In another embodiment of the invention B' is a valence bond or —O-

In another embodiment of the invention B' is a valence bond or —-S- in another embodiment of the invention B' is a valence bond or -N(R®)-

In another embodiment of the invention B! is -O-or -S-

In another embodiment of the invention B' is -O-or -N(R®)-

In another embodiment of the invention B is -S-or -N(R®)-

In another embodiment of the invention B' is a valence bond in another embodiment of the invention B' is -O-

In another embodiment of the invention B' is -S-

In another embodiment of the invention B is -N(R®)-

In another embodiment of the invention B? is a valence bond, C;-Cie-alkylene, C2-Cis- alkenylene, C,-C,g-alkynylene, arylene, heteroarylene, -C,-Css-alkyl-aryl-, -C(=0)-Cy-C1a- akyl-C(=0)-, -C(=0)-C,-Css-alkyl-O-C;-Cys-alkyl-C(=0)-, -C(=0)-C;-C1g-alkyl-S-C;-Cg-alkyl-

C(=0)-, -C(=0)-C1-Cs-alkyl-NR®-C,-Cys-alkyl-C(=O)-; and the alkylene and arylene moieties are optionally substituted as defined in claim 1 in another embodiment of the invention B? is a valence bond, C;-Cis-alkylene, Co-Ce- alkenylene, C,-Cys-alkynylene, arylene, heteroarylene, -C1-Cyg-alkyl-aryl-, -C(=0)-Cy-Crs- alkyl-C(=0)-, -C(=0)-C;-C,g-alkyl-O-C4-C1s-alkyl-C(=0)-, and the alkylene and arylene moie- ties are optionally substituted as defined in claim 1

In another embodiment of the invention BZ is a valence bond, C;-Cqg-alkylene, C-Cis- alkenylene, C,-Cig-alkynylene, arylene, heteroarylene, -Cy-Cye-alkyl-aryl-, -C(=0)-C4-Cys- alkyl-C(=0)-, and the alkylene and arylene moieties are optionally substituted as defined in claim1 : :

In another embodiment of the invention B? is a valence bond, C-Cis-alkylene, arylene, het- eroarylene, -C4-Cg-alkyl-aryl-, -C(=0)-C,-Cg-alkyl-C(=0}-, and the alkylene and arylene moieties are optionally substituted as defined in claim 1

In another embodiment of the invention B? is a valence bond, C,-Cs-alkylene, arylene, het- ‘ eroarylene, -C,-Cys-alkyl-aryl-, and the alkylene and arylene moieties are optionally substi- tuted as defined in claim 1 :

In another embodiment of the invention B? is a valence bond, C,-C,s-alkylene, arylene, -C+-

C,s-alkyl-aryl-, and the alkylene and arylene moieties are optionally substituted as defined in claim 1

In another embodiment of the invention B? is a valence bond or -C,-Cys-alkylene, and the al- kylene 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 inde- pendently 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 —NH,

In another embodiment of the invention X is -NH;

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 con- sisting of B29-N°-myristoyl-des(B30) human insulin, B29-N*-paimitoyl-des(B30) human insu- lin, B29-N°-myristoyl human insulin, B29-N"-palmitoyl human insulin, B28-N"-myristoy Lys***

Pro? human insulin, B28-N°-paimitoyl Lys®? Pro®% human insulin, B30-N°-myristoyl-

Thi®®LysB* human insulin, B30-N°-palmitoyl-Thr®®Lys®* human insulin, B29-N°*-(N- palmitoyl-y-giutamyl)-des(B30) human insulin, B29-N°-(N-lithocholyl-y-glutamyl)-des(B30) human insulin, B29-N°-(o-carboxyheptadecanoyl)-des(B30) human insulin and B29-N°*~(o- carboxyheptadecanoyl) human insulin.

In another embodiment of the invention the insulin derivative is B29-N°-myristoyl-des(B30) human insulin.

In another embodiment of the invention the insulin hexamer as defined above further com- prises at ieast 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 prepara- tion which comprises adding a zinc-binding ligand as defined above to the insulin prepara- tion.

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

His®'%-Zn?" site « optionally attaching a fragment consisting of 0 to 5 neutral o- or $-amino acids e attaching a fragment comprising 1 to 20 positively charged groups independently se- lected from amino or guanidino groups

The compounds of the present invention may be chiral, and it is intended that any enanti- omers, 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 di- astereomers may be formed. It is intended that any diastereomers, as separated, pure of partially purified diastereomers or mixtures thereof are included within the scope of the inven- - tion.

Furthermore, some of the compounds of the present invention may exist in different tauto- meric 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, pharma- ceutically 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-, diethyi-, 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 dia- betes in a patient in need of such a treatment comprising an R-state hexamer of insulin ac- cording 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 in- sulin.

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/m! of insulin.

Zinc ions may be present in an amount corresponding to 10 to 40 pg Zn/100 U insulin, more preferably 10 to 26 pg 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, chioro-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 pre- . sentinvention.

The action of insulin may further be slowed down in vivo by the addition of physiologically : acceptable agents that increase the viscosity of the pharmaceutical preparation. Thus, the pharmaceutical preparation according to the invention may furthermore comprise an agent which increases the viscosity, such as polyethylene glycol, polypropylene glycol, copolymers thereof, dextrans and/or polylactides.

In a particular embodiment the insulin preparation of the invention comprises between 0.001 % by weight and 1 % by weight of a non-ionic surfactant, for example tween 20 or 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 refer- ring 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% TFA in water

B: 0.01% TFA in acetonitrile

The analysis was performed at 40 °C by injecting an appropriate volume of the sample (pref- erably 1 pL) 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.

MS lonisation mode: API-ES

J er

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 pg/mL of the com- pound 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 pL 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 pl/min through approx. 1 m. 75 p fused silica capillary to the API interface of APt 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 spec- trometer, 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.

MS Experiment: Start: 100 amu Stop: 800 amu Step: 0.2 amu

Dwell: 0.571 msec

Co 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 o Hewlett Packard series 1100 G1315A DAD diode array detector o 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:

The analysis is performed at 40 °C by injecting an appropriate volume of the sample (pref- erably 1 pl) onto the column which is eluted with a gradient of acetonitrile.

Gradient 5% - 100% acetonitrile linear during 7.5 min at 1.5 )

I en

Detection 210 nm (analogue output from DAD)

CE [Seen

MS ionisation mode API-ES

I x

After the DAD the flow is divided yielding approximately 1 mi/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 controlied 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 pg/ml of the com- pound 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 pu! 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 pl/min through approx. 1 m 75 p fused silica capillary to the API interface of

S API 150 spectrometer.

The remaining 1.48 m/min was passed through the UV detector and to the ELS detector.

MS Experiment: Start: 100 amu Stop: 800 amu Step: 0.2 amu :

Dwell: 0.571 msec

EEL

EXAMPLES

Example 1 1H-Benzotriazole -

N

N,

N

H

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

N CH,

CII

N CH,

Example 3 1H-Benzotriazole-5-carboxylic acid

H

N

N,

OH

Example 4 4-Nitro-1H-benzotriazole

H

N

N,

N

NI oO "0

Example 5 5-Amino-1H-benzotriazole :

N NH,

CY

N

H

Example 6 5-Chloro-1H-benzotriazole

N Cl

IT

N

H

Example 7 5-Nitro-1H-benzotriazole

H

N

IL N +0

Oo

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

N

JL x 0 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 hydrox- ide (35 mL). The mixture was stirred at room temperature for 16 hours and then 1N hydro- chloric acid (45 mL) was added. The mixture was added water (200 mL) and extracted with ethyl acetate (2 x 500 mL). The combined organic phases were evaporated in vacuo to afford 0.44 g of 4-{(1H-benzotriazole-5-carbonyl)amino]benzoic acid. By filtration of the aqueous phase a further crop of 4-[(1H-benzotriazole-5-carbonyl)amino]benzoic acid was isolated (0.529). "H-NMR (DMSO-dg): & 7.97 (4H, s), 8.03 (2H, m), 8.66 (1H, bs), 10.7 (1H, 5), 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 14:

TN i LI i /\ _H

N’ Sl + mi Ny _— SLY ! l wherein U, J and R® are as defined above, and J is optionally containing up to three sub- stituents, R%, R® and R* 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, pentafluoro- phenoxy, N-succinyloxy 3,4-dihydro-4-oxo-3-(1,2,3-benzotriazinyl)oxy, benzotriazole 5-COO0, or any other leaving group known to act as a leaving group in acylation reactions. The acti- vated benzotriazole-5-carboxylic acid is then reacted with R?-(CH,),-B' in the presence of a base. The base can be either absent (i.e. R?>-(CH,),-B’ acts as a base) or triethylamine, N- ethyl-N,N.-diisopropylamine, N-methylmorpholine, 2,6-lutidine, 2,2.6,6-tetramethylpiperidine, potassium carbonate, sodium carbonate, caesium carbonate or any other base known to be useful in acylation reactions. The reaction is performed in a solvent solvent such as THF, di- oxane, toluene, dichloromethane, DMF, NMP or a mixture of two or more of these. The reaction is performed between 0 °C and 80 °C, preferably between 20 °C and 40 °C. When the acylation is complete, the product is isolated by extraction, filtration, chromatography or other methods known to those skilled in the art. i

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

Example 9 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid phenylamide

H .

N

N, H

N N

Ng

Benzotriazole-5-carboxylic acid (856 mg), HOAt (715 mg) and EDAC (1.00 g) were dissolved in DMF (17.5 mL) and the mixture was stirred at room temperature 1 hour. A 0.5 mL aliqot of this mixture was added to aniline (13.7 pL, 0.15 mmol) and the resulting mixture was vigor- ously shaken at room temperature for 16 hours. 1N hydrochloric acid (2 mL) and ethyl ace- tate (1 mL) were added and the mixture was vigorously shaken at room temperature for 2 hours. The organic phase was isolated and concentrated in vacuo to afford the title com- pound.

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-Renzotriazole-S-carboxylic acid (4-methoxyphenyllamide

H

N

N, H

TTA

0] oH

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)aminojphenyl}carbamic acid tert-butyl ester

N

SON

RON $=)

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

N

N 0 4) Q, A,

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

H :

N

N IDE

N N F ag

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

N

N, H Cl

N N

0

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

Example 15 (General Procedure (A)) 4-[(1H-Benzotriazole-5-carbonyl)aminolbenzoic acid methyl ester

Jy

N H

Seu

FOL, 5 3

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 0 | GP

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

J tI xO

O CH,

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

Example 18 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid benzytamide

JEN N

SOUPS

0

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

IIH LT

0

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

H

N

Ny a

N N

0 Cl

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

H

N o. cH, sole;

[0]

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

H

N

SO

[o]

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

H

N .

SO

}

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

H :

N,

I

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

Example 25 (General Procedure (A)) 4-{{(1H-Benzotriazole-5-carbonyl)amino}jmethyl}benzoic acid ’ fo)

N eV a.

N N

0

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

Example 26 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid phenethylamide

H

: N (SOY

N 0 ©

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

Example 27 (Generali Procedure (A)) 1H-Benzotriazole-5-carboxylic acid [2-(4-chlorophenyl)ethyljamide

I

N H

Tiny ° @

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)ethyllamide

A

0 XQ oH

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)ethylJamide

Jt

Sey o. ge

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)ethyllamide

N

Sey a

Se

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 y (J

N

JO x

Se

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

N

N, ME “N CC © ci

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

Example 33 (General Procedure (A)) : “1H-Benzotriazole-5-carboxylic acid methylphenylamide

N

N N ag

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

Example 34 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid benzylmethylamide

H

N

SOS Je

N

0

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)ethyljmethyl-amide

H

N o TC en,

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

Example 36 (General Procedure (A)) 1H-Benzotriazole-5-carboxylic acid methylphenethylamide

H

NS 2K “N N

X 0

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)ethyljmethylamide

H .

N

( RG :

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

H .

Ne CH, OH “N N 0 C ]

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 gl

N

H

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

N

N : .

H

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 .

EY

N. H

N :

H

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 o ox

N O )

N SR

N

H

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 oF te

N N

N

N H

H

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 2 JIA

N

N SR of

N

H

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

0] 0 Os Mom

N N H,C CH,

N H

N 3

H

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 lo} lo] lo] OY on oR H

N

. H

H

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 : 0) lo) oy or

N

SY

N

H

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 o od

N

STH

. N

H «

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 0 Las

N

H

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 2, J 0

N N or

N H

HN

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 0 yo

N

STH

N

H

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

OF -

RIFF

N Br

N

H

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

Cl

N

H

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 0

N N

H oO

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 rte

N

H

HPLC-MS (Method A): m/z: 329 (M+1); Rt = 2.34 min. 15 .

Example 56

N-(1H-Benzotriazol-5-yl)acetamide

H

OLS

N N” “CH,

H .

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 q ] 0

General procedure (B) for preparation of compounds of general formula I:

X X

>~y vy Eon wl)

HN » + Te Me NE o 0) Rr . i, wherein X, Y, E and R' are as defined above and E is optionally containing up to four op- tional substituents, R™, R", R*®, and R'"" 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 hetero- cyclic ring (eg thiazolidine-2,4-dione (X = O; Y = 8), rhodanine (X = Y = 8) and hydantoin (X = 0; Y = NH) in the presence of a base, such as sodium acetate, potassium acetate, ammo- nium 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 0)

Ps 0

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 uL, 0.6 mmal) and the resulting mixture was shaken at 115 °C for 16 hours. After cooling, the mixture was con- centrated 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 com- pounds 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 (0) ’ CH, .

Ms N-ch,

HN A

0]

HPLC-MS (Method C): m/z: 249 (M+1); Rt = 4.80 min

Example 60 (General procedure (B)) 5-Naphthalen-1-ylmethylenethiazolidine-2,4-dione 0]

Ps

HN -

I~

HPLC-MS (Method A): m/z: 256 (M+1); Rt = 4,16 min.

Example 61 (General procedure (B)) . 5-Benzylidene-thiazolidine-2,4-dione

0)

Ms

HN S

6

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 0] 5 cn,

HN, I le}

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 0

Ys cl

HN ~ 0

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 0 0.

Ms 0

HN a 0

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

Oo

Ps OH :

HNC AS oC :

Oo .

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 >

S

S “CH w I ’ 0

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 0

HN -

F

0

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 o Ho, »~s CH, X

HN x 0

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)phenyljacetamide

L H

N._CH,

IT T

0

HPLC-MS (Method A): m/z: 263 (M+1); Rt = 3,90 min.

Example 70 (General procedure (B)) 5-Biphenyi-4-yimethylene-thiazolidine-2,4-dione 3 ® >s

WIC

0 .

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 lo)

Po o ww J |® lo} - 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 lo}

WIL fo} ] Ae

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

CH,

WIT

J

HPLC-MS (Method A): m/z: 312 (M+1); Rt = 6,87 min. :

Example 74 (General procedure (B)) 5-Naphthalen-2-ylmethylene-thiazolidine-2,4-dione 6)

UC

HN So

Oo

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

Ye 0

S

HN >

ICL

O

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

S ho Cl

S w AT 0

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

S CH,

Ra Nach,

N lo)

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 s ?.

N

S 0) oO

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 ? s

Ys “CH,

HN 1 0

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 .

S

Na Och,

N F o -

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 }

S

SUPP

HN a 0 .

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

CH s Ce

Ys N._ CH, 3 0

HPLC-MS (Method A): m/z: 293 (M+1); Rt = 5,99 min.

Example 83 (General procedure (B)) 5-Biphenyl-4-yimethylene-2-thioxothiazolidin-4-one

J

Ms 0)

HNL o]

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

Ss

Ms jg

HN AN 0 . o}

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

Ss hp

HN. 1 . 0

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 8 0 J w IMT )

HPLC-MS (Method A): m/z: 328 (M+1); RT = 6,89 min.

Example 87 (General procedure (B)) 5-Naphthalen-1-yimethylene-2-thioxothiazolidin-4-one

S .

SC

HN ~

Fe

HPLC-MS (Method A): m/z: 272 (M+1); Rt = 6,43 min.

Example 88 (General procedure (B)) 5-(3-Methoxybenzyi)thiazolidine-2,4-dione 0}

Ms

HN oC oO

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

3 pores " ~ o

HPLC-MS (Method A): nvz: 392 (M+23), Rt = 4.32 min. i

Example 90 (General procedure (D)) 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenoxyl-butyric acid 0 0 hu oA,

Br 0}

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 ’ 0}

Js

N Br oO

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

Oo

MM Br

S wl ICT

Oo .

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

0]

I

H

NN

Cl 0

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

Yo 3 \

HN

AP

O

HPLC-MS (Method A): m/z: 212 (M+1); Rt = 3.09 min.

Example 95 (General procedure (B)) 5-(4-Bromothiophen-2-yimethylene)thiazolidine-2,4-dione

Oo Br

Ps \

HN sya

O

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 0 Cl

Ys

HN

= cl 0)

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 oO CH;

Ys N

J .

HPLC-MS (Method A). m/z: 259 (M+1); Rt = 3.55 min.

Example 98 (General procedure (B)) 5-(1H-Indol-3-yimethylene)thiazolidine-2,4-dione oO 5

HN so As NH : 0]

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 0) >=s

HN

IAN C

B® - 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

Oo >

HN

~N

O CH,

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 0 i s

HN

AT

O CH,

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 0)

HN

A

O CH,

HPLC-MS (Method A): m/z: 270 (M+1); Rt = 4,30 min.

Example 103 (General procedure (B)) 51 -(4-Bromophenyl)-ethylidene}-thiazolidine-2,4-dione 0) ha Br

S

AMT

>

O CH,

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 3. [J

Ps

HN

I~

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 0]

OS

Nn os

O CH,

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 0 Hs see)

HN x oO CH,

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

Q seas

HN :

IS

O CH,

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 o}

Ps Oven,

HN

= F

O CH,

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 0] -

HN

~~ Br 0 CH,

HPLC-MS (Method A): m/z: 300 (M+1); Rt = 4,13 min.

Example 110 (General procedure (B)) 5-Anthracen-9-yimethylenethiazoiidine-2,4-dione 3,

Ps

HN

NS dU © HPLC-MS (Method A): m/z: 306 (M+1); Rt = 4,64 min.

Example 111 (General procedure (B)) 5-(2-Methoxynaphthalen-1-yimethylene)-thiazolidine-2 J4-dione o ho Oo

QQ

HN a rT U

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 3 0}

S “CH ‘ee dU

HPLC-MS (Method A). m/z: 286 (M+1); Rt = 4,31 min.

Example 113 (General procedure (B)) 5-(4-Dimethylaminonaphthalen-1-yimethylene)-thiazolidine-2,4-dione : 3, LL

No

NS © CH, ]

N

©

HPLC-MS (Method A): m/z: 299 (M+1); Rt = 4,22 min.

Example 114 (General procedure (B)) 5-(4-Methylinaphthalen-1-yimethylene)-thiazolidine-2,4-dione 3

HN S (1) CH,

AN : .

RS

; :

HPLC-MS (Method A): m/z: 270 (M+1); Rt = 4,47 min.

Example 115 (General procedure (B)) 5-Pyridin-2-yimethylene-thiazolidine-2,4-dione

0) ~s A

HN

A

N

Oo

Example 116 5-Pyridin-2-ylmethyl-thiazolidine-2,4-dione 0) sD

HN ~

N

0) 5-Pyridin-2-yimethylene-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: R; = 0.30 (SiO,; EtOAc: heptane 1:1)

Example 117 (General procedure (B)) 5-(1 H-imidazol-4-yimethylene)-thiazolidine-2,4-dione 0)

Ys n=

AAS

O

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

3 LI

SYA

HN Js lo} .

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

F

: oI

Se}

HN. J lo}

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

J

Ss

Ms YC

AN Ax = i

Example 121 (General procedure (B)) 5-(3-Methoxybenzylidene)thiazolidine-2,4-dione lo]

Ps

HNC AS oCHs 0

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

SH

HN

SYOW CH,

O

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 0)

H

MN Och,

HN ~~ (0)

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

O

»S in Se 0) Cl

Example 125 (General procedure (B)) 5-Benzofuran-7-ylmethylenethiazolidine-2,4-dione o = »>S ©

HN x ’ . 0)

HPLC-MS (Method C): m/z: 247 (M+1); Rt = 4,57 min.

Example 126 (General procedure (B)) 5-Benzo[1,3]dioxol-4-yimethylenethiazolidine-2,4-dione

0 $9 *S

Oo .

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

CH o) 03 *S 0) CH,

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

Oo *S

HN Js oCHs

O O

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

Os in SL) oO OH

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

O Cl >S 0) Cl "H-NMR (DMSO-ds): 7.60 (2H, "s"), 7.78 (1H, s), 7.82 (1H, s).

Example 131 (General procedure (B)) 5-(2-Chlorobenzylidene)thiazolidine-2,4-dione %s 0) Cl "H-NMR (DMSO-ds): 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

As an SL) 0) Br "H-NMR (DMSO-ds): 7.33 (1H, t), 7.52 (1H, t), 7.60 (1H, d), 7.71 (1H, 5), 7.77 (1H, d).

Example 133 (General procedure (B)) 5-(2,4-Dimethoxybenzylidene)thiazolidine-2,4-dione

CH,

Os 0) 0 Och,

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 oO >S

HN_ 0 Och,

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

Oo »sF 0) F

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 oO CH *S 3

HN As e) CH,

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

CH GH, ®) 3.0 ~g©

HN Js

Oo Och,

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

Q H

»S ©

HN

O OcH,

HPLC-MS (Method C): m/z: 252 (M+1); Rt = 3,64 min.

Example 139 (General procedure (B)) 5-(4-Hydroxynaphthalen-1-ylimethylene)thiazolidine-2,4-dione 0 Clo

S88

HN. La

Oo "H-NMR (DMSO-d): 6 = 7.04 (1H, d), 7.57 (2H, m), 7.67 (1H, 1), 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 jthiazolidine-2,4-dione

As in SU) @) OE

F

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

Oo >S gy °° O

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

Q

0

HN 1 o} 0]

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

L

HN S$

EN

SY

HPLC-MS (Method A): m/z: 250 (M+1); Rt = 4,30 min.

General procedure (C) for preparation of compounds of general formula I:

X

> Oy Eh wl iN + R'" > NE 10 0 0 R

I, wherein X, Y, E, and R' are as defined above and E is optionally containing up to four op- tional substituents, R™, R™, R", and R"** 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

0

Js Br

SCL, 0)

A mixture of thiazolidine-2,4-dione (90%, 65 mg, 0.5 mmol), 3,4-dibromobenzaldehyde (132 mg, 0.5 mmol), and piperidine (247 uL, 2.5 mmol) was shaken in acetic acid (2mL) 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 com- pound. 'H NMR (Acetone-dq): 61 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 com- pounds 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

RK | OH

AN o CMs ©

Mp = 256 °C; 'H NMR (DMSO-d) 6 = 12.5 (s,broad, 1H), 10.5 (s,broad,1 H), 7.69 (s,1H), 7.51 (d, 1H), 7.19 (d,1H)3.88 (s,3H), 3C NMR (DMSO-ds) é¢c = 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

0

Vdc OH

WIT

0 CH,

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

OH

; Jou

Ps ©

SIOLY

0 Br HPLC-MS (Method C): m/z: 506 (M+23); Rt.= 4.27 min.

Example 148 (Genera! procedure (C)) 5-(4-Bromo-2,6-dichlorobenzylidene)thiazolidine-2,4-dione 2 Ci

HN 1 0 cl

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 ) OH i

Ss

Wl ICT o }

Mp 310-314 °C, 'H NMR (DMSO-d): én = 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). 3C NMR (DMSO-ds): 6c = 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 hy- dride (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

H,SO, (10% solution, 15 mL) was added followed by water until all salt was dissolved. The resulting solution was extracted with ethyl acetate (3x), the combined organic phases were dried with MgSO,, evaporated to dryness to afford 0.89 g of 6-hydroxy-2- naphtalenecarbaldehyde.

Mp.: 153.5-156.5 “©; HPLC-MS (Method A): m/z: 173 (M+1); Rt = 2.67 min; 'H NMR (DMSO- ds): Su = 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 OC. 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 mix- ture 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 Na,SO, 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-lodo-4-methoxybenzylidene)thiazolidiene-2,4-dione 0) = I 0 . 'H NMR (DMSO-d): 8+ 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); °C NMR (DMSO-d;): 8c 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). lodine (1.19 g, 4.7 mmol) was added in small por- tions and the mixture was stirred overnight at room temperature under nitrogen. The mixture was subsequently filtered and the residue washed with DCM. The combined filtrates were treated with an acqueous sodium thiosulfate solution (1 M) until the colour disappeared.

Subsequent extraction with dichloromethane (3 x 20 mL) followed by drying with MgSO, and evaporation in vacuo afforded 0.94 g of 3-iodo-4-methoxybenzaldehyde.

Mp 104-107 °C; HPLC-MS (Method A): m/z:263 (M+1), Rt = 3.56 min.;'H NMR (CDCl): 6, = 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 0)

SN

0 Br

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

H,C

J

0 0°

I 2

S ST )

O H

'H NMR (DMSO-d): 8 = 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-yImethylene) thiazolidine-2,4-dione lo] .

Ps =N 0 }

TH NMR (DMSO-d): 61 = 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 0]

Ms = N

HN SA

0]

HPLC-MS (Method A): m/z: 257 (M+1); Rt = 2.40 min.

Example 155 (General procedure (C)) 5-(6-Methylpyridin-2-yimethylene)thiazolidine-2,4-dione

0 CH, }

HN NaN . 0) 'H NMR (DMSO-d;): dy = 12.35 (s,broad, 1H), 7.82 (t,1H), 7.78 (s,1H), 7.65 (d,1H), 7.18 (d,1H), 2.52 (s,3 H); HPLC-MS (Method A): m/z: 221 (M+1); Rt = 3.03 min.

Example 156 (General procedure (C)) 5-(2,4-dioxothiazolidin-5-ylidenemethyl)-furan-2-yimethylacetate 0 c oI" A 0 o 0 'H NMR (DMSO-ds): 84 = 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-CH,COO); Rt = 2.67 min.

Example 157 (General procedure (C)) 5-(2,4-Dioxothiazolidin-5-ylidenemethyl)furan-2-sulfonic acid 0] ws /B\ 0 ys 3%

Oo 0

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-yimethylene)-thiazolidine-2,4-dione

He

Oy _ \ rN

HN 0

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

Oo eS

HN 1 7 o

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

Se

S i IA

S

0 OH

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 0 8

Sr

HN, x N 0]

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

N

~

Ves Ns 0

HPLC-MS (Method A): m/z: 272 (M+1); Rt = 1.38 min.

Example 163 (General procedure (C)) 5-(9-Ethyl-9H-carbazol-3-yimethylene)thiazolidine-2,4-dione or 0} N '

HN, 1 o :

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 0 HGH by (1

HN. 1 le} CH,

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 0) H

Vs HCN ol 30 0)

HPLC-MS (Method A): m/z: 259 (M+1); Rt = 3.24 min.

Example 166 (General procedure (C)) 5-(2-Ethylindol-3-yimethylene)thiazolidine-2,4-dione ° CH, eng

DP.

J : 2-Methylindole (1.0 g, 7.6mmol) dissolved in diethyl ether (100 mL) under nitrogen was treated with n-Butyl lithium (2 M in pentane, 22.8 mmol) and potassium tert-butoxide (15.2 mmol) with stirring at RT for 30 min. The temperature was lowered to —70 C and methyl lo- dide (15.2 mmol) was added and the resulting mixture was stirred at —70 for 2h. Then 5 drops of water was added and the mixture allowed to warm up to RT. Subsequently, the mix- ture 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 Na;SO4 and evaporated to dryness. The residur was purified by column chromatography on silica gel using heptane/ether( 4/1) as eluent. This afforded 720 mg (69 %) of 2-ethylindole. 'H NMR (DMSO-d; ): 5 = 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,1). 2-Ethylindole (0.5 g, 3.4mmol) dissolved in DMF (2 mL) was added to a cold (0 °C) premixed (30 minutes) mixture of DMF (1.15 mL) and phosphorous oxychloride (0.64 g, 4.16 mmol).

After addition of 2-ethylindole, the mixture was heated to 40 °C for 1 h, water (5 mL) was added and the pH adjusted to 5 by means of 1 N sodium hydroxide. The mixture was subse- quently extracted with diethyl ether, the organic phase isolated, dried with MgSO, and evapo- rated 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 gen- eral 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

Br fe) > cH,

SN

0]

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-yimethylenejthiazolidine-2,4-dione cl : d “A

NY

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 o 0]

ITC x Br o

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

A [

S oe

HN 1 N

Oo 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

QO cls at

HN =

[0]

HPLC-MS (Method A): m/z: 418 (M+1); Rt = 5.13 min.

Example 172 (General procedure (C)) 5-[4-(4-Nitrobenzyloxy)-benzylidenelthiazolidine-2,4-dione lo) nN .

Q o)

Ys

WILT

0

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 o 2, sy

ROE

8)

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 0 H

Ms "

HN SN

0

Re

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 o :

J OH

S wl IT 0)

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

Oo ws /

SON o) CH, 'H NMR (DMSO-d): 81 = 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-yimethylene)thiazolidine-2,4- dione ®) H 3 N

NIL

0) + 3¥o .Mp 330-333 °C, 'H NMR (DMSO-ds): 8 = 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 0] H.C »s 0 an SC

Oo

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

CH

®) ~Fl3 = Br

Oo .

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)phenylJamino}propionitrile

N

J

QL N

LSS ORY lo} SN

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 0 H ~s (N o

HN. J [ a

Oo O-CH,

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.

A, A

LSS aw, ; 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-6-carboxylic acid methyl ester (example 181, 59 mg; 0.195mmol) was stirred in pentanol (20 mL) at 145 °C for 16 hours. The mixture was evaporated to dryness affording the title compound (69 mg).

HPLC-MS (Method C): m/z: 359 (M+1); Rt.= 4.25 min.

Example 183 (General procedure (C)) 3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7- carboxylic acid 0 H HO sg N Oo

HN YN

0)

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

QL N

Oo

HPLC-MS (Method A): m/z: 335 (M+1); Rt = 4.55 min.

Example 185 (General procedure (C)) 5-(1-Benzenesulfonylindol-3- yimethylene)thiazolidine-2,4-dione o 950

MAL oO

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 0) N:N *s NG]

HN_ J 0)

HPLC-MS (Method A): m/z: 290 (M+1); Rt = 3.45 min.

Example 187 (General procedure (C)) 5-[4-(4-Nitrobenzyloxy)-benzylidenelthiazolidine-2,4- dione 0, te

Oo *s O 0)

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 d1,C. 0,0 gs (N

HN XJ

0 . HPLC-MS (Method A): m/z: 317 (M+1); Rt = 4.35 min.

Example 189 (General procedure (C)) 5-[2-(4-Pentylbenzoyl)-benzofuran-5- yimethylenejthiazolidine-2,4-dione

Q o 0

Ms Sgn oP

J CH,

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

F

3 A

S iN AY

O + oN,

HPLC-MS (Method A): (Anyone 1) m/z: 398 (M+1); Rt = 4.42 min.

Example 191 (General procedure (C)) 5-(4-Benzyloxyindol-3-yimethylene)thiazolidine-2,4- dione 0] H sg N

RENEE) c

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 o H,C. CH,

HN S

Oo

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

FF o FF 0 $0 *~s 0 an SIC dU

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 mix- ture was cooled to 0-5 °C. With stirring, trifluoromethanesulfonic acid anhydride (11.7 ml, 70 mmol) was added drop-wise. After addition was complete, the mixture was allowed to warm up to room temperature, and diethyl ether (200 ml) was added. The mixture was washed with water (2 x 250 ml), hydrochloric acid (3N, 200 ml), and saturated aqueous sodium chloride (100 ml). After drying (MgS04), 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-yimethylene)-thiazolidine-2,4-dione ®) H gs N

HN SO

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 o Br on

Ys

HN. A

Br 10] '"H NMR (DMSO-d; ): 6 = 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.

Q 0)

Nn

Ps B

HN ~

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 ace- tone (200 mL) at RT until a solution was obtained indicating full conversion to the indole po- tassium 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 mi. 20 mL portions of this solution were mixed with equimolar amounts of arylmethylbromides in R 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 pro- cedure C.

Example 197 (General procedure (C)) 4-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1- ylmethyl]lbenzoic acid methyl ester 0—CH,

Q Ar 0

Ps mo o}

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-yimethylene]thiazolidine-2,4-dione 0 k ve

Sg [2 o}

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 q ete p= N “5 0

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-ylmethyi]benzonitrile.

o! MN as HG ~ n/N o t 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.

Q ~

Pg N lo]

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 es- ter

GH,

O40

L H.C

S N

HNL o

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- yimethylene)thiazolidine-2,4-dione 0) CH,

Ch

ST

N

H SA

0

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 0) pu OH

S

HN So

Oo

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 ? OH

Ms

HN NS

0]

HPLC-MS (Method C): m/z: 348 (M+1); Rt. = 3.13 min 'H-NMR: (DMSO-d, ): 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 0 . Cl .

Ps

HN a 0)

Cl

H PLC-MS (Method C): m/z: 309 (M+1); Rt.= 4.07 min

Example 207 (General procedure (C)) 5-(2,6-Dichlorabenzylidene)thiazolidine-2,4-dione ? Ci

Ps

HN x

O Cl

Mp. 152-154°C.

HPLC-MS (Method C): m/z: 274 (M+1), Rt.= 3.70 min

H-NMR: (DMSO-d): 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-triflusromethylphenyl)-2,5-dimethyl-1H-pyrrol-3-yimethylene]thiazolidine- 2,4-dione

FF

F o wa)

Ne HC © 0

HPLC-MS (Method C): m/z: 436 (M+1); Rt. 4.81 min

Example 209 (General procedure (C)) 5-[1-(3,5-Dichloropheny!)-5-(4-methanesulfonylphenyl)-2-methyl- 1H-pyrrol-3-yimethylene]- thiazolidine-2,4-dione cl yo

Jghe N 0 0 CH,

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-methyi-1H-pyrrol-3-yimethylene}- thiazolidine-2,4-dione

HC, Lo 3 HCN

A O~-Lo lo] CH,

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-yllbenzoic acid :

HO o] a

Pg CNN > 0 1

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 o fH

W s © OH

HN “ o lo) : “CH,

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 0) fue

HN a o CH,

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 o

Ms ©

HN So 0

Och,

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 o - a o o}

J = SR)

OCH,

Mp: 2565-256 °C

HPLC-MS (Method C): m/z: 435 (M+1), Rt4.13 min,

Exampie 216 (General procedure (C)) 5-Benzofuran-2-yimethylenethiazolidine-2,4-dione 0 > 0 6]

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

Hs 3. 1

CH,

IST lo]

HPLC-MS (Method C): m/z:276(M+1); Rt.= 3.63, mp = 259-263 °C

'H-NMR: (DMS0-0s) 6= 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-Methyi-3-phenylallylidene)thiazolidine-2,4-dione i : lo)

AR

0

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 lo)

PN lo}

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

HN Ao lo!

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.

0 OH

He g Br Br

HN A

: ¢} Br

HPLC-MS (Method C): m/z: 4569 (M+1);Rt.= 3.65 min.

Example 222 (General procedure (C)) 5-(5-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione. 8) gC N

HN. J 0

Br

HPLC-MS (Method C): m/z: 339 (M+1), Rt = 3.37min.

Example 223 (General procedure (C)) 5-(7-Bromo-2-methylindol-3-ylmethylene)thiazolidine-2,4-dione. lo}

PghOs Noa

AALS ’ 0

HPLC-MS (Method C): m/z: 319 (M+1); Rt = 3.48min.

Example 224 (General procedure (C)) 5-(6-Bromoindol-3-yimethylene)thiazolidine-2,4-dione. 0 H

Ms (

WIS

0]

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-yimethylene)thiazolidine-2,4-dione.

o uy SH jog oH

HNC LG

0 .

HPLC-MS (Method C): m/z: 287 (M+1); Rt = 2.86 min. -

Example 226 (General procedure (C)) 5-(6-Methoxy-2-ox0-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione. 0 H

Na ON

AN 0 0 CH,

HPLC-MS (Method C): m/z: 303 (M+1); Rt = 2.65 min.

Example 227 (General procedure (C)) 5-Quinolin-3-yimethylenethiazolidine-2,4-dione.

Q bog oN

HNC o}

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. lo)

Ns ~~

HN J Sy 0 OH

HPLC-MS (Method C): m/z: 273 (M+1); Rt = 3.44 min.

Example 229 (General procedure (C)) ‘ 5-Quinolin-8-yimethylenethiazolidine-2,4-dione.

0 >s

HN

0 Nas

HPLC-MS (Method C): m/z: 257 (M+1); Rt = 3.15 min.

Example 230 (General procedure (C)) 5-(1-Bromo-6-methoxynaphthalen-2-yimethylene)thiazolidine-2,4-dione. 0)

HNL

¢) Br

HPLC-MS (Method C): m/z: 366 (M+1); Rt = 4.44 min.

Example 231 (General procedure (C)) 5-(6-Methyl-2-ox0-1,2-dihydroquinolin-3-ylmethylene)thiazolidine-2,4-dione. lo) o. XH ne "

SPAN CH, o

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.

Se a N $

WIT HO

0 0]

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:

0 q Shs =o

HN 1 0 ci

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

Q H

Ms N

HN - 0}

Br

Example 234 5-Pyridin-4-yimethylenethiazolidine-2,4-dione e}

Ys N wl II 0

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

HN x Br 0)

HPLC-MS (Method A):

Example 236 5-(3-Nitrobenzylidene)thiazolidine-2,4-dione :

0]

Ra

NN id

O oO

HPLC-MS (Method A):

Example 237 5-Cyclohexylidene-1,3-thiazolidine-2,4-dione 0) s

HN

Oo

HPLC-MS (Method A):

Example 238 5-(3,4-Dihydroxybenzylidene)thiazolidine-2,4-dione o ~s OH

S10 dé aad “OH o

Example 239 + 5-(3-Ethoxy-4-hydroxybenzylidene)thiazolidine-2,4-dione lo) ph OH . S © Sen,

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

H.C. o) >To ws > x NO 0 Oo

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

J" le) oO

Ng OH

HN a 0]

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

HN Ss OH

NX

Y ?

CH,

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

Yq OH

HN

TR

0)

CH,

Example 244 5-(3-Ethoxy-4-hydroxy-5-nitrobenzylidene)thiazolidine-2,4-dione o 0:

OH

= 0 BY 0 Sen,

Example 245

HG

[o] 0 ~<)- ° 4 —

HN, _S 0— )—CH,

T

Example 246

HO

ES Dan

SOPs 0

Example 247

N 0 °

S

<LI

Example 248

S pa

N s [¢] . o

He”

N

I

0

Example 249

H s N 0 ~ n 3 NN

N

\ New,

Example 250 () [ ~

NH

Ss

OH

Ss

Example 251

HC oO 0

Oo 0 0,

HN CH, =0

Ps

Example 252

LICL

0 @

Example 253

ALK l L

Example 254

HS

N

1 i J

Sah

Example 255

H,

CH,

H,C s

HO. A -

HG NH

ZZ .

HC

CH, °

Example 256

CH

73 ge o)

S

~ i ’

Example 257 5-(3-Hydroxy-5-methyi-phenylamino)-thiazolidine-2,4-dione 0 CH,

Ps

HN

N OH

0 H

Example 258 o 0)

S .

JH

S

Example 259

A ~S <7

S A Br

Example 260 o \: fo) o” = NH

N Ss

H CH,

Ss

Example 261 oh

O

Heo YY

NS

X»_-S

S=s 0” HN

Example 262 {

NH

=

Ci 0

Example 263 ci S yr lo] N : 0

Example 264

FF

F OA

JOL= oo"

Example 265 :

H

N S o= 1

S

H.C

Oo

N

H

Example 266 0) OH

HN _

As

Example 267

OH

0)

HN

PN

Example 268

O

OH Sy

Ss

Example 269 0 = NH [A

S

N CH,

Example 270 3) H

N

=

Ss

Ci

Example 271

S

= = | S Va

N

N 4 \ }

H o [e]

Example 272 ol : HN = Br rt s Ji

HC

Example 273

Hy o ¥ *

N S

67 =

N o H

Example 274 0.

H

N

— ps

Br

N\

N

H

Example 275 %\ H

J

Orel

N'=0 /- 0)

Example 276 .

Cl [@]

Ss

T-

Example 277

CH,

H.C. 3 N N\ o

N—~( NA

Cosy

S

Example 278

H Oo Br

Ss = g

CH,

Example 279

H,C

N-cH,

H £ :

N Say s=A =

S 4

Example 280

H 0

N s=A 7 =

Example 281 0

H,C—~# )

Oo —¢ NH

N \ 54g 5

General procedure (D) for preparation of compounds of general formula la:

CH,) CH,) (CH,) (Cth), (CH),

Oy E- JN" sept O EO Step2 og. / \ OH

Yo H+ |e Nor IE Y. 0 R —— "YO

R Rr" O 10 fe) R 0)

Step 3 \-

Y (CH,),

HN

NE J oH oO rR 'e) ly wherein X, Y, R' are as defined above, nis 1 or 3-20,

E is arylene or heterarylene (including up to four optional substituents, R'®, R', R", and R'™ as defined above),

R'is a standard carboxylic acid protecting group, such as C,-Ce-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 R'°-C(=0)-

E-OH with an w-bromo-alkane-carboxylic acid ester (or a synthetic equivalent) in the pres- ence of a base such as sodium or potassium carbonate, sodium or potassium hydroxide, so- dium hydride, sodium or potassium alkoxide in a solvent, such as DMF, NMP, DMSO, ace- tone, 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 mare 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:

SUBSTITUTE SHEET (RULE 26)

Example 282 (General procedure (D)) 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxylbutyric acid o Oo ole ad. )

S

J .

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. "H-NMR (DMSO-ds): 6 1.21 (3H, 1), 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); R; = 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. "H-NMR (DMSO-ds): § 1.99 (2H, p), 2.42 (2H, 1), 4.13 (2H, 1), 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); R;= 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. } "H-NMR (DMSO-ds): § 1.97 (2H, p), 2.40 (2H, t), 4.07 (2H, 1), 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)phenoxylacetic acid 0) )—s

I OH oY 0 0}

Step 3: Thiazolidine-2,4-dione (3.9 g, 33 mmol), 3-formylphenoxyacetic acid (6.0 g, 33 mmol), anhy- drous sodium acetate (13.6 g, 165 mmol) and acetic acid (100 mL) was refluxed for 16 h. Af- ter 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)phenoxyjacetic acid as a solid. "H-NMR (DMSO-d): § 4.69 (2H, s), 6.95 (1H, dd), 7.09 (1H, 1), 7.15 (1H, d), 7.39 (1H, t).7.53 (1H, s); HPLC-MS (Method A): m/z = 280 (M+1) (poor ionisation); Ry = 2.49 min.

The compounds in the following examples were similarly prepared.

Example 284 (General procedure (D)) 3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyljacrylic acid o 0] x

Oo "H-NMR (DMSO-ds): 66.63 (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)phenoxylacetic acid 0 Oo

SIT

NN z

Triethylamine salt: "H-NMR (DMSO-ds): & 4.27 (2H, s), 6.90 (2H, d), 7.26 (1H, 5), 7.40 (2H, d).

Example 286 (General procedure (D)) 4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzoic acid o O >= oH

HN ~~ :

Oo

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

Q . -

LANGS OH

0 ©

H-NMR (DMSO-dg): 6 7.57 (1H, s), 7.60 (1H, t), 7.79 (1H, dt), 7.92 (1H, dt), 8.14 (1H, 1).

Example 288 (General procedure (D)) 4-[2-Chloro-4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenoxy]butyric acid 0 0)

HN x cl 0 "H-NMR (DMSO-ds): § 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

0 oO

Ra o~Hon x Br

Oo "H-NMR (DMSO-ds): 6 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

Ss 0 0S On xX Br : 0)

H-NMR (DMSO-ds): 5 1.99 (2H, p), 2.45 (2H, t), 4.18 (2H, 1), 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 8 (J i >s @ o~ Hon

HN So 15- ©

H-NMR (DMSO-ds): § 2.12 (2H, p), 2.5 (below DMSO), 4.28 (2H, 1), 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

3 Clo 0

Ss ~TNNY

HN CC OH

0]

HPLC-MS (Method A): m/z: 394 (M+23); Rt = 3.62 min. . "H-NMR (DMSO-dg): § 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. ? C 0 0

Ys ~NN

HNL OH

Br 6) 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 bro- minated compound and unchanged starting material. Purification by preparative HPLC on a

C18 column using acetonitrile and water as eluent afforded 8 mg of the title compound.

HPLC-MS (Method C): m/z: 473 (M+23), Rt. = 3.77 min

Example 294 4-[2-Bromo-4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyric acid. 2 ) x

ST

HN. 1 a © }

Starting with 4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxy]-butyric acid (ex- ample 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)phenoxylacetic acid 0

Oo 0)

Na Aon x Br

O

"H-NMR (DMSO-d;): § 4.90 (2H, s), 7.12 (1H, d), 7.52 (1H, dd), 7.65 (1H, s) 7.84 (1 H, 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 0) a 2 Ee 0 0) "H-NMR (DMSO-ds): § 1.98 (2H, p), 2.42 (2H, t), 4.04 (2H, t), 7.05 (1H, dd), 7.15 (2H, m), 7.45 (1H, 1), 7.77 (1H, 8), 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 o . 0) cS ong,

HN 1 0]

O-ch,

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-yloxylacetic acid o) Q 1 Se

A .

Oo

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

O

Ra 4

N

5 :

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- yllacetic acid 0)

A

Ns N OH “AS

Oo 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 nitrogenand with external cooling, keeping the temperature below 15 °C, sodium hydride (60% in mineral oil, 3.0 g, 76 mmol) was added in portions. Then a solution : of ethyl bromoacetate (8.4 mL, 76 mmol) in 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 wa- ter (300 mL) and ethyl acetate (2 x 150 mL). The combined organic extracts were washed with a saturated aqueous solution of ammonium chloride (100 mL), dried (MgSO.) and con- centrated in vacuo to afford 15.9 g (quant.) of (3-formylindol-1-yl)acetic acid ethyl ester as an oil

H-NMR (CDCl,): 6 = 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 SN hydrochloric acid and extracted with ethyl acetate (250 + 150 mL). The combined organic extracts were dried (MgSO.,) and concentrated in vacuo to afford 10.3 g (73%) of (3-formylindol-1-yl)acetic acid as a solid. "H-NMR (DMSO-d;): 84 = 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

Oo

OH

LT

Ps N “SNE

Oo

HPLC-MS (Method A): m/z: 317 (M+1); Rt = 3.08 min. : 25

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 con-

centrated in vacuo to afford 17.5 g (quant.) of 3-(3-formylindol-1-yi)propionic acid ethyl ester as a solid. "H-NMR (DMSO-d): 6x = 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 de- scribed above to afford 12.5 g (83%) of 3-(3-formylindol-1-yl)propionic acid as a solid.

H-NMR (DMSO-ds): 64 = 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 oO OH 8) gn oO | O »~g

HN i

O

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 o 0}

WN OA, w IMC lo]

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 so- dium 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 MgSO, 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. o [8]

Oo wl SC

[0]

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-yloxyJundecanoic acid. 0 0 OH

WIC Md ¢ [e}

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-yloxyjpentadecanoic acid. fo}

Ys ONS

EAN CO 0

I

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. lo} OH ph Ona ‘sl oes 0

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. :

Q cNsenas

HN, A

Qo

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-yloxylhexanoic acid ethyl ester. 1

O

HN J

0

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. 0 ? > oH oan 3

WC

HPLC-MS (Method C): m/z: 408 (M+23); Rt.= 4.28 min.

Example 311

N-(3-Aminopropy!)-4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-naphthalen-1-yloxyl- butyramide 3, J x

Vg OA,

HN. J H 0

To a mixture of 4-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxyjbutyric 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 resuiting 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-yloxylbutyrylamino}- 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:

2 0) oo

Mg OH

HN 1

Oo oO) on = Oy

HN fo) 0 2 ) PP

Ys ) OH

HN a 0 a Ul, on >~s OS

HN 1 : o 0)

Q OP

Ms oN oH

HN lo pz 0) oO

O OH

Ms NNN

HN a fe} x

Ms ® OH

HN 1 (8)

I ) 0 OH

Ss SONNY

HN x ® Oo @] .

? () Oe eee

Ms ® OH

HN So 0 o. Ulo on

Ys SONNY

HN. J 0 0]

Ms C © OH

HN a

Oo

The following compounds are commercially available and may be prepared according to general procedure (D): § Example 312 = > [o) ry

HN

OLX

L

CH,

Example 313

Ss

NN [e] hd 0 OH

Example 314

[0] =

Hi

OL ° [+] : YY

OH

Example 315

HE h 0 0.

BRN

Ory v ®

HO. o

Example 316 ~ 9] ae OH

ANS O-CH,

Ss

Example 317 0) so

HNL

0 "Neo

HO

Example 318

H ,S

N— ye" 0 So

The following salicylic acid derivatives do all bind to the His B10 Zn?" site of the insulin hexamer:

Example 319

Salicylic acid

O

HO

Example 320

Thiosailicylic acid (or: 2-Mercaptobenzoic acid) 0

HS

Example 321 2-Hydroxy-5-nitrobenzoic acid

OH o

No _-

Sen

HO

Example 322 3-Nitrosalicyclic acid 0

HO

HO )

NT

Oo” "0

Example 323 5,5-Methylenedisalicylic acid

Oy OH Os OH

HO. . L OH

Example 324 2-Amino-5-trifluoromethylbenzoesyre

OH F E

HN

Example 325 2-Amino-4-chlorobenzoic acid 0

H,N Cl 2 :

Example 326 2-Amino-5-methoxybenzoesyre

OH oO.

SY CH,

H,N

Example 327 0

H,N

Example 328

O

H,N

Example 329

OH

Nee

HO

0=5=0

NH,

Example 330

OH

“0.

HN cl

Example 331 0 © IC

HO .

O=s-y" o ©

Example 332 0 <1

H,N 07 CH,

Example 333 5-lodosalicylic acid 0

HO

Example 334 5-Chiorosalicylic acid 6)

Cl

HO

Example 335 1-Hydroxy-2-naphthoic acid

OH OH

C0

Example 336 3,5-Dihydroxy-2-naphthoic acid 0] ©

HO

OH

Example 337 3-Hydroxy-2-naphthoic acid 0

Hee

HO

Example 328 3,7-Dihydroxy-2-naphthoic acid 0

OH

= YOY

HO

Example 339 2-Hydroxybenzo[a]carbazole-3-carboxylic acid — 0 © I

HO

1-2

H

Example 340 7-Bromo-3-hydroxy-2-naphthoic acid 0

HO .

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 0

Br

HO CO

Br

HPLC-MS (Method A): m/z: 346 (M+1); Rt: = 4,19 min.

Example 342 7-Formyl-3-hydroxynaphthalene-2-carboxylic Acid le} 0

HO

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 result- ing solution was heated to 50 °C for 2 minutes and then allowed to cool to ambient tempera- ture 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-Dimethyiformamide (8.7 mL, 8.2 g, 112 mmol) was added after 90 minutes additional stirring. The cooling was discontinued, and the reaction mixture was stirred at room temperature for 17 hours before it : was poured into 1 N hydrochloric acid (aq.) (750 mL). The organic solvents were evaporated in vacuo, and the resulting precipitate was filtered off and rinsed with water (3 x 100 mL) to yield the crude product (16.2 g). Purification on silica gel (dichloromethane / methanol / ace- tic acid = 90:9:1) furnished the title compound as a solid. "H-NMR (DMSO-ds): § 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 0

HO

Example 344 4-Amino-2-hydroxybenzoic acid 0

HO” 7 “NH,

Example 345 5-Acetylamino-2-hydroxybenzoic acid i N CH © hi 0

HO

Example 346 2-Hydroxy-5-methoxybenzoic acid

O CH, 0)

HO ’

The following compounds were prepared as described below:

Example 347 4-Bromo-3-hydroxynaphthalene-2-carboxylic acid

Oo

Moe

HO

Br 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 pL, 15.9 mmol) in acetic acid (10 mL) was added drop wise during 30 minutes. The suspension was stirred at room tem- perature 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. ‘H-NMR (DMSO-d;): & 7.49 (1H, 1), 7.75 (1H, 1), 8.07 (2H, “t"), 8.64 (1H, s). The substitution pattern was confirmed by a COSY experiment, showing connectivities between the 3 (4 hy- drogen) “triplets”. HPLC-MS (Method A): m/z: 267 (M+1), Rt = 3.73 min.

Example 348 3-Hydroxy-4-iodonaphthalene-2-carboxylic acid

O oe

HO

I

3-Hydroxynaphthalene-2-carboxylic acid (0.5 g, 2.7 mmol) was suspended in acetic acid (5 mL) and with stirring iodine monochloride (135 ul, 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. :

"H-NMR (DMSO-ds): 6 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

CH,

J

H

HO 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 ammo- nium 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 wa- ter containing 0.1% trifluoroacetic acid to afford the title compound.

HPLC-MS (Method A): m/z: 274 (M+1); Rt = 1.77 min. "H-NMR (methanol-d,): § 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

CH,

O i DX ry °N

HO

A solution of 5-chirosulfonylsalicylic 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 result- ing mixture was stirred at room temperature for 16 hours. The mixture was added dichloro- methane (50 mL) and was washed with water (2 x 100 mL). Drying (MgSQ,) 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. "H-NMR (DMSO-ds): 6 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 l,: 0 R wo oe Lea o Pd catalyst ) .

CO + BT — “OY H

HO Oo H

R HO

15 . wherein Lea is a leaving group such as Cl, Br, | or OSO,CF3, R is hydrogen or C,-Cg-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 0) 0] 4$ CH,

HO CC

HO

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 sus- pension of palladium(Il) acetate (4 mg, 0.02 mmol) in acetone (0.5 mL) was filtered and added to the above solution. The mixture was purged with N, and stirred vigorously for 24 hours at room temperature. The reaction mixture was poured into 1 N hydrochioric acid (aqg.) (60 mL) and the precipitate was filtered off and rinsed with water (3 x 40 mL). The crude product was dissolved in acetone (25 mL) and dried with magnesium sulfate (1 h). Filtration followed by concentration furnished the title compound as a solid (92 mg). "H-NMR (DMSO-ds): 512.60 (1H, bs), 8.64 (1H, s), 8.42 (1H, s), 8.08 (2H, d), 7.97 (2H, d), 7.92 (2H, m), 7.33 (1H, s), 2.63 (3H, s); HPLC-MS (Method (A): m/z: 307 (M+1); Rt = 3.84 min.

The compounds in the following examples were prepared in a similar fashion. Optionally, the compounds can be further purified by recrystallization from e.g. ethanol or by chromatogra- phy.

Example 352 (General Procedure (E)) 3-Hydroxy-7-(3-methoxyphenyl)naphthalene-2-carboxylic acid i $

Ary o

HO CH,

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 :

i ¢ 0

HO :

HPLC-MS (Method (A)): m/z: 265 (M+1); Rt = 4.6 min.

Example 354 (General Procedure (E)) 3-Hydroxy-7-p-tolyinaphthalene-2-carboxylic acid o g CH,

I

HO

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 0] . 0 $ H

Mee

HO

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 i $

LU

HO

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

0 0 4$ OH

HO CO

HO

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

HO oo 0

HO

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 o) a Och, oend

HO

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 i J ooh

HO CH,

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

Ses

MOO

HO : ‘HPLC-MS (Method (A)): m/z: 308 (M+1); Rt = 5.61 min.

Example 362 (General Procedure (E)) 7-Biphenyl-3-yl-3-hydroxynaphthalene-2-carboxylic acid i 9

BOO A®

HO

HPLC-MS (Method (A): m/z: 341 (M+1); Rt = 5.45 min.

General procedure (F) for preparation of compounds of general formula ls: 0 0) o ’

H ’

CSR . T-NEROH oY ET

HO HO ls wherein R* is hydrogen or C,-Ce-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.

CH, 9) or CH,

N :

HO

7-Formyi-3-hydroxynaphthalene-2-carboxylic acid (40 mg, 0.19 mmol) (example 342) was suspended in methanol (300 pL). Acetic acid (16 pL, 17 mg, 0.28 mmol) and 4-(2- propylianiline (40 pL, 40 mg, 0.30 mmol) were added consecutively, and the resulting mix- ture was stirred vigorously at room temperature for 2 hours. Sodium cyanoborohydride (1.0

Min tetrahydrofuran, 300 ul, 0.3 mmol) was added, and the stirring was continued for an- other 17 hours. The reaction mixture was poured into 6 N hydrochloric acid (aq.) (6 mL), and the precipitate was filtered off and rinsed with water (3 x 2 mL) to yield the title compound (40 mg) as its hydrochloride salt. No further purification was necessary. "H-NMR (DMSO-ds): 610.95 (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 x

HO

HPLC-MS (Method C): m/z: 372 (M+1); Rt = 4.31min.

Example 365 (General procedure (F)) 7-{[(3,5-Dichlorophenyl)aminolmethy!}-3-hydroxynaphthalene-2-carboxylic Acid !

Cl

HO

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 i LD

H

HO }

HPLC-MS (Method C): m/z: 351 (M+1); Rt = 3.43 min.

Example 367 (General procedure (F)) 3-Hydroxy-7-{[(quinolin-6-yl)Jaminojmethyl}naphthalene-2-carboxylic Acid

N

YH XX

H

HO

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 0 or Och, ‘S008

HO

Example 369 (General procedure (F)) 7-{[(2.3-Dihydrobenzofuran-5-ylmethyl)amino]methyl}-3-hydroxynaphthalene-2-carboxylic

Acid lo]

HO 0 : 20° HPLC-MS (Method C). m/z: 3560 (M+1); Rt = 2.22 min.

Example 370 (General procedure (F)) 7-{[(4-Chlorobenzyl)amino)methyl}-3-hydroxynaphthalene-2-carboxylic Acid

QO .

H

HO Cl

HPLC-MS (Method C): m/z: 342 (M+1); Rt = 2.45 min.

Example 371 (General procedure (F)) 3-Hydroxy-7-{[(naphthalen-1-yimethyl)amino]methyi}naphthalene-2-carboxylic Acid lo] sseape

H

®

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

Oo "OCC

HO ®

HPLC-MS (Method C): m/z: 384 (M+1), Rt = 2.90 min.

Example 373 (General procedure (F)) 3-Hydroxy-7-{[(4-phenoxybenzyl)amino]methyljnaphthalene-2-carboxylic Acid 0]

CLO)

HO o

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 0

TY WL :

HO ots

HPLC-MS (Method C): m/z: 338 (M+1); Rt = 2.32 min.

General procedure (G) for preparation of compounds of general formula lg: 0 0

SORE + (C,-C,-alkanoyl),0——» LS 00S

HO H HO PNG, ak

Is wherein T is as defined above and the moiety (C,-Ce-alkanoyl),O 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)methyljnaphthalene-2-carboxylic Acid

CH, "HO 0” CH, 3-Hydroxy-7-[(4-(2-propyl)phenylamino)methyljnaphthalene-2-carboxylic acid (25 mg, 0.07 mmol) (example 363) was suspended in tetrahydrofuran (200 pL). A solution of sodium hy- drogencarbonate (23 mg, 0.27 mmol) in water (200 pL) was added followed by acetic anhy- dride (14 pL, 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 fil- . tered off and rinsed with water (3 x 1 mL) to yield the title compound (21 mg). No further puri- fication was necessary.

"H-NMR (DMSO-dq): 610.96 (1H, bs), 8.48 (1H, s), 7.73 (1H, 5), 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

Br i ig

HO Pen,

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]methyi}-3-hydroxynaphthalene-2- carboxylic Acid 0

OY OCD

R 0

HO 0” "CH,

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 0]

HO 0” “CH, cl

HPLC-MS (Method C): m/z: 384 (M+1), Rt = 3.67 min.

Example 379 5-(3-(Naphthalen-2-yloxymethyl)-phenyl)-1H-tetrazole :

AJ 1

I

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 MgSO, 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 C4gH1aNO:

C, 83.37 %; H, 5.05 %; N, 5.40 %, Found

CC, 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 : 20 125 °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 C,8H14N4O:

C, 71.51 %; H, 4.67 %; N, 18.54 %; Found

C, 72.11%; H, 4.65 %; N, 17.43 %.

TH NMR (400 MHz, DMSO-d¢) 84 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

Q IN

NN

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 tem- perature for 30 minutes. Water (50 mL) was added and the volatiles was exaporated in_ vacuo. The resulting mixture was filtered and the filter cake was washed with water followed by heptane (2 x 25 mL). Drying in vacuo at 50 °C for 16 hours afforded 15.0 g (95 %) of N-(3- cyanophenyl)-2-naphtoic acid amide.

M.p. 138-140°C

The above naphthoic acid amide (10 g, 37 mmol) was dissolved in N,N-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 ammo- nium chloride (0.98 g) were added and the mixture heated at 125 °C for 16 hours. After cool- ing, the mixture was poured into water (1.5 1) 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. 'H NMR (200 MHz, DMSO-dg): 84 7.58-7.70 (m, 3H), 7.77 (d, 1H), 8.04-8.13 (m, 5H), 8.65 (d, 1H), 10.7 (s, 1H).

Calculated for C45H43NsO, 0.75 HO:

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

NSN

\ NH .

To a solution of 4-phenylphenol (10.0 g, 59 mmol) in dry N,N-dimethyl-formamide (45 mL) kept under an atmosphere of nitrogen, sodium hydride (2.82 g, 71 mmol, 60 % dispersion in oil) was added in portions and the reaction mixture was stirred until gas evolution ceased. A solution of m-cyanobenzyl bromide (13 g, 65 mmol) in dry N,N-dimethylformamide (45 mL) was added dropwise and the reaction mixture was stirred at room temperature for 18 hours.

The reaction mixture was poured on to ice water (150 mL). The precipitate was filtered of and washed with 50 % ethanol (3 x 50 mL), ethanol (2 x 50 mL), diethyl ether (80 mL), and dried in vacuo at 50 °C for 18 hours affording crude 17.39 g of 3-(biphenyl-4-yloxymethyl)-benzonitrile as a solid. : 'H NMR (200 MHz, CDCI) 84 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 x 100 mL), 50 % ethanol (3 x 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. '"H NMR (200 MHz, DMSO-ds) 84 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 }

eye,

N-N 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 wa- ter (400 mL) and extracted with ethyl acetate (2 x 200 mL). The combined organic extracts were washed with water (2 x 100 mL), dried (MgSQ,) and evaporated in vacuo to afford 5.19 g (97 %) 3-(phenoxymethyl)benzonitrile as an oil.

TLC: R; = 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 ageous 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 2.05 g (33 %) of the title compound as a solid. 'H NMR (200 MHz, CDCl; + DMSO-de) 8, 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-yimethoxy)phenyl}-1H-tetrazole

HSN

=N

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-

Phenylbenzy! chloride (9.26 g, 44.79 mmol) and potassium iodide (0.2 g, 1.21 mmol) were added and the reaction mixture was stirred at room temperature for 60 hours. The reaction mixture was poured on to a mixture of saturated sodium carbonate (100 mL) and ice water (300 mL). The precipitate was filtered of and washed with water (3 x 100 mL), n-hexane (2x 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 reac- tion mixture stirred for 0.75 hour. The precipitate was filtered off and washed with water, 96 % ethanol (2 x 50 mL), and dried in vacuo at 50°C for 18 hours affording 5.13 g (56 %) of the title compound. 'H NMR (200 MHz, DMSO-d¢) 8x 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 oA o H

CH,

This compound was made similarly as described in example 383.

Example 385 0)

Sapew 0) \ N .

N-N’ :

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

SSR

H

This compound was prepared similarly as described in example 379, step 2.

Example 387 5-(1-Naphtylmethyl)-1H-tetrazole

N-N.

IN

N

This compound was prepared similarly as described in example 379, step 2.

Example 388 5-[4-(Biphenyl-4-yloxymethyl)phenyl]-1H-tetrazole

N-

O00

H

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 mix- ture was stirred at room temperature for 1 hour. The precipitate was filtered off and washed with thoroughly with water. Drying in vacuo over night at 50 °C afforded 7.09 g (97 %) of 4- (biphenyl-4-yloxymethyl)benzonitrile as a solid.

The above benzonitrile (3.00 g, 10.5 mmol) was dissolved in N,N-dimethylformamide (50 mL), and sodium azide (1.03 g, 15.8 mmol) and ammonium chloride (0.84 g, 15.8 mmol) were added and the mixture was stirred 16 hours at 125 °C. The mixture was cooled to room temperature and water (50 mL) was added. The suspension was stirred overnight, filtered, washed with water and dried in vacuo at 50 °C for 3 days to give crude 3.07 g (89 %) of the title compound. From the mother liquor crystals were colected and washed with water, dried by suction to give 0.18 g (5 %) of the title compound as a solid.

HNMR (200 MHz, DMSO-d): 814 5.21 (s, 2H), 7.12 (d, 2H), 7.30 (t, 1H), 7.42 (1, 2H), 7.56- 7.63 (m, 6H), 8.03 (d, 2H).

Calculated for C,oH:6N4O, 2H,0:

C, 65.92 %; H, 5.53 %; N, 15.37 %. Found:

C, 65.65%; H, 5.01 %; N, 14.92 %.

Example 389 =

N

0)

Cho,

H N

This compound was prepared similarly as described in example 383.

Example 390 + N-N.

N or

Example 391

N=N " Te hel

Example 392

Za

SEN

™ N H of

HN" TN

N=N

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

HN

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

N=N ] :

This compound was prepared similarly as described in example 379, step 2.

Example 395 5-[3-Methoxy-4-(4-methylsulfonylbenzyloxy)phenyl]-1 H-tetrazole

NN H,

Aer (0) 0 7 cn, e]

This compound was made similarly as described in example 383.

Example 396 5-(2-Naphthyl)-1H-tetrazole

N-N.

IN

N

H

This compound was prepared similarly as described in example 379, step 2.

Example 397 2-Amino-N-(1H-tetrazol-5-yl)-benzamide 0 NN

PE N

N N

H H

NH,

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

N=N

Ns NH

H,C- 0

OH

This compound was prepared similarly as described in example 379, step 2. :

Example 399 4-(2H-Tetrazol-5-ylmethoxy)benzoic acid 0 or

N

HN STO

N=N

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 Na,SO, and concentrated in vacuo. The residue was dissolved in acetone and chioroacetonitrile (6.04 g,0.08 mol), so- dium iodide (12.0 g, 0.08 mol) and potassium carbonate (11.1 g, 0.08 mol) were added and the mixture was stirred for 16 hours at RT and at 60 °C. More chloroacetonitrile was added until the conversion was 97%. Water was added and the mixture was acidified with 1N hy- drochloric acid and the mixture was extracted with diethyl ether. The combined organic lay- ers were dried over Na,SO, and concentrated in vacuo to afford methyl 4- cyanomethyloxybenzoate in quantitative yield. This compound was used without further puri- fication 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 N,. 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 Na,SO, and concentrated in vacuo, to afford methyl 4-(2H-tetrazol-5- yimethoxy)benzoate. This compound was used as such in the following step.

Methyl 4-(2H-Tetrazol-5-yimethoxy)-benzoate was refluxed in 3N sodium hydroxide. The re- action 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 result- ing 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. "H-NMR (DMSO-ds): 4.70 (2H, s), 7.48 (2H, d), 7.73 (2H, d), 13 (1H, bs).

Example 400 4-(2H-Tetrazol-5-yimethylsulfanyl)benzoic acid ] 0]

Ns

HN YS

N=N

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 so- lution 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 N,. The mixture was filtered and washed with heptane. The aque- ous phase was acidified with 3N hydrochloric acid and the product was filtered off, washed with water and dried, affording 4-cyanomethylsulfanylbenzoic acid (27.2 g, 88%). This com- pound 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 chioride (9.7 g, 0.18 mol) in DMF (1000 mL) was refluxed over- night under N,. 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 Na,SO, 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 chromatogra- phy using DCM:MeOH = 9:1 as eluent, affording the title compound (5.2 g, 16%). "H-NMR (DMSO-d): 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

Ns .

HNN re

N

H :

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 mix- ture was poured on to water (600 ml) and the precipitate was filtered off and washed with ethyl acetate (3 x 50 ml). The filtrate was extracted with ethyl acetate (3 x 250 ml) and the combined ethyl acetate extracts were washed with water (150 mi), brine (150 mi), dried (MgSO0,) and concentrated in vacuo. The residue was crystallised from heptanes and recrys- tallised from acetonitrile (70 ml) affording 7.16 g (40 %) of 3-cyano-9H-carbazole as a solid.

M.p. 180-181 °C. 3-Cyano-9H-carbazole (5.77 g, 30 mmol) was dissolved in N,N-dimethylformamide (150 mi), 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 suspen- sion was stirred for 0.5 h, and the precipitate was filtered off and washed with water (3 x 200 mi). and dried in vacuo at 50 °C. The dried crude product was suspended in diethyl ether (500 mi) and stirred for 2 h, filtered off and washed with diethyl ether (2 x 200 ml) and dried in vacuo at 50 °C affording 5.79 g (82 %) of the title compound as a solid. "H-NMR (DMSO-d): 511.78 (1H, bs), 8.93 (1H, d), 8.23 (1H, d), 8.14 (1H, dd), 7.72 (1H, d), 7.60 (1H, d), 7.49 (1H, 1), 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 Zn?" site of the insulin hexamer:

Example 402 5-(3-Tolyl)-1H-tetrazole

CH, oy

N XN :

NN

Example 403 5-(2-Bromophenyl)tetrazole

N

Br NT .N

N

Example 404 5-(4-Ethoxalylamino-3-nitrophenyli)tetrazole § (0)

NE

HN

RS

=A

[8] HN

Example 405

Nz, cl

Example 406

N=N

No NH lo) 8

CI] “0

Example 407

N——N /

Nd

H

. ci

Example 408

F. f =

NH

Or

Example 409

Tetrazole

H

N

1} //

N—N

Example 410 5-Methyltetrazole : :

N

H.C .

FN

N-N

Example 411 5-Benzyl-2H-tetrazole

Las

N

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

O yon

HT

Example 413 5-Phenyl-2H-tetrazole

HN

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

O° 8

HN

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

No

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

H

MN=N 0}

N,

N

TL

Example 417 ct —N ’ ~ Lo)

H, d RNY 2 N

Example 418

F of —N

HN \

IN NZ

Example 419

HC, )

HC 0 0 —

J

Example 420 ‘Om

Ny

H 0 CH,

Example 421

H,

N~—

N

NT

H

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

N

NT

SY)

N—

H

Example 423 cl \

HN

WN

TIC /

N

Example 424

H y _—N Sr / 0

N— [o] /

H,C

Example 425

Sa Welas

Q [+]

Example 426 o NN

HC NH

CO

Example 427 o /

ON’

H

Nn :

HO \

Na } )

HC

Example 428 aq

N—_ o 74 I wa

H

Example 429 [& Nn

J

AN

N

H

Example 430

N,

O-- om) fF

Example 431

N

Ty Se

HC

Example 432

Ct, o H

N A

. east A Pp

Example 433 {0}

H n" / (

I —) on he /

N% /

H,C

Example 434 o 0 m= N\ “ APU Py :

H

Example 435

[o

Le] [8] / {7

N +

H \_w

Example 436

Br

N

NT

I

Ney

H

HO Br

Example 437 —N

N

[> ~n 7

H

0 []

Example 438 o / o—Nn’

N

HO

Ne \

CH, :

Example 439

N H -

N ig NT NL

N—N

H 0

CH,

Example 440

Cl. fo) H

N—N

Try

N XN N nd H N

Example 441 in ny N ~ Nd

F

Example 442 nd

NH

R

Ny \

NN

Example 443

H

Se oO \ J \\ © []

Example 444 ox," £3 CY \ J \\ ° [o]

Example 445 0 N N

AY, 4

Example 446 =

F “7 N joe

Example 447

AN

HN NN N

0 / —N

N

~~ H

N

\_

Example 448 lo)

No" .

N cl v4 Jl a

H

N

H AVR

Example 449

NN

[\ “~ "

Su cl o

Example 450

WQ 03/027081 PCT/DKO02/00595 _N

TH

N—_ y 7] . S o i

Br

Example 451 cl cl

TL

HN y hd AN \ nt :

Example 452 lo}

He” N—N

I \

H,C

So nN

H

General procedure (H) for preparation of compounds of general formula ;:

NaCBH,

HOAc ~-N 0] -N

HN 1 DMF HN

LOA H >A H

NsN" ARLNH, ¥ Hoar > a AR-NTAR?

H

I; wherein A, AR", and AR? are as defined above. d

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, dichloro-

methane, 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, chro- matography 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)phenyljamine

UN

H

HN

A solution of 5-(3-aminophenyl)-2H-tetrazole (example 589, 48 mg, 0.3 mmol) in DMF (250 ul) was mixed with a solution of 4-biphenylylcarbaldehyde (54 mg, 0.3 mmol) in DMF (250 pL) and acetic acid glacial (250 pL) was added to the mixture followed by a solution of so- dium cyano borohydride (15 mg, 0.24 mmol) in methanol (250 pL). The resulting mixture was shaken at room temperature for 2 hours. Water (2 mL) was added to the mixture and the re- sulting 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)phenyllamine

N=N

AN. 2 J

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)phenyllamine

HPLC-MS (Method D): m/z: 282,2 (M+1); Rt = 3,57min.

Example 456 (General procedure (H)) 4-{[3-(2H-Tetrazol-5-yl)phenylamino]jmethyl}phenol

A 0 CT ”

HN, _

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)phenyljamine 0}

ND

HN, _

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)phenyljamine

¢

Nap c

HN, _ y

N

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)phenyljamine

N=N

HN. _ H ta)

Cl

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)phenyllamine

N=N y oy

HN

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)phenyllamine

SNe!

OTC

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)phenyllamine

Pp 0 WO 03/027081 PCT/DK02/00595

N=N wi

OU

HPLC-MS (Method D): m/z: 302 (M+1); Rt = 4,70 min.

Example 463 (General procedure (H))

Naphthalen-2-yimethyl-[3-(2H-tetrazol-5-yl)phenylJamine

N=

HN. POY

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

Oo

N=N OH

HN. = Be

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)benzyllamine ‘N

ADO

F F

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)phenyllamine

N=N

WN. 7 PG Q

N 0]

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 0]

N=N

NZ CT 1S

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 o

VBS a

HN 2 N

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)phenyljamine

H

TES C Pe]

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

0

N= "0H ow ! Tene

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

SU, a H 4 N-ch,

HPLC-MS (Method D): m/z: 345 (M+1); Rt = 3,07 min. :

Example 472 (General procedure (H)) (4-Methoxybiphenyi-4-yimethyl)-[3-(2H-tetrazol-5-yl)phenyljamine

CH, qf

N=n

HN _ or aes

HPLC-MS (Method D): m/z: 358 (M+1); Rt = 4,97 min.

Example 473 (General procedure (H)) : (2'-Chlorobiphenyl-4-yimethyl)-[3-(2H-tetrazol-5-yl)phenyllamine

N=N g 1 aT

HPLC-MS (Method D): m/z: 362 (M+1); Rt = 5,27 min.

Example 474 (General procedure (H))

Benzyl-[4-(2H-tetrazol-5-yl)phenyljamine

N=N

HN

N

H i

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)phenyljamine

N=N

HN, _

N 0.

H CH, “N

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

N=N

HN. _

N OH

H

0 CV 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)phenyllamine

N=N

HN. “CL A

N N. -

SOA

N

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]lamine

N=N

HN

N cl

H

N Se :

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

N=N

HN, _

N

0)

N

Cl

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)phenyllamine

N=N

HN _ “OL Br

H

0 CT

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)phenyllamine ll

H C i

ROARS

HPLC-MS (Method D): m/z: 358 (M+1), Rt = 5,07min. '

Example 482 (General procedure (H))

Naphthalen-1-yimethyl-[4-(2H-tetrazol-5-yl)phenyljamine

N=n

HN.

N

H 4 ih

HPLC-MS (Method D): m/z: 302 (M+1); Rt = 4,70 min.

Example 483 (General procedure (H))

Naphthalen-2-yimethyl-[4-(2H-tetrazol-5-yl)phenyljamine

N=N

HN. _ “0

I)

N

HPLC-MS (Method D): m/z: 302 (M+1); Rt=4,70 min.

Example 484 (General procedure (H))

Biphenyl-4-yimethyl-[4-(2H-tetrazol-5-yl)phenyllamine

N=N

HN,

N

1

N

HPLC-MS (Method D): m/z: 328 (M+1); Rt = 5,07 min.

Example 485 (General procedure (H)) 4-{[A-(2H-Tetrazol-5-yl)phenylamino]methyl}benzoic acid

N=N

N

Pen >

N

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)benzyllamine

N=n

N

LOL

N ° i

F .

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)phenyljamine

N=N

HN _

N

Oo

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

N=n

HN,

N 1 J Oo

CY 1®

N

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

N=N

HN. _

N

1 . ;

OH

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

N=n : HN, lo]

N

Ro! Jens:

H

N

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)phenyllamine

N=n je 0

N lo}

Ca

N

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

N=n

HN. lo}

N xn

Oy or o"

N

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-yjamine

N=N

HN $ CH,

N N.

J

N

HPLC-MS (Method D): m/z: 345 (M+1), Rt = 3,10 min.

Example 494 (General procedure (H)) (4'-Methoxybiphenyl-4-yimethyl)-[4-(2H-tetrazol-5-yl)phenyljamine cH,

N=p (0) - “ 0 “OL 1 J

N «

HPLC-MS (Method D): m/z: 358 (M+1); Rt = 5,04 min.

Example 495 (General procedure (H)) (2'-Chiorobiphenyi-4-yimethyl)-[4-(2H-tetrazol-5-yl)-phenyl}-amine

N=N u» 0

N

H

N $ Cl

HPLC-MS (Method D): m/z: 362 (M+1); Rt = 5,30 min.

General procedure (1) for preparation of compounds of general formula lg:

HOA

HNN yn HNN ¢N Al O H \ A' H

Ne ARH + PNR? -— Ney AR ARE

OH fo) lg wherein A’, AR’, and AR? are as defined above.

This procedure is very similar to general procedure (A), the only difference being the carbox- ylic acid is containing a tetrazole moiety. When the acylation is complete, the product is iso- lated by extraction, filtration, chromatography or other methods known to those skilled in the art.

The general procedure (1) is further illustrated in the following example 496:

Example 496 (General procedure (1) 4-[4-(2H-Tetrazol-5-yl)benzoylamino]benzoic acid

N=N

HN, _

N

O

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 hydrochlo- ride (4.2 mmol) and the resulting mixture was stirred at room temperature for 1 hour. An alquot of this HOAt-ester solution (0.45 mL) was mixed with 0.25 mL of a solution of 4- aminobenzoic acid (1.2 mmo! 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 tempera- ture. 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 ex- traction) 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 (1)) 3-[4-(2H-Tetrazol-5-yl)benzoylamino}benzoic acid

N=N

HN, _

N H O oe 0

HPLC-MS (Method D): m/z: 310 (M+1); Rt = 2.89 min.

Example 498 (General procedure (1)) 3-{4-[4-(2H-Tetrazol-5-yl)benzoylaminojphenyl}acrylic acid

N=N

HN

N H

Oy

HPLC-MS (Method D): m/z: 336 (M+1), Rt = 3.10 min.

Example 499 (General procedure (1) 3-{4-[4-(2H-Tetrazol-5-yl)benzoylamino]phenyl}propionic acid

N=N

HN, _ ast

N oO Tor 0

HPLC-MS (Method D): m/z: 338 (M+1); Rt = 2.97 min.

Example 500 (General procedure (1)) 3-Methoxy-4-[4-(2H-tetrazol-5-yl)benzoylamino]benzoic acid

N=N

HN. — CH

OL (0) 3

N

©

HPLC-MS (Method D): m/z: 340 (M+1); Rt = 3.03 min.

Example 501 (General procedure (1)

N-(4-Benzyloxypheny!)-4-(2H-tetrazol-5-yl)benzamide

N=N

HN

OL

“CL

HPLC-MS (Method D): m/z: 372 (M+1); Rt = 4.47 min.

Example 502 (General procedure (1))

N-(4-Phenoxypheny!)-4-(2H-tetrazol-5-yl)benzamide

N=

HN J) “OL © QO 0 o

HPLC-MS (Method D): m/z: 358 (M+1); Rt = 4.50 min.

Example 503 (General procedure (})) N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-yl)benzamide

N=

HN, J)

Fn

HPLC-MS (Method D). m/z: 354 (M+1); Rt = 4.60 min.

Example 504 (General procedure (1)) N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-yl)benzamide

N=N

HN,

OL CH,

S N N

0]

HPLC-MS (Method D): m/z: 383 (M+1); Rt = 4.60 min.

Example 505 (General procedure (1)) N-Phenyl-4-(2H-tetrazol-5-yl)benzamide

N=N

HN, _

N H

N

FO

HPLC-MS (Method D): m/z: 266 (M+1); Rt = 3.23 min.

Example 506 (General procedure (1)) 4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic acid

N=N

HN, Ao ! 6: 0 TL on 0

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 (1) 3-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]benzoic acid

N=N

No H 0 “OY or . lo} HPLC-MS (Method D): m/z: 340 (M+1); Rt = 2.90 min.

Example 508 (General procedure (1)) 3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}acrylic acid my

NTO " 1 PY N

Ne Thor lo} HPLC-MS (Method D): m/z: 366 (M+1); Rt = 3.07 min.

Example 509 (General procedure (1)) 3-{4-[4-(2H-Tetrazol-5-ylmethoxy)benzoylamino]phenyl}propionic acid ay

OL

N . o Thon lo]

HPLC-MS (Method D): m/z: 368 (M+1); Rt = 2.97 min.

Example 510 (General procedure (1) 3-Methoxy-4-[4-(2H-tetrazol-5-yimethoxy)benzoylamino]benzoic acid

N=N

HN Ao yg oC ) [OE o} OH o}

HPLC-MS (Method D): m/z: 370 (M+1); Rt = 3.07 min.

Example 511 (General procedure (1))

N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-yimethoxy)benzamide

Hi oy

N A_o y

CL N

FC, 0

HPLC-MS (Method D): m/z: 402 (M+1); Rt = 4.43 min.

Example 512 (General procedure (1))

N-{4-Phencxyphenyl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

N=

HN AO

N

“tao

HPLC-MS (Method D): m/z: 388 (M+1); Rt = 4.50 min.

Example 513 (General procedure (1))

N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide

N=N

HN, Ao y

N

- § Cn

HPLC-MS (Method D): m/z: 384 (M+1); Rt = 4.57 min.

Example 514 (General procedure (l))

N-(9-Ethyl-QH-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethoxy)benzamide aden as)

OU SNF

N lo) Cn {)

HPLC-MS (Method D): m/z: 413 (M+1); Rt= 4.57 min.

Example 515 (General procedure (1))

N-Phenyl-4-(2H-tetrazol-5-ylmethoxy)benzamide

N=N

NA oN

N

A |G

HPLC-MS (Method D): m/z: 296 (M+1); Rt = 3.23 min.

Example 516 (General procedure (1)) 4-[4-(2H-Tetrazol-5-yimethylsulfanyl)benzoylamino}benzoic acid

H LN

NAS Ny

N lo}

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 (})) 3-[4-(2H-Tetrazol-5-yimethylsulfanyl)benzoylaminolbenzoic acid

ND

. H NPS y ] o :

HPLC-MS (Method D): m/z: 356 (M+1); Rt = 3.00 min.

Example 518 (General procedure (1)) 3-{4-[4-(2H-Tetrazol-5-yimethylsulfanyl)benzoylamino]phenyl}acrylic acid

N=

HN, ACs. ®

N

0 Thor lo}

HPLC-MS (Method D): m/z: 382 (M+1); Rt = 3.26 min.

Example 519 (General procedure (1)) 3-{4-[4-(2H-Tetrazol-5-ylmethylsulfanyl)benzoylamino]phenyl}propionic acid

Ne

HN As

CL N oO Tor 0

HPLC-MS (Method D): m/z: 384 (M+1); Rt = 3.10 min.

Example 520 (General procedure (1)) 3-Methoxy-4-[4-(2H-tetrazol-5-yimethylsulfanyl)benzoylaminolbenzoic acid

N=N

HN As , oC

Cs lo} OH 0

HPLC-MS (Method D): m/z: 386 (M+1); Rt = 3.20 min.

Example 521 (General procedure (1)

N-(4-Benzyloxyphenyl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

N=N

HN A_s [ pi N . 0 Copy

HPLC-MS (Method D): m/z: 418 (M+1); Rt = 4.57 min.

Example 522 (General procedure (1))

N-(4-Phenoxyphenyl)-4-(2H-tetrazol-5-yimethylsulfanyl)benzamide

N=N

OL

N

Sole

HPLC-MS (Method D): m/z: 404 (M+1); Rt = 4.60 min.

Example 523 (General procedure (1))

N-(9H-Fluoren-2-yl)-4-(2H-tetrazol-5-yimethyisulfanyl)benzamide

N=

NPs H

CL N lo)

HPLC-MS (Method D): m/z: 400 (M+1); Rt = 4.67 min.

Example 524 (General procedure (l))

N-(9-Ethyl-9H-carbazol-2-yl)-4-(2H-tetrazol-5-ylmethylsulfanyl)benzamide

H Loy

OL CH,

N N

0

HPLC-MS (Method D): m/z: 429 (M+1); Rt = 4.67 min.

Example 525 (General procedure (1))

N-Phenyl-4-(2H-tetrazol-5-yimethylsulfanyl)benzamide

N=N

HN, _

N

“TO

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 lo: 0, N=N N=N

N. _ HN

Sher) — "eo

C) N N H

H \_ A? ly 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-y1)-9H-carbazole

N=N

HN _

Ors

N == 2 c 3-(2H-Tetrazol-5-yl)-9H-carbazole (example 401, 17 g, 72.26 mmol) was dissolved in N,N- dimethylformamide (150 mL). Triphenylmethy! chloride (21.153 g, 75.88 mmol) and triethyl- amine (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 ad- ditional 1 hour. The crude product was filtered off and dissolved in dichloromethane (500 mL). The organic phase was washed with water (2 x 250 mL) and dried with magnesium sul- fate (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. "H-NMR (CDCl): 68.87 (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 ul, 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 (3x10 mL) to furnish 9-(3-chlorobenzyl)-3-[2-(triphenyimethyl)-2H-tetrazol-5-yl]-9H-carbazole, which was: ‘ dissolved in a mixture of tetrahydrofuran and 6 N hydrochloric acid (ag.) (9:1) (10 mL) and stirred at room temperature for 18 hours. The reaction mixture was poured into water (100 mL). The solid was filtered off and rinsed with water (3 x 10 mL) and dichloromethane (3 x 10 mL) to yield the title compound (127 mg). No further purification was necessary. "H-NMR (DMSO-ds): $8.89 (1H, d), 8.29 (1H, d), 8.12 (1H, dd), 7.90 (1H, d), 7.72 (1H, d), 7.53 (1H, 1), 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

N=n

HN. _

Re

N

~-o

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

N=N

HN. _

Re

N on,

HPLC-MS (Method C): m/z: 340 (M+1); Rt = 4.26 min.

Example 529 (General Procedure (J)). 3-(2H-Tetrazol-5-yi)-9-(4-trifluoromethylbenzyl)-9H-carbazole

N=N

HN. _ “U0

N

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 ’

N=pn

HN. “0

N

“©

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

N=pN

HN. “0 “Oo

CH, 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

N=n

HN. . “0

N . "H-NMR (DMSO-d): 8.91 (1H, dd), 8.30 (1H, d), 8.13 (1H, dd), 7.90 (1H, d), 7.73 (1H, d), 7.53 (1H, 1), 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

N=nN

HN, _

YO

“0-0 ‘H-NMR (DMSO-ds): 58.94 (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

N=n

HN. _ ers ~O

O-c

Hy

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

N=N

HN, _ “C pte

HPLC-MS (Method C): m/z: 376 (M+1); Rt = 4.48 min.

Example 536 (General Procedure (J)). 9-(3-Bromobenzy!)-3-(2H-tetrazol-5-yl)-9H-carbazole

N=N

HN : he B ne

Br

HPLC-MS (Method C): m/z: 404 (M+1); Rt = 4.33 min.

Example 537 (General Procedure (J)). 9-(Biphenyl-2-yimethyl)-3-(2H-tetrazol-5-yl)-8H-carbazole

N=N

HN

“UO >

HPLC-MS (Method C): m/z: 402 (M+1); Rt = 4.80 min.

Example 538 (General Procedure (J)). 3-(2H-Tetrazol-5-yl)-9-{4-(1 ,2,3-thiadiazol-4-yl)benzyl]-9H-carbazole

N=N

HN _ “UO

N

ROE

Ss .

Example 539 (General Procedure (J)). 9-(2'-Cyanobiphenyl-4-yimethyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

N=N

HN, _ “0 y/ | :

NT

"H-NMR (DMSO-ds): 58.91 (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-lodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

N=n

HN. _ ae

N

~~

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

N=N

HN. _ “UC

N o

CF,

HPLC-MS (Method C): m/z: 462 (M+1); Rt = 4.70 min.

Example 542 (General Procedure (J)). 9-(4-Bromobenzyi)-3-(2H-tetrazol-5-yi)-9H-carbazole

N=n

HN, _ ae

N .

De "H-NMR (DMSO-d): 5 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, 1), 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

N=N

HN

Oa pr!

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

N=N

HN. “TC

N

O°

OH N

3.6 fold excess sodium hydride was used. "H-NMR (DMSO-d): 512.89 (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, 1), 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

N=N

HN, “0 ne,

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

N=N

HN )

TT

N

—O- "H-NMR (DMSO-d): 68.88 (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, 1), 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

N=N

HN. “TC “Go

F

H-NMR (DMSO-d): 68.89 (1H, d), 8.29 (1H, d), 8.12 (1H. dd), 7.90 (1H. d), 7.72 (1H, d), 7.53 (1H, 1), 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-lodobenzyl)-3-(2H-tetrazol-5-yl)-9H-carbazole

N=N

HN

“0 “©

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

N=N

HN. _ “0

N

H-NMR (DMSO-ds): § 12.97 (1H, bs), 8.90 (1H, bs), 8.30 (1H, d), 8.12 (1 H, bd), 7.89 (1H, d), 7.82 (1H, m), 7.77 (1H, bs), 7.71 (1H, d), 7.53 (1H, 1), 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

N=N

HN _ aS

N

Oo

CH, "H-NMR (DMSO-de): 68.87 (1H, d), 8.27 (1H, d), 8.10 (1H, dd), 7.87 (1H, d), 7.71 (1H, d), 7.51 (1H, 1), 7.31 (1H, 1), 7.15 (2H, d), 7.12 (2H, d), 5.68 (2H, 5), 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

N=p

HN, _ aS

N en

O-CH,

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

N=N

HN as

N F

~

F

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-ylJacetamide

N=N

HN he pe

N

H,C

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

N=N

HN ae

N

“(Oa

CH,

H-NMR (DMSO-dq): 68.86 (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, 1), 7.18 (2H, d), 6.84 (2H, d), 5.66 (2H, s), 3.67 (3H, 5); 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

N=N

HN. _ “TU <0

Q :

CH, "H-NMR (DMSO-ds): 8.87 (1H, d), 8.27 (1H, d), 8.09 (1H, dd), 7.77 (1H, d), 7.60 (1H, d), 7.49 (1H, 1), 7.29 (1H, 1), 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

N=n

HN, _ “0

N

~

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)-OH-carbazole

N=N

HN

“TU

N

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 :

N=n

HN.

Ne

N Ci

Q

CH,

H-NMR (DMSO-d;): 58.87 (1H, d), 8.35 (1H, d), 8.10 (1H, dd), 7.73 (1H, d), 7.59 (1H, d), 7.49 (1H, 1), 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-yllacetamide

N=N :

HN. _ ord ee

N

0 "H-NMR (DMSO-d): 510.54 (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, 1), 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-yljacetamide

N=N

HN, _ “0 ee

N

CH,

"H-NMR (DMSO-d): 58.85 (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

N=N

HN

“TU

N po

Cl "H-NMR (DMSO-de): 68.92 (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, 1), 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

N=N “0 <3

H,C "H-NMR (DMSO-d,): 68.92 (1H, d), 8.32 (1H, d), 8.08 (1H, dd), 7.72 (1H, d), 7.55 (1H, d), 7.48 (1H, 1), 7.32 (1H, 1), 7.26 (1H, d), 7.12 (1H, 1), 6.92 (1H, 1), 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

N=N

HN _ ae he!

NO,

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

N=N

HN _

Re

N .

Cl

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

N=N

HN. _ he

N n-

F

H-NMR (DMSO-d): 68.89 (1H, d), 8.29 (1H, d), 8.11 (1H, dd), 7.88 (1H, d), 7.69 (1H, d), 7.52 (1H, 1), 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

N=n

HN, _

RS

N “3

F t "H-NMR (DMSO-ds): 68.90 (1H, bs), 8.31 (1H, d), 8.13 (1H, bd), 7.90 (1H, d), 7.73 (1 H, d), 7.54 (1H, t), 7.34 (1H, 1), 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-tetrazoi-5-yl)-9H-carbazole

N=N

HN,

YC

N

~~

F

"H-NMR (DMSO-d): 58.89 (1H, bs), 8.29 (1H, d), 8.12 (1H, bd), 7.92 (1H, d), 7.74 (1H, d), 7.54 (1H, 1), 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-lodobenzyl)-3-(2H-tetrazol-5-yl)-8H-carbazole

N=N er

TC

3

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

N=N

HN he he

CF,

H-NMR (DMSO-ds): 68.89 (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-ylJacetamide

N=N

HN. _ “0 \°

HO

H

OH

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-yllacetamide

N=N “AD “OC °

CH, i

CH, : :

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-yfJacetamide

N=y

HN. he 0 »

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

N=n

HN. “CL ®

Le

OO"

H

Me Me

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]Jacetamide

N

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

N=N

HN _ he

Be

F,C

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

N=N

HN

YC

F

N

>

F.C

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

N=N

HN

“0

N cr,

F.C 3

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

N=n

HN he

NHC en

H,C

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

N=N

HN, _

Te

N y CH,

HC bn,

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)-SH-carbazole

N=N “AO he pe

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 X procedure (J) includes:

AD ed Aer

N

J ® CL,

Pp pe 3 m2) oO wl, Bi O) wo O)

N

Cr ig CC 4 CL 5 b

Me

F.C 5 Me “Aer MAD “Aer ® N R F @® N O @® N

Fo OH ed ef [Yep

N

® N @® N ® N C) . ola (pon

F

New “Ne

N

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 refer- ences cited therein), or using the methodology described in general procedure (E), optional! y changing the palladium catalyst to bis(tri-fert-butylphosphine)palladium (0).

N=p N=p N=n h . ™

N N N

CL, oe CL,

Oar RON GW [o]

oN 0) wi oN O N=N Oo

HN A Co LA CI HNL C

N N N kK;

CH, OH 0 *

General procedure (K) for preparation of compounds of general formula yo: ~N,

N N NaN, HNN \ AR cl \ NH,CI N= y yy ue N

N NaH N N \. AR? ( AR?

H lo H : 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

N=N

HN

N \ “Oo 5-Cyanoindole (1.0 g, 7.0 mmol) was dissolved in N,N-dimethyiformamide (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 x 25 mL). The combined organic phases were washed with brine (30 mL) and dried with so- dium 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 dichioromethane/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

N=N

HN :

N A .

N

~ a

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

N=N

HN. _

N \

N

H-NMR (200 MHz, DMSO-dq): 6 = 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 CoH 7Ns, HO: 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

N=N

HN. _

N N

N

H

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

H

N-N / \\

NaN on

N

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:

Neng N=n N=y

HN HN, _| HN,

N N .

CO <Q “O

CF, O~cH, *

Nay N= Nan

HN HN _ HN,

N N O-~cH,

Oo “O- ae

O-cH,

N=y N=pn N=y

HN, HN, _ HN ae O 00 [»] 1 . () nN’

N=n N=n N=n

HN. HN, _ HN

N (7) N

CO pe

CH, HC

N=y N=N N=N

HN, HN, _] HN)

N N

“Chen —O ae! ° Br

N=py N=N N=Nn

HN, _| HN, HN.

N N

C- 0 “-

F F

N=p N=py N=N

HN, HN HN

N N cl 1. CH,

N= N=n N=N

HN HN. _ HN,

N N F

F ( ) ~Ct C )

F Ci F

The following preferred compounds of the invention may be prepared eg. from 1-(4- bromobenzyl)-5-(2H-tetrazol-5-yl)-1H-indole (example 582) 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 refer- ences cited therein), or using the methodology described in general procedure (E), optionally changing the palladium catalyst to bis(tri-tert-butylphosphine)palladium (0).

N=N Nay

HN, HN,

N N

OG Oo

Y .

N=n N=n N=N

HN] HN, HN, :

CH, g OH

General procedure (L) for preparation of compounds of general formula l44:

N N ne | : \ CIn__AR? \ NaN, A \ hig ~u \ NHC N

DE N Licl \

N Et,N/DMAP N H N H

H 3 /

I~AR’ AR? o lo} by

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

N=N

HN

N A\

N av

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 concentra-

tion 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-yi}-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)-1 H-indole

H

N-N

NN

0

N al 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-benzoyi-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 Zn? site of the insulin hexamer:

Example 5881-(4-Fluorophenyl)-5-(2H-tetrazol-5-yl)-1H-indole

N=n

HN _

N A

F

Example 5891-Amino-3-(2H-tetrazol-5-yl)benzene

N=n

HN er

Example 5901-Amino-4-(2H-tetrazol-5-yl)benzene

N=n

HN, _

N

NF,

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 mix- ture was filtered and the solid was dried in vacuo at 40 °C for 16 hours to afford 5-(4- aminophenyl)-2H-tetrazole. 'H NMR DMSO-de): 6 = 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 5911-Nitro-4-(2H-tetrazol-5-yl)benzene

N=N

HN.

N

NO

Example 5921-Bromo-4-(2H-tetrazol-5-yl)benzene

N=N

HN.

N

5 Br

General procedure (M) for solution phase preparation of amides of general formula bz: 0 PY 0 astetlon + HYTR —» a-sletlNTr

R' R' h2 wherein A, B', B2 are as defined above, R is hydrogen, optionally substituted aryl or C,.-alkyl and R’ is hydrogen or C,4-alkyl.

A-B'-B%-CO,H 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)-1 H-indol-1-yllacetamide oO

Q Ay & Ae ~s | NH

Ee ©

[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indol-1-yljacetic 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-dimethylamino- propyl)carbodiimide, hydrochloride (86.4 mg, 0.45 mmol) and 1-hydroxybenzotriazol (68.8 mg, 0.45 mmol) in NMP (1 mL). The resulting mixture was shaken at RT for 2 h. 4- Chlorobenzylamine (51 mg, 0.36 mmol) and DIPEA (46.4 mg, 0.36 mmol) in NMP (1 mL) were added to the mixture and the resulting mixture shaken at RT for 2 days. Subsequently ethyl acetate (10 mL) was added and the resulting mixture washed with 2x10 mL water fol- lowed by saturated ammonium chloride (5 mL). The organic phase was evaporated to dry- ness 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 0]

TY OL

N, H

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)phenoxylbutyramide o 0

Ys o~ NX, w ICL TOL 0

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 0 Q : sus naa as x ct

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)phenoxyl-N-(4-chlorobenzyl)acetamide o lo) x Br Ci 0]

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)phenoxylacetamide

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)phenyljacrylamide o 0

WS CCL

= cl 0

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 o lo] ph ou~Ay -

LI COTO

Br Ci lo)

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 " lo) ss uag ie!

HN J H

Br Ci 0]

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 -yloxylacetamide oO

Oo . a

Ms ©

HN. A

FOC

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-yljpropionamide

N

: H «A sn

A

0

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 3. OJ i

SIT x cl lo]

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 3, OJ i >~s C) och,

HNL H lo]

HPLC-MS (Method C): m/z: 441 (M+1); Rt = 4.31 min.

Example 607 (General procedure (M))

N-(4-Chlorobenzyi)-4-[3-(2H-tetrazol-5-yl)carbazol-9-yimethyllbenzamide

Ns

HNN

“or

N

. Qa Cl

So

N oO ’

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-3-yimethyllbenzamide

Ns ’

HN TN

“or

N "CL re o

HPLC-MS (Method C). m/z: 493 (M+1); Rt = 4.20 min.

Example 609 4-({[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)indole-7-carbonyllamino}methyl)benzoic acid methyl ester a 0-CH,

Ys N °

HN

The commercially available compounds in the following examples do all bind to the HisB10

Zn*'site:

Example 610 1-(4-Bromo-3-methylphenyl)-1,4-dihydrotetrazole-5-thione

N=N

HN

N

IC

S

Br .

CH,

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

N=N

HN N

LGN

S

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

N=N

NN cl r XX

Cl Ci

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

N=N cH,

HN A

S

H.C

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

N=N CH,

HNL A

S

H,C CH,

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

N=N

ANN

Eh, oo

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

N=N

NN

S g "Cl

Cl

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

N=N "Ny N

S Tl. CH,

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

N=N

HNN

S C ]

Cl

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

N=N

NN

I 1

F

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

N=N

U aN

I 1

Cl Cl

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

N=N

HN

"QJ

S or

F

Example 622

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

N=N

HNL

Fi by

H,C” "0

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

N=N

HNN

IC

Cl

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

N=N

ANA .

Ss | @ o-CHs

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

N=N

HNN

S

F

Preparation of 1-aryl-1,4-dihydrotetrazole-5-thiones (or the tautomeric 1-aryitetrazole-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: 0, 0 o* N Step 1 oN Step 2 HN . CH of —= Tg

OH oY o No 0 0

Step 3

N=n

Myon Step 4 SCN (CH) - (CH),

HS CL / A oH TL 4 2)m 0 Ng 0 YO" 6) 0]

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. SnClz, H; 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 CS,

CSCl,, 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: = N=n on A @ HN A re

N=N N=N :

HNN HNN

I'L I'l oy o>"

Oo oO

N=N N=N

HN | HN 3

Oo

N=N

HNN

0]

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

Ho—( a

NH

N= .

Phenylsulphony! acetonitrile (2.0 g, 11.04 mmol) was mixed with 4-hydroxybenzaldehyde (1 354, 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 di- ethyl ether and toluene. The solid formed was filtered to afford 2.08 g (66%) of 2- benzenesuifonyi-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 so- dium azide (0.47g,7.3 mmol) in DMF (50 mL) was heated at reflux temperature 2 hours. After cooling, the mixture was poured on ice. The mixture was evaporated in vacuo to almost dry- ness 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-dg): 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. = 1.83 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

FE

Xf

F

0

I {

Ny SY

H

Example 628

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

N= AN

Wi ¢ [e}

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

F

~=N nS = \

AN

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

N

Nn ) ~~ 4

NN

N

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

N

Ci = . ~~

Nt

Example 632

4-Thiophen-2-yl-1H-[1,2,3]triazole-5-carbonitrile s .

JN

NL — .

N IN

H

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

CH o \ : N

CH, O =

NH

N=N

Example 634 3,3-Dimethyl-butyric acid 4-(5-cyano-1H-{1,2,3]triazol-4-yi)phenyl ester a

NC Ill [o} ~~ ba

FN

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

N

< N\

NSN

N i RN) \ lo] AN

CH, .

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

N

“ NN

AN

\ N \ 0) 0)

Cl

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

N=N © =N

Example 638 4-(5-Bromo-2-methoxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile ,CH, 0 NEN oo ~=N

Br

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

Fo NN ~ NH =N

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)-phenyljterephthalamic acid ) 4-(4-Octyloxyphenyl)-1H-[1,2,3]triazole-5-carbonitrile 4-(4-Styrylphenyl)-1H-[1,2,3]triazole-5-carbonitrile. 4-(4"-Trifluoromethylbiphenyl-4-yl)-1 H-[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-Anthrany!)-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 l43:

CH,) (CH,)

AR! (CH,) AR ( 2/n AR’ 2/n 0 J\Z" Step! 0 $Y . step2 oR /\_oH

Y OH + | ea Nor Y 0 RF — Y 0

H 0 H oO H 0

Step 3

SO.Ph |, (CH.) = Step 4 21 q 2/n

N=N 1(CH In AR /

N la wherein nis 1or 3-20,

AR is as defined above, :

R” is a standard carboxylic acid protecting group, such as C;-Ce-alkyl or benzyl and Lea is a leaving group, such as chioro, 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 & Gunther, Chem. Ber., 106, 2758-66 (1973)

Step 3 is a Knoevenagel condensation of the aldehyde obtained in step 2 with phenylsuifon- ylacetonitrile and step 4 is a reaction of the vinylsulfonyl compound obtained in step 3 with sodium azide. This reaction is usually performed in DMF at 90 — 110 °C. 10 .

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:

N=N : “OL “se

OH

HRS

0)

N=N

HN, $ on

N oY ©

N=N

HN___

OH

{ Clay 0] 2-(4-(5-Cyano-1H-[1,2,3]triazol-4-yl)phenoxy)acetic acid:

N=N

OH ery 0) 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 ,2,3triazol-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 I;:

H,N-PS D

HN-(Arg);N-PS D

HN-(Gly)7 (Arg); N-PS

H,N-(Abz); (Gly); (Arg);N-PS J j J

N - (Cl) -N—

N OR (4-Abz){Gly); (Arg); N-PS

N

H ) o .

OR Le

N

H

7 wherein PS is polymeric support, a Tentagel S RAM resin, nis 1-20, mis 0-5, andpis 0 or 1.

The compounds of the invention of general formula (I.) can be prepared by means of stan- dard 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 exam- ples 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 substitu- tion 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 pl 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 (No- vabiochem) 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. TH-benzotriazole-5-carboxylic acid on Abz or 4-Apac:

Due to the lower nucleophilicity of the amino group in Abz the following procedure was nec- essary. To 4 mmol of A-OH, e.g. 1H-benzotriazole-5-carboxylic acid was added 6,66 ml ofa solution of 0,6M HOA, 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 us- ing SnCl, 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 mi 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 x 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 % (“/) TFA in HO and B-

Buffer (87.5 % (*/w) MeCN, 0,13 % (*/) TFA in H;0).

The products were purified on preparative RP-HPLC C18 column (2x25 cm) using a gradient (variable, see e.g examples 640 to 643643643), temperature 25°C, wavelength 214 nm and flow rate 6 mi/min with A-buffer 0,15 % (*/,)) TFA in H,O and B-Buffer (87,5 % (*/) MeCN, 0,13 % ("/w) :

TFAin H,0) and verified by mass spectrometry (MALDI).

Table 1:

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

Ti i el hn 0 [wo feo 10 00 io Joo [00 “000 100 Joo [1000 #500 [10 [0 [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 (0).

Example 640 (General Procedure (O))

Benzotriazol-5-ylcarbonyl-Gly,-Args-NH; (BT-GzR3).

SORRNE N Ay NH,

N H H : H

N lo] lo] 0}

H id

HN

HN SH

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

HPLC gradient:

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

Example 641 (General Procedure (O))

Benzotriazol-5-ylcarbonyl-Gly,-Args-NH: (BT-G2R4).

FINS NH HN NH

NH NH

N Te NA NA,

N. Hg H § i H o J

HN HN

HN Sh nS

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

HPLC gradient:

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

EL CE LC EC

ERE CR 2 J LC

Example 642 (General Procedure (O))

Benzotriazol-5-yicarbonyl-Gly,-Args-NH, (BT-G.Rs).

HN NH, HN NH HN NH;

NH NH NH

0] [0] 0 fo)

H H H

N ax Cit Cag Co

N H § H og 2 H go 2 H o : JES

HN HN

HN SNH Sh

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

HPLC gradient

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

EEC CC LC EN

Example 643 (General Procedure (O)) Benzotriazol-5-ylcarbonyl-Gly,-Args-NH; (BT-G2Rs). /

HNo NH, HN NH, HN NH, @ Ho 9 Ho Ho$ Ho §

N x Cas Cat, NA,

N, H o H § 2 H oo 2 H § 2 ; I

HN HN HN

HS HAS Sh :

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

HPLC gradient:

I ON I I o® Jeo [mo [0

CEC I EC

Example 644 (General Procedure (O))

Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Args-NH; (BT-4-Abz-G2Rs).

HN NHz HN NH, HN NF,

NH NH NH

0 0 0 lo]

ST I i

HNTSNH HN NH

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

HPLC gradient:

Time (min) Flow %A %B i oor jew [wo [50

EN LN J EC a LC LU EJ

EE LC I Kc a LI LCI

EE LC LB

Example 645 (General Procedure (0) Benzotriazol-5-ylcarbonyl-4-Abz-Gly-Args-NH; (BT-4-Abz-GRs).

H NH, HN NH, HN NH,

N, Ls

N 0 fo} [0

H H H

° Cl, ES ak 0 Hoo 7 Hoo je 0

HN HN )

NS HSH

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

HPLC gradient:

Time (min) Flow %A %B (ml/min) 000 ~~ Je00 50 [80 4000 600 Jeeo 400 45.00 600 ~~ jeoo [400 50.00 60 [00 |1000 55.00 600 Joo [1000 60.00 600 Jss0 [SO

Example 646 (General Procedure (O))

Benzotriazol-5-ylcarbonyl-4-Abz-Args-NH, (BT-4-Abz-Rs).

H,N.__NH H,N.__NH

H YY YY

N HN HN

NG §

Ho HS Hf 0 RAHA,

ER UE WR

NH NH NH

HNN, HNN, HNP NH,

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

HPLC gradient: :

Time (min) Flow %A %B (ml/min) : oo [em [wo [80

General procedure (P) for preparation of compounds of general formula ls:

HN-PS D

HN-(Arg);N=PS )

H N= (Gly) (Arg);N-PS

H,N~(Abz); (Gly) (Arg);N-PS b)

Ney )

ANE +B NPS ~~ ~ —- — —_ ~N]— l Te B TN (Abz)5 (Gly) (Arg); N P ; J vy

HN E B2 = B rN (Ab2); (Gly) (Ara); NH, 0 R Oo lg wherein X, Y, R™, E, B', B? are as defined above, pisQort, m is 0-5 and nis 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): > J 2 a J 0 RY 0)

Example 647 (General Procedure (P)) 4-{2-[3-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxylacetylamino}benzoyl-Gly,-Args-NH:

HN.__NH HN.___NH lo} YY ad . HN HN

S w ICL ~

Oo 0 0) 0 0 .

H H H H o] 0] N N . Y ~~ + NAN, 0 0 hl 0 Ng 0 al by NH NH

HN” “NH, HNN, HN NH,

Example 648 (General Procedure (P)) 3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyllacryloyl-Args-NH;

HNN HN NH. HN ANH,

NH NH NH

0} Oo 0] 0) H H

Yes x Lesa i

HN” SNH HN” “NH

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)phenyllacryloyl-Arg,-NH,

HN NH, HN

NH NH

0] Oo 0 0) H H

N

)~s S Ie EY wn

HASH H,N” “NH

MS (MALDI): m/z: 899.1 g/mo}; calculated: 901.6 g/mol.

Example 650 (General Procedure (P)) 3-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenyi]acryloyl-Args-NH;

HN NH; HNN

NH NH

0) 0

H

Vos x EE

HSH

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)phenoxylbutyryl-Args-NH.

HN NF HN NH HN Ne

NH NH NH

0 0 0) 0) H H ee ia it

HN a H fo) = H 0 > 0 ,

L J

HN BY

HN SH HN” “NH

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-Args-NH..

HN NH: HN He

NH NH

Oo 10) oO o H H

HN x H 0] > H Oo Ie

L J

HN "I

HN SNH H,N7 NH

MS (MALDI): m/z: 933.0 g/mol; calculated: 931 g/mol.

Example 653 (General Procedure (P)) 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)phenoxylbutyryl-Args-NH,.

HN NH HN NH,

NH NH o) 0 Hof

Yes oI AANA AN : : mw AT H § : H 4 lo} ig

HN

HN SNH

MS (MALDI): m/z: 776.9 g/mol; calculated: 774.0 g/mol.

Example 654 (General Procedure (P)) 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxylbutyryl-Arg-NH,.

HN NH HN NF HN NH HN NH: HN NF: HN He

HN Sh HSH HS HS HSN Sh

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-yloxylbutyryl-Args-NH,.

HN. NH HN_NH HN. NH HNy NH

SARA St St

NH NH NH NH

Ys o~N AA NA NI NAN ® N TN ~"N < "N "NH,

HN. 1 H og ¢: H og 2 H go > H o ~ k Soy

HN HN HN HN

HS HS HN Sh HS

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-Args-NH,.

HN NH: HN NH: HN NH

NH NH NH

0) (0) oO . 3. C BoE

NH

Vg alas Cand 2

HN Ng H fe) ped H e) = 0 A : J

HN "TL

HS HN" “NH

MS (MALDI): m/z: 1141.9 g/mol; calculated: 1137.4 g/mol. : : Example 657 (General Procedure (P)) 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Args-NH,.

HN NH, ANN,

NH NH

Oo 0] 0 0) H H i CO) 2 Lad Cad,

HS HN NH

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-Args-NH..

HN NH, HN NH,

NH NH o Oo H 0

Yes ou ~ IANA ANH, :

HS

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-Gly,-Args-NH;

HN NF HN Ne HN Ns ) NH NH NH

NA N N NH,

N en HN HN

N=N ew No

MS (MALDI): m/z: 1203.8 g/mol; calculated: 1203.8 g/mol.

Example 660 4-[3-(2H-Tetrazol-5-yl)carbazol-9-yimethyllbenzoyl-Args-NH: nN

N=

HN N HN NH, HN NH,

De TS dh A 4

N NH NH NH

OC us Cat

NH,

NYY NYY jd o Is

HN HN an NH nS NH

MS (MALDI): m/z: 1152.5 g/mol; calculated: 1149.3 g/mol.

Example 661 4-[3-(2H-Tetrazol-5-yl)carbazol-9-ylimethyllbenzoyl-Args-NH;

HN

Or HN yt A i a

N NH NH NH NH o .

ESS Se

HN HN HN HN

EN Sw EN aS

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-Arg:>-NH,

Ny

HNN

“OO HNS NH, HS NH, HN NH, HN NH, HNN, HN NH,

N NH NH NH - NH NH NH

H o : H 0 z 0 : 0 H 0 HS 0 i wd oJ ol oJ oJ +

HSN HN SNH HN SH NSH HASH HN" SNH

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 pro- cedure (O) and / or general procedure (P) includes:

Building block from example 291: 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg,,-NH; 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Argg-NH; 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Args-NH; 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg;-NH. 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Args1-NH, 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg,,-NH, 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxy]butyryl-Arg;-NH, } 4-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)naphthalen-1-yloxylbutyryl-Args-NH,

2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3- 2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3-

2-{5-[4-(2,4-Dioxothiazolidin-5-ylidenemethyl)benzylidene]-4-oxo-2-thioxothiazolidin-3- oo

4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyllbenzoyl-Args- 4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyllbenzoyl-Args- 4-[5-Bromo-6-(2,4-dioxothiazolidin-5-ylidenemethyl)naphthalen-2-yloxymethyllbenzoyl-Args-

Example 663

Equilibrium Solubility. For pH-solubility profiles, a 0.6 mM human insulin stock solution con- taining 0.2 mM Zn?*, 30 mM phenol, 0.2 M mannitol, 2 mM phosphate and Zn*" -binding ligand as required were prepared and the pH was adjusted to the desired value correspond- ing 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 con- tents in the supernatant by SEC HPLC analysis

The effect of various concentration of the ligand BTG,Rs on the pH-dependence of insulin solubility is illustrated in Figure 1.

Example 664

The effect of increasing concentrations of the ligand BTG;R, on the pH-dependence of insu- lin solubility is illustrated in Figure 2. The solubility was determined as in example 663. Solu- tion conditions: 0.6 mM human insuiin, 0.2 mM mM Zn?*, 30 mM phenoi, 0.2 M mannitol, 2 mM phosphate, 23 C.

Example 665

The slow release (prolonged action) properties of certain formulations of the present inven- . tion was characterized by the disappearance rate from the subcutaneous depot following subcutaneous injections in pigs. Tse is the time when 50% of the A14 Tyr(*®1) insulin has disappeared from the site of injection as measured with an external y-counter (Ribel et al.,

The Pig as a Model for Subcutaneous Absorption in Man. In: M. Serrano-Rtios and P.J. Le- febre (Eds): Diabetes (1985) Proceedings of the 12™ congress of the International Diabetes

Federation, Madrid, Spain, 1985 (Excerpta Medica, Amsterdam (1986), 891-896). The com- position of a series of protracted formulations is given in the table below together with the

Tsoy, values. The disappearance curves are illustrated in Figure 3 a-d. For comparison, the

Tso 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 fur- ther advantages in terms of versatility regarding the choice of insulin species and release patterns. Consequently, human or mutant insulins such as Asp, Lys®?*Pro®, or Gly*2'Lys®*GIu®? may be formulated as slow- or dual-release preparations by adding vari- able amounts of His3™® ZnZ-site ligand. This is illustrated below for Asp®®® human insulin em- ploying two different levels of the ligand TZD-Abz-G.Rs (example 647). As shown in the table and in Figure 3 panels e-f, addition of this ligand in slight excess of the Zn?" concentration produces a slow release preparation with Tse, about 14.8. In contrast, when the ligand is added in concentrations lower than that of Zn?*, a distinctly dual-release formulation results.

I-Prep. I-Prep. 0.6 0.6 0.6 0.6 B28 0.6

Insulin 0.6 Asp . B28 el human human human insu- human k . Asp (mM) - Co Co . insulin Co insulin insulin lin insulin insulin

Ea CN ACEI IC CE WC 30mM

Phenolic 30mM 30mm 30mM phe- 7 30 mM 30 mM ligand phenol phenol nol . phenol phenol hydroxyindole 0.4 mM 0.15 mM 6mM mM 2mM BT- 2mM BT-

Zn?" ligand BTGR, BTGRs AbzG,R AbzG,R TZD- T0- n' liga gan 2 2 2% ih AbzG,Rs | AbzG,Rs (Ex. 641) | (Ex.643) (Ex. 644) (Ex. 644) (Ex. 647) | (Ex. 647)

Phosphate 2 2 2 2 2 2 buffer (mM)

ANALYTICAL METHODS

Assays to quantify the binding affinity of ligands to the metal site of the insulin Re hexamers:

AH3N-assay:

The binding affinity of ligands to the metal site of insulin Rs 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 Rs 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 Rs hexamers with 4H3N mounted in the metal site allows the binding affinity of these ligands to be determined follow- ing the reduction of absorption at 444 nm.

A stock solution with the following composition 0.2 mM human insulin, 0.067 mM Zn-acetate, 40 mM phenol, 0.101 mM 4H3N is prepared in a 10mL quantum as described below. Buffer is always 50mM tris buffer adjusted to pH=8.0 with NaOH/CIO,". 1000 pl of 2.0mM human insulin in buffer 66.7 pL of 10mM Zn-acetate in buffer 800 pL of 500mM phenol in HO 201 pl of 4H3N in HO 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 addi- tion the UV/vis spectrum is measured. The titration points are listed in Table 3 below.

Table 3 ligand | ligand addition | conc. | dilution (a) | (mM) | factor 0.010 | 1.0005 0.020 | 1.0010 0.030 | 1.0015 0.050 | 1.0025 0.100 | 1.0050 0.198 | 1.0100 0.392 | 1.0200 0.583 | 1.0300 : 0.769 | 1.0400 0.952 | 1.0500

The UVNis spectra resulting from a titration of the compound 3-hydroxy-2-naphthoic acid is shown in Figure 5. Inserted in the upper right corner is the absorbance at 444nm vs. the con- centration of ligand.

The following equation is fitted to these datapoints to determine the two parameters Kp(obs), the observed dissociation constant, and abs. the absorbance at maximal ligand concentra- tion. abs ([ligand]yee) = (absmax * lligand]eee)/ (Ko(obs) + [ligand]iee)

The observed dissociation constant is recalculated to obtain the apparent dissociation con- stant

Ko(app) = Kp(obs) / ( 1+[4H3N}/ Kanan)

The value of Keuan=50 uM is taken from Huang et al., 1997, Biochemistry 36, 9878-9888.

TZD-assay:

The binding affinity of ligands to the metal site of insulin Rg hexamers are measured in a fluo- rescense based displacement assay. The fluorescence of 5.(4- dimethylaminobenzylidene)thiazolidine-2,4-dione (TZD) which is a ligand for the metal site of insulin Rg is quenched upon displacement from the metal site to the solution. Titration of a ligand to a stock solution of insulin Rs hexamers with this compound mounted in the metal site allows the binding affinity of these ligands to be determined measuring the fluorescence at 455nm upon excitation at 410nm. 5)

Preparation

Stock solution: 0.02 mM human insulin, 0.007 mM Zn-acetate, 40 mM phenol, 0.01 mM TZD in 50mM tris buffer adjusted to pH=8.0 with NaOH/CIO,".

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 OM.

Measurements

Fluorescence measurements were carried out on a Perkin Eimer Spectrofiuorometer

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

AF(455nm)) = AF max * [ligand}iee/( Ko(app) * ( 1+{TZD)/Krzp )+ [ligandliee)) Kp(app) is the apparent dissociation constant and Fay is the fluorescence at maximal ligand concentration. The value of Kyzp is measured separately to 230 nM

Two different fitting-procedures can be used. One in which both parameters, Ko(app) and

F..ex, are adjusted to best fit the data and a second in which the value of Frmax is fixed (Fmax=1) and only Kp(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 general formula (ill) A-B-C-D-X (li) wherein: A is a chemical group which reversibly binds to a His®'® Zn?" site of an insulin hexamer; Bis a linker selected from ¢ A valence bond « A chemical group G® of the formula -B'-B%-C(0)-, -B'-B%-S0,-, -B'-B-CHg-, or -B'- B2-NH-; wherein B' is a valence bond, -O-, -S-, or -NR°-, B? is a valence bond, C-Cys-alkylene, C,-Cis-alkenylene, C,-Cis-alkynylene, arylene, heteroarylene, -C,-Cg-alkyl-aryl-, -C,-Cig-alkenyl-aryl-, -C,-Cqg-alkynyl-aryl-, -C(=0)- C1-Cig-alkyl-C(=0)-, -C(=0)-C4-Cyg-alkenyl-C(=0)-, -C(=0)-C;-Cs-alkyl-O-C4-C1s- alkyl-C(=0)-, -C(=0)- C4-C4s-alkyl-S-C;-Cyg-alkyl-C(=0)-, -C(=0)-C,-Cys-alkyl-NR®-C;- Cie-alkyl-C(=0)-, -C(=0)-aryl-C(=0)-, -C(=0)-heteroaryl-C(=0)-; wherein the alkylene, alkenylene, and alkynylene moieties are optionally substituted by —CN, -CF3, -OCF3, -OR®, or -NR°R’ and the arylene and heteroarylene moieties are optionally substituted by halogen, -C(O)OR®, -C(O)H, OCORS®, -S0O,, -CN, -CF;, - OCF3, -NO,, -OR®, -NR°R’, C,-C4g-alkyl, or C-Cis-alkanoyl; R®and R’ are independently H, C;-C4-alkyl; Cis 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, -NH, 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.

2. A zinc-binding ligand according to claim 1 wherein A is a chemical structure selected from the group consisting of carboxylates, dithiocarboxylates, phenolates, thiophenolates, alkyl- thiolates, sulfonamides, imidazoles, triazoles, 4-cyano-1,2,3-triazoles, benzimidazoles, ben- zotriazoles, purines, thiazolidinediones, tetrazoles, 5-mercaptotetrazoles, rhodanines, N- } hydroxyazoles, hydantoines, thiohydantoines, barbiturates, naphthoic acids and salicylic ac-

ids.

3. A zinc-binding ligand according to claim 2 wherein 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.

4. A zinc-binding ligand according to any one of the claims 1 to 3 wherein Ais No \ : Y — Y HN E or "Ns ec 10 N Rr? R R" \_12 0] R® oO R . wherein Xis=0, =S or =NH Y is -S-, -O- or -NH- R® and R"! are independently hydrogen or C;-Cg-alkyl, R? is hydrogen or C,-Cs-alky! or aryl, R® and R? may optionally be combined to form a double bond, R'™ and R™ are independently hydrogen, aryl, C,-Ce-alkyl, or -C(O)NR"R"’ E and G are independently C,-Cg-alkylene, arylene, -aryl-C,-Ce-alkyl-, -aryl-C,-Cg-alkenyl- or : heteroarylene, wherein the alkylene or alkenylene is optionally substituted with one or more substituents independently selected from halogen, -CN, -CF3, -OCF;3, aryl, -COOH and —NH,, and the arylene or heteroarylene is optionally substituted with up to four substituents RR", RS and R15A E and R' may be connected through one or two valence bonds, G and R'? may be con- nected through one or two valence bonds;

. R® RY R'" and R"™ are independently selected from

« hydrogen, halogen, -CN, -CH,CN, -CHF, -CF3, -OCF3, -OCHF,, -OCH,CFs3, -OCF,CHF,, -S(0).CF3, -OS(0),CFs, -SCF3, -NO,, -OR™, -NR™R", -SR™, -NR™S(0),R", -S(0);NR™R", -S(O)NR™R", -S(O)R", -S(0),R"®, -0S(0), R", -C(O)NR™R", -OC(O)NR™R", -NR™C(O)R"", -CH,C(O)NR"R", -OC,-Ce-alkyl-C(OINR™R"7, -CH,OR¢, -CH,OC(O)R", -CH,NR'*R"’, -OC(O)R", -OC,-Ce-alkyl-C(O)OR®, -OC;-Cg-alkyl-OR'®, -SC,-Cs-alkyl-C(O)OR™®, -C,-C¢-alkenyl-C(=O)OR'®, -NR'6-C(=0)-C;-Cs-alkyl-C(=O)OR'®, -NR'6.C(=0)-C;-Ce-alkenyl-C(=O)OR™® , -C(O)OR', or —-C,-C¢-alkenyl-C(=O)R™, =O, or -C-Ce-alkenyl-C(=0)-NR'"R", » C,-Ce-atkyl, Co-Ce-alkenyl or C,-Cg-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCFs, -OR', and -NRR" « aryl, aryloxy, aryloxycarbonyl, aroyl, arylsuifanyl, aryl-C,-Ce-alkoxy, aryl-C4-Ce-alkyl, aryl-C,-Cg-alkenyl, aroyl-C,-Cs-alkenyl, aryl-C,-Cg-alkynyl, heteroaryl, heteroaryl-C- Ce-alkyl, heteroaryl-C>-Ce-alkenyl or heteroaryl-C,-Ce-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR’®, -CH,C(O)OR', -CH,0OR, -CN, -CF3, -OCF;, -NO,, -OR'6, -NR'"®R', S(0),R'®, aryl and C,-Ce-alkyl, R' and R' independently are hydrogen, OH, C,-Cx-alkyl, aryl-C-Ce-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3;, -OCF3, -OC,-Cg-alky!, -C{(O)OC;-Ce-alkyl, -COOH and —NH;, and the aryl groups may optionally be substituted by halogen, -C(O)OC;-Cs-alkyl, -COOH, -CN, -CF,, - OCF, -NO,, -OH, -OC4-Ce-alkyl, -NH,, C(=0) or C,-Ce-alkyl; R'® and R"” 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

5. A zinc-binding ligand according to claim 4 wherein X is =O or =S

6. A zinc-binding ligand according to claim 5 wherein X is =O

7. A zinc-binding ligand according to claim 5 wherein X is =S

8. A zinc-binding ligand according to any one of the claims 4 to 7 wherein Y is -O- or -S-

9. A zinc-binding ligand according to claim 8 wherein Y is -O-

10. A zinc-binding ligand according to claim 8 wherein Y is -S-

11. A zinc-binding ligand according to any one of the claims 4 to 10 wherein E is arylene op- tionally substituted with up to four substituents, R*, R™, R'®, and RA.

12. A zinc-binding ligand according to claim 11 wherein E is phenylene or naphtylene option- ally substituted with up to four substituents, R", R", R'®, and R"A.

13. A zinc-binding ligand according to claim 12 wherein E is R" rR" or Joc] 14 Rr" R R 13 R'

14. A zinc-binding ligand according to claim 13 wherein E is RS rR! s ra 13 RY 14 R Rr" R

15. A zinc-binding ligand according to claim 12 wherein E is phenylene

16. A zinc-binding ligand according to any one of the claims 4 to 10 wherein E is heteroary- lene optionally substituted with up to four substituents, R'®, R*, R'®, and R**,

17. A zinc-binding ligand according to claim 15. A zinc-binding ligand according to claim 12 wherein E is phenylene 16 wherein E is benzofuranylidene optionally substituted with up to four substituents R', R", R', and R"™A

18. A zinc-binding ligand according to claim 17 wherein E is RL] hy ® 13 0 RY 0] 14 0 13 rR"? R R }

19. A zinc-binding ligand according to claim 15. A zinc-binding ligand according to claim 12 wherein E is phenylene

16 wherein E is carbazolylidene optionally substituted with up to four substituents R" R™, RY and RA :

20. A zinc-binding ligand according to claim 19 wherein E is R'S rR" / Ia rR”

21. Azinc-binding ligand according to claim 15. A zinc-binding ligand according to claim 12 wherein E is phenylene 16 wherein E is quinolylidene optionally substituted with up to four substituents R', R', R™, and R*™*.

22. A zinc-binding ligand according to claim 21 wherein E is R" R' oF i N or RE rR" 13 rR" R

23. A zinc-binding ligand according to claim 15. A zinc-binding ligand according to claim 12 wherein E is phenylene 16 wherein E is indolylene optionally substituted with up to four substituents R™®, R™, R", and R",

24. A zinc-binding ligand according to claim 23 wherein E is RS R" rR R™ RY R™ RR R" u L _ NH § - Rr" R'

25. A zinc-binding ligand according to any one of the claims 4 to 24 wherein R® is Hydrogen.

26. A zinc-binding ligand according to any one of the claims 4 to 25 wherein R® is Hydrogen.

27. A zinc-binding ligand according to any one of the claims 4 to 24 wherein R® and R® are combined to form a double bond.

28. A zinc-binding ligand according to any one of the claims 4 to 27 wherein RY is C-Ce- alkyl.

29. A zinc-binding ligand according to claim 28 wherein R' is methyl.

30. A zinc-binding ligand according to any one of the claims 4 to 10 wherein G is phenylene optionally substituted with up to four substituents, R™®, R', R'®, and R™.

31. A zinc-binding ligand according to any one of the claims 4 to 10 or 30 wherein R'" is Hy- drogen.

32. A zinc-binding ligand according to any one of the claims 4 to 10 or 30 to 31 wherein R' is Hydrogen.

33. A zinc-binding ligand according to any one of the claims 4 to 32 wherein R™, R*, R'® and R'** are independently selected from « hydrogen, halogen, -NO,, -OR®, -NR"R", -SR¢, -NR°S(0),R"", -S(0).NR"R", -S(O)NR™R", -S(O)R®, -S(0),R™, -0S(0), R*®, -NR'®C(O)R"’, -CH,OR"™, - CH,OC(O)R®, -CH,NR™R", -OC(O)R'®, -OC;-Cs-alkyl-C(O)OR™, -OC;-Cs- alkyl-C(O)NR'®R", -OC,-Cs-alkyl-OR'®, -SC,-Cs-alkyl-C(O)OR'®, ~C,-C;-alkenyl- C(=O)OR’®, -C(O)OR™, or —C,-Cs-alkenyl-C(=O)R"®, ¢ C,-Cg-alkyl, C-Cs-alkenyl or C,-Cs-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF;, -OR'®, and -NR"R"’ « aryl, aryloxy, aroyl, arylsulfanyl, aryl-C,-Ce-alkoxy, aryl-C-Ce-alkyl, aryl-Co- Ce-alkenyl, aroyl-C,-Ce-alkenyl, aryl-C,-Cs-alkynyl, heteroaryl, heteroaryl-C,-Ce-alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR*, -CH,C(O)OR', -CH,OR’, -CN, -CF3, -OCF3, -NO;, -OR", -NR"R"” and C4-C¢-alkyl.

34. A zinc-binding ligand according to claim 33 wherein R', R', R"® and R'** are independ- ently selected from « hydrogen, halogen, -NO,, -OR®, -NR"R'?, -SR', -S(0),R®, -0S(0), R", - CH,OC(O)R'®, -OC(O)R, -OC,-Cs-alky!-C(O)OR", -OC;-Ce-alkyl-OR', -SC+-Cs- alkyl-C(O)OR'®, -C(O)OR', or -C,-C¢-alkenyl-C(=O)R'®, :

« C,-Ce-alkyl or C,-Cs-alkenyl which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF3, -OR™, and -NR"R" «aryl, aryloxy, aroyl, aryl-C,-Cs-alkoxy, aryl-C+-Ce-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR', -CH,C(O)OR', -CH,OR', -CN, -CF3, -OCFs, -NO,, -OR'®, -NR'"®R'” and C,-C¢-alkyl.

35. A zinc-binding ligand according to claim 34 wherein R'®, R*, R™ and R'** are independ- ently selected from « hydrogen, halogen, -NO,, -OR®, -NR*R", -SR, -S(0).R™, -0S(0): R'S, - CH,OC(O)R', -OC(O)R", -OC,-Ce-alkyl-C(O)OR'®, -OC;-Cg-alkyl-OR™®, -SC;-Ce- alkyl-C(O)OR'®, -C(O)OR", or ~C,-Ce-alkenyl-C(=O)R', « C;-Ce-alkyl or C,-Cs-alkenyl which may optionally be substituted with one or more substituents selected from halogen, -CF3, -OR'®, and -NR"R" « aryl, aryloxy, aroyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, C(O)OR', -CN, -NO,, -OR'®, -NR"R"" and C,-Ce-alkyl.

36. A zinc-binding ligand according to claim 35 wherein R™®, R*, R' and R"™" are independ- ently selected from « hydrogen, halogen, -OR®, -OC;-Cs-alkyl-C(O)OR'™, or -C(O)OR’, « C,-Cs-alkyl which may optionally be substituted with one or more substituents se- lected from halogen, -OR', and -NR"R"’ e aryl, aryloxy, aryl-C,-Ce-alkoxy, of which the cyclic moieties optionally may be substituted with one or more substity- ents selected from halogen, C(O)OR'", OR, and C,-Ce-alkyl.

37. A zinc-binding ligand according to any of the claims 4 to 36 wherein R® and R' inde- pendently are hydrogen, C-Cx-alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CFa, -OCF3, -OC;-Ce-alkyl, -COOH and ~NH,, and the aryl groups may optionally be substituted by halogen, -COOH, - CN, -CF,, OCF, -NO,, -OH, -OC-Ce-alkyl, -NH,, C(=0) or C;-Cs-alkyl; R* and R"” 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

38. A zinc-binding ligand according to claim 37 wherein R'® and R" independently are hy- drogen, C,-Cx-alkyl, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CF3, -OC;-Cg-alkyl, -COOH and -NH;, and the aryl groups may optionally be substituted by halogen, -COOH, -CN, -CF;, -OCF3;, -OH, -NH,,0r C4-Cg-alkyl.

39. A zinc-binding ligand according to any one of the claims 1 to 3 wherein Ais R'® N N ? A N RM CTE o NH J or CIA N 9 N L200 ~~ N L R H R H © wherein R® is hydrogen or C,-Cs-alkyl, R?' is hydrogen or C;-Ce-alkyl, U and V are a valence bond or C;-Cg-alkylene optionally substituted with one or more hy- droxy, C1-Ce-alkyl, or aryl independently, J is C,-Ce-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is option- ) ally substituted with up to three substituents R%, R® and R*, L is C,-Ce-alkylene, arylene or heteroarylene, wherein the arylene or heteroarylene is option- ally substituted with up to three substituents R%, R®* and RZ,

R', R" RZ R®, R* R®, R® and R” are independently selected from « hydrogen, halogen, -CN, -CHzCN, -CHF, -CF3, -OCF;, -OCHF,, -OCH.CF;, -OCF,CHF, -S(0),CF3, -SCF3, -NO,, -OR®, -NR*R?, -SR*, -NR%®S(0).,R%, -S(O);NR®R®, -S(O)NR®R?, -S(O)R?, -S(0),R?, -C(O)NR*R®, -OC(ONR®R?, -NRZC(0)R?, -NR®C(O)OR®, -CH,C(O)NR*R?, -OCH,C(O)NR®R?, -CH.OR?, -CH,NRZR?, -OC(0)R?, -OC,-Cs-alkyl-C(O)OR?, -SC,-Cq-alkyl-C(O)OR®, ~C2-Ce- alkenyl-C(=0)OR?, -NR?-C(=0)-C,-Ce-alkyl-C(=0)OR?, -NR*-C(=0)-C;-Ce- alkenyl-C(=0)OR?, -C(=0)NR?-C;-C¢-alkyl-C(=O)OR?, -C-Ce-alkyl-C(=0)OR?,0r -C(O)OR?, C,-Ce-alkyl, Co-Ce-alkenyl or C-Ce-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR?, and -NR*®R* « aryl, aryloxy, aryloxycarbonyl, aroyl, aryi-C-Ce-alkoxy, aryl-C,-Ce-alkyl, aryl-Co- Cg-alkenyl, aryl-C,-Cs-alkynyl, heteroaryl, heteroaryl-C;-Ce-alkyl, heteroaryl-C,-Ce- alkenyl or heteroaryl-C,-Cg-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, -CF3, -OCF3, -NO,, -OR®, -NR?®R? and C;-Ce-alkyl, R? and R® independently are hydrogen, C-Cs-alkyl, aryl-C,-Cg-alkyl or aryl, or R® and R® when attached to the same nitrogen atom together with the said nitrogen atom may forma 3 to 8 membered heterocyclic ring optionally containing one or two further heteroatoms se- lected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds

40. A zinc-binding ligand according to claim 39 wherein U is a valence bond

41. A zinc-binding ligand according to claim 39 wherein U is C;-Cq-alkylene optionally substi- tuted with one or more hydroxy, C4-Cg-alkyl, or aryl

42. A zinc-binding ligand according to any one of the claims 39 to 41 wherein J is arylene or heteroarylene, wherein the arylene or heteroarylene is optionally substituted with up to three substituents R%, R® and R*

43. A zinc-binding ligand according to claim 42 wherein J is arylene optionally substituted with up to three substituents R*, R* and R*

44. A zinc-binding ligand according to claim 43 wherein J is phenylene optionally substituted with up to three substituents RZ, R* and R*

45. A zinc-binding ligand according to claim 44 wherein J is R=: 0 jg N N 22 CE R

46. A zinc-binding ligand according to any one of the claims 39 to 45 wherein R%, R® and R?* are independently selected from : « hydrogen, halogen, -CHF,, -CF3, -OCF3;, -OCHF,, -OCH,CF3, -OCF,CHF,, -SCF;, - NO,, -OR%, -NR?®R?, -SR?, -C(O)INR®R?, -OC(0)NR?*R?, -NR®C(O)R>, -NRZ®C(0)OR?, -CH,C(O)NR®R?, -OCH,C(O)NR?®R?, -CH,0OR?, -CH,NR**R?, -OC(O)R?, -OC,-Cs-alkyl-C(OYOR?, -SC,-Cs-alkyl-C(O)OR?, —C,-Cq-alkenyl- C(=0)OR?, -NR?-C(=0)-C,-Ce-alkyl-C(=0)OR?, -NR?*-C(=0)-C1-Cs- alkenyl-C(=0)OR%-, -C(=0)NR?-C,-Cs-alkyl-C(=0)OR?, -C,-Ce-alkyl-C(=0)OR?, or -C(O)OR?%, » C,-Ce-alkyl, C>-C¢-alkenyl or C,-Cg-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR?, and -NR*R% « aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C;-Ce-alkoxy, aryl-C,-Ce-alkyl, aryl-C,- Ce-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C,-Ce-alkyl, heteroaryl-C,-C- alkenyl or heteroaryl-C,-Ce-alkynyl, :

of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, CFs, OCF, -NO,, -OR?, -NR**R? and C,-Cg-alkyl

47. A zinc-binding ligand according to claim 45. A zinc-binding ligand according to claim 44 wherein J is RrR% o) Jag OY N L290 R% N R H 46 wherein RZ, R? and R* are independently selected from « hydrogen, halogen, -OCF;, -OR®, -NR*R®, -SR%, -NR*C(O)R*, -NRZC(0)OR?, -OC(O)R?, -OC,-Ce-alkyl-C(O)OR?, -SC,-C¢-alkyl-C(O)OR?, —C,-Cq-alkenyl- C(=0)OR?, -C(=0)NR?-C,-Ce-alkyl-C(=0)OR?, -Cs-Ce-alkyl-C(=O)OR®, or : -C(O)OR®, « C,-Cg-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CF, OCF, -OR®, and -NR*R? « aryl, aryloxy, aroyl, aryl-C;-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-C;-Ce- alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, -CF3, -OCF3, -NO, -OR?, -NR*R® and C¢-Ce-alkyl o5 48. A zinc-binding ligand according to claim 47 wherein R%, R* and R* are independently selected from « hydrogen, halogen, -OCF3, -OR?, -NR?*R%, -SR®, -NRZC(O)R?, -NR®C(0)OR?, -OC(O)R?%, -OC,-Ce-alkyl-C(O)OR?, -SC,-Ce-alkyl-C(O)OR?®, —C,-Cs-alkenyl- C(=0)OR?, -C(=0)NR%-C,-Cs-alkyl-C(=O)OR?, -C;-Ce-alkyl-C(=O)OR?, or -C(O)OR?,

* C,-C¢-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, or -CF5 «aryl, aryloxy, aroyl, aryl-C,-Cs-alkoxy, aryl-C;-Ce-alkyl, heteroaryl, heteroaryl-C1-Ce- alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OH, -CN, -CF3, -NO,, or C,-Ce-alkyl

49. A zinc-binding ligand according to any of the claims 39 to 48 wherein R* is hydrogen or methyl

50. A zinc-binding ligand according to claim 49 wherein R® is hydrogen

51. A zinc-binding ligand according to any one of the claims 39 to 50 wherein R*® is Hydro- gen, C-Cg-alkyl or aryl

52. A zinc-binding ligand according to claim 51 wherein R? is Hydrogen or C;-Ce-alkyl

53. A zinc-binding ligand according to any one of the claims 39 to 52 wherein R* is Hydro- gen or C,-Cg-alkyl

54. A zinc-binding ligand according to claim 39 wherein V is a valence bond

55. A zinc-binding ligand according to claim 39 wherein V is C,-Cg-alkylene optionally substi- tuted with one or more hydroxy, C,-Cg-alkyl, or aryl

56. A zinc-binding ligand according to any one of the claims 39 or 54 to 55 wherein L is C,- Cs-alkylene or arylene, wherein the arylene is optionally substituted with up to three substitu- ents R®, R® and RY

57. A zinc-binding ligand according to claim 56 wherein L is C,-Cg-alkylene

58. A zinc-binding ligand according to claim 56 wherein L is phenylene optionally substituted with up to three substituents R?®, R?® and RY

59. A zinc-binding ligand according to any one of the claims 39 to 58 wherein R%, R® and RY are independently selected from e hydrogen, halogen, -CHF,, -CF3;, -OCF3;, -OCHF,, -OCH,CF;, -OCF,CHF 5, -SCF;, - NO,, -OR%, -NR?R?, -SR%, -C(O)NR*RZ, -OC(O)NRZ®R?, -NR**C(0)R*, : -NRZC(0)OR?, -CH,C(O)NR*R%, -OCH,C(O)NR®R?, -CH,0R?, -CH,NR*R?, -OC(0O)R%, -OC;-Cq-alkyl-C(O)OR?, -SC,-Cs-alkyl-C(O)OR?®, —C,-Cg-alkenyl-

C(=0)OR%, -NR?-C(=0)-C,-Cq-alkyl-C(=0)OR?, -NR*-C(=0)-C;-Ce- alkenyl-C(=0)OR?-, -C(=0)NR?-C,-Cs-alkyl-C(=0)OR%, -C;-Cs-alkyl-C(=0)OR?, or -C(O)OR?, e C,-Cg-alkyl, C,-Cs-alkenyl or C»-Ce-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF3, -OR%, and -NR*®R* «aryl, aryloxy, aryloxycarbonyl, aroyl, aryl-C,-Cg-alkoxy, aryl-C,-Ce-alkyl, aryl-C,- Ce-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C;-Ce-alkyl, heteroaryl-C;-Ce- alkenyl or heteroaryl-C,-Cg-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, -CF3, -OCF3, -NO,, -OR?, -NR*R and C,-Ces-alkyl

60. A zinc-binding ligand according to claim 59 wherein R*, R* and R* are independently selected from « hydrogen, halogen, -OCFs, -OR%, -NR®R?, -SR?, -NR*C(O)R*, -NR*C(0)OR*, -OC(O)R?, -OC,-Cs-alkyl-C(O)OR?, -SC;-Ce-alkyl-C(O)OR?, —Co-C-alkenyl- C(=0)ORZ, -C(=O)NR?-C,-Cq-alkyl-C(=0)OR?, -C;-Ce-alkyl-C(=0)OR?, or -C(O)OR?, « C,-Ce-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CF;, -OCF3, -OR?®, and -NR*R* « aryl, aryloxy, aroyl, ary!-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-C,-Cg- alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR?, -CN, -CF, OCF, -NO,, -OR?, -NR*R” and C;-Cs-alkyl!

61. A zinc-binding ligand according to claim 60 wherein R?, R® and R¥ are independently selected from «hydrogen, halogen, -OCFs, -OR?, -NRZ®R?, -SR%, -NR®C(0)R%, -NR®C(0)OR®, -OC(O)R%, -OC;-Cs-alkyl-C(O)OR?, -SC4-Ce-alkyl-C(O)OR?, ~C,-Cs-alkenyl- C(=0)OR?, -C(=O)NR?-C,-Cg-alkyl-C(=O)OR?, -C,-Cq-alkyl-C(=O)OR?, or -C(O)OR?, « C,-Cs-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, or -CF; e aryl, aryloxy, aroyl, aryl-C-Ce-alkoxy, aryl-C,-Cg-alkyl, heteroaryl, heteroaryl-C,-Cs- alkyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OH, -CN, -CF3, -NO;, or C,-Cg-alkyl

62. A zinc-binding ligand according to any of the claims 39 or 54 to 61 wherein R?' is hydro- gen or methyl :

63. A zinc-binding ligand according to claim 62 wherein R*' is hydrogen

64. A zinc-binding ligand according to any one of the claims 39 or 54 to 63 wherein R% is Hydrogen, C;-Ce-alkyl or aryl

65. A zinc-binding figand according to claim 64 wherein R? is Hydrogen or C,-Ce-alkyl

66. A zinc-binding ligand according to any one of the claims 39 or 54 to 65 wherein R¥ is Hydrogen or C,-Cg-alkyl

67. A zinc-binding ligand according to claim 39 wherein R™ and R'® are independently se- lected from «hydrogen, halogen, -CN, -CF3, -OCF;, -NO,, -OR%, -NR®R?, -SR?, -S(O)R”, -S(0),R?, -C(O)NR®*R¥, -CH,OR?, -OC(0)R?, -OC-Cg-alkyl-C(O)OR?, -SC1-Cs- alkyl-C(O)OR?, or -C(O)ORZ, o C,-Cs-alkyl, C,-C¢-alkenyl or C,-Ce-alkynyl, }

which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF3, -OR?, and -NR?R? « aryl, aryloxy, aryl-C;-Cs-alkoxy, aryl-C,-Ce-alkyl, heteroaryl, heteroaryl-C;-Cg-alkyl - of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)ORZ, -CN, -CF;, OCF, -NO,, -OR%, -NR*?R” and C;-Ce-alky!

68. A zinc-binding ligand according to claim 67 wherein R'® and R' are independently se- lected from « hydrogen, halogen, -CN, -CFs, -NO,, -OR®, -NR®R?, or -C(O)OR?, « C,-C¢-alkyl optionally substituted with one or more substituents selected from halo- gen, -CN, -CF, -OCF;, -OR%, and -NR*®R* | ’ e aryl, aryloxy, aryl-C-Ce-alkyl, heteroaryl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR%, -CN, -CFs, -OCFs, -NO,, -OR?, -NR*R* and C;-Ce-alkyl

69. A zinc-binding ligand according to claim 39 wherein A is “r N, N

70. A zinc-binding ligand according to any of the claims 1 to 3 wherein A is of the form M-Q-T- : wherein M is o HO. Oo ° HO 1 B e 2S SY or or WwW’ Ww Ww? (¥ - b wherein

W!, W2, and W? are independently OH, SH or NH, and the phenyl, naphthalene or benzocar- bazole rings are optionally substituted by one or more R* independently Q is selected from the following: = a valence bond o —CH,N(R®)- or —-SON(R*)- —zt-N—Hn NE « A compound of the formula wherein Z' is S(O); or CH,, Z%is N,-O-or -S-,and nis 1 or 2; Tis « C,-Cs-alkylene, C,-Cs-alkenylene or C,-Ce-alkynylene, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCFs, -OR¥, and -NR*R*? « Arylene, arylene-oxy, -aryl-oxycarbonyl-, -aroyl-, -aryl-C4-Cg-alkoxy-, -aryl-C4-Ce- alkyl-, -aryl-C,-Ce-alkenyl-, -aryl-C,-Ce-alkynyl-, heteroarylene, -heteroaryl-C-Ce¢- alkyl-, -heteroaryl-C,-Cs-alkenyl- or -heteroaryl-C,-Ce-alkynyl-, wherein the cyclic moieties are optionally substituted by one or more substituents selected from halo- gen, -C(O)OR®, -C(O)H, -CN, -CF3, -OCF3, -NO,, -OR¥, -NR¥R*, C,-C¢-alkyl or C;- Ce-alkanoyl, ¢ A valence bond R® and R® independently are hydrogen, C,-Ce-alkyl, aryl-C,-Ce-alky! or aryl, or R*? and R¥ 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 se- lected from nitrogen, oxygen and sulphur, and optionally containing one or two double bonds, R* and R* are independently hydrogen, C,-Cg-alkyl or C4-Ce-alkanoyl.

R* is hydrogen, halogen, -CN, -CH,CN, -CHF;, -CF3, -OCF3, -OCHF, -OCH,CF3, -OCF,CHF,, -S(0),CF3, -SCF3, -NO,, -OR%, -C(O)R*, -NR®R*, -SR¥, -NR¥S(0),R*, -5(0),NR¥R®, -S(O)NR*ZR, -S(0)R*, -S(0).R¥, -C(O)NR¥R®, -OC(O)NR*R¥,

NRZC(O)R®, -CH,C(O)NRZR®, -OCH,C(OINR¥R™, -CH;OR®, -CH,NR¥*R%, -OC(O)R%, - OC;-Cs-alkyl-C(O)OR®, -SC,-Ce-alkyl-C(O)OR™ —C,-Ce-alkenyl-C(=0)OR®, -NR¥-C(=0)-C;-Cs-alkyl-C(=0)OR%, -NR32-C(=0)-C+-Ce-alkenyl-C(=O)OR*-, C-Ce-alkyl, C,-Cs-alkanoyl or -C(O)OR¥,

71. A zinc-binding ligand according to claim 70 wherein Mis oO O or w' W?2

72. A zinc-binding ligand according to claim 71 wherein M is 0 ans w'

73. A zinc-binding ligand according to claim 71 wherein Mis o I W2

74. A zinc-binding ligand according to claim 71 wherein M is HO (0)

75. A zinc-binding ligand according to claim 71 wherein Mis Oo oS HO

76. A zinc-binding ligand according to any one of the claims 70 to 75 wherein Q is a valence bond, —CHN(R¥)-, or ~SO,N(R*")-

77. A zinc-binding ligand according to claim 76 wherein Q is a valence bond

78. A zinc-binding ligand according to any one of the claims 70 to 77 wherein T is oA valence bond « C,-Cs-alkylene, C,-Ce-alkenylene or Co-Ce-alkynylene, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR®, and -NR¥R™ « Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70

79. A zinc-binding ligand according to claim 78 wherein T is » A valence bond « Arylene, or heteroarylene, wherein the cyclic moieties are optionally substituted as defined in claim 70

80. A zinc-binding ligand according to any one of the claims 70 to 75 wherein T is phenylene or naphthalene

81. A zinc-binding ligand according to any one of the claims 70 to 80 wherein the cyclic moi- ety in T is optionally substituted by halogen, -C(O)OR®, -CN, -CFs, -OR%, -NR¥R¥, C;- Cs-alkyl or C;-Ce-alkanoy!

82. A zinc-binding ligand according to claim 81 wherein the cyclic moiety in T is optionally substituted by halogen, -C(O)OR%, -OR?*, -NR*R*, C,-Cg-alkyl or C;-Ce-alkanoyl

83. A zinc-binding ligand according to claim 81 wherein the cyclic moiety in T is optionally substituted by halogen, -C(O)OR® or -OR*

84. A zinc-binding ligand according to any one of the claims 70 to 75 wherein T is a valence bond

85. A zinc-binding ligand according to any one of the claims 70 to 84 wherein R* and R*' are independently hydrogen or C;-Ce-alkyl

86. A zinc-binding ligand according to any one of the claims 70 to 85 wherein R* is hydro- gen, halogen, -CN, -CF, -OCFs, -SCFs, -NO;, -OR%, -C(O)R%, -NR¥R¥, -SR*, _C(OINRZR®, -OC(O)NR¥R¥, -NR¥C(0)R®, -OC(0)R®, -OC;-Ce-alkyl-C(O)OR®, -SC1-Ce- alkyl-C(O)OR® or -C(O)OR*

87. A zinc-binding ligand according to claim 86 wherein R* is hydrogen, halogen, -CF3, - NO, -OR%, -NR¥R¥®, -SR¥%, -NR¥C(O)R®, or -C(O)OR*

88. A zinc-binding ligand according to claim 87 wherein R* is hydrogen, halogen, -CF3, - NO,, -OR®, -NR¥R®, or -NR*C(0O)R*

89. A zinc-binding ligand according to claim 88 wherein R* is hydrogen, halogen, or -OR® -

90. A zinc-binding ligand according to any of the claims 70 to 89 wherein R* and R* inde- pendently are hydrogen, C,-Ce-alkyl, or aryl

91. A zinc-binding ligand according to claim 90 wherein R*? and R* independently are hy- drogen or C,-Ce-alkyl

92. A zinc-binding ligand according to any of the claims 1 to 3 wherein Ais N Al 1 "\ a MRT ON pe N—N wherein A' is a valence bond, C;-Ce-alkylene, -NH-C(=0)-A%, -C;-C¢-alkyl-S-, -Cy- Ce-alkyl-O-, -C(=0)-, or -C(=0)-NH-, wherein any C;-Ce-alky! moiety is optionally substituted by R™ Ais a valence bond, C,-Cs-alkylene, C,-Ce-alkenylene, or -C;-Ce-alkyl-O-; R"™ is C,-Ce-alkyl, aryl, wherein the alkyl or aryl moieties are optionally substituted by one or more halogen, cyano, nitro, amino; AR'is a valence bond, arylene or heteroarylene, wherein the aryl or heteroaryl moieties are optionally substituted by one or more R'8 independently R'® is selected from « hydrogen, halogen, -CN, -CH,CN, -CHF, -CF3, -OCF;, -OCHF,, -OCH.CF3, -OCF,CHF,, -S(0),CF3, -OS(0),CFs, -SCF3, -NO,, -OR'®, -NR'R'®, -SR'C, _NR'CS(O),R™®, -S(0):NR'°R™, -S(O)NR'°R™®, -S(O)R'’, -S(0)R'®, -0S(0) RS, -C(O)NR'°R™, -OC(O)NR'°R™, -NR'°C(O)R™, -CH,C(ONR'°R', -OC,-Cs- alkyl-C(O)NR'°R'®, -CH,OR'®, -CH,OC(O)R', -CH,NR'CR™, -OC(O)R'®, -OC,-Cs- alkyl-C(O)OR'®, -OC,-Ce-alkyl-OR'®, -S-C,-Cg-alkyl-C(O)OR'®, —C,-Cs-alkenyl- C(=0)OR'®, -NR'C-C(=0)-C;-Ce-alkyl-C(=0)OR'®, -NR'®-C(=0)-C1-Ce- alkenyl-C(=O)OR'® , -C(O)OR'C, ~C,-Ce-alkenyl-C(=O)R'®, =O, -NH-C(=0)-0-C;- Ce-alkyl, or -NH-C(=0)-C(=0)-O-C;-Ce-alky! o C,-Ce-alkyl, Co-Ce-alkenyl or Co-Ce-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OR'C, and -NR'“R™

« aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C,-Ce-alkoxy, aryl-C,-Ce-alkyl, aryl-C,-Cs-alkenyl, aroyl-C,-Ce-alkenyl, aryl-Co-Cg-alkynyl, heteroaryl, heteroaryl-Cs- Ce-alkyl, heteroaryl-C,-Ce-alkenyl or heteroaryl-C,-Ce-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR'C, -CH,C(O)OR'®, -CH,OR'®, -CN, -CF3, OCF;, -NO,, -OR'C, -NR'“R'® and C;-Ce-alkyl, R'C and R'° independently are hydrogen, -OH, Cs-Ce-alkyl, C4-Ce-alkenyl, aryl-C,-Ce-alkyl or aryl, wherein the alkyl moieties may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -O-C,-Ce-alkyl, -C(0)-0-C,-Ce-alkyl, -COOH and — NH,, and the aryl moieties may optionally be substituted by halogen, -C(0O)OC-Ce-alkyl, - COOH, -CN, -CFa, OCF, -NO,, -OH, -OC,-Cs-alkyl, -NH,, C(=0) or C;-Cs-alkyl; R'® and R™ 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 heteroa- : toms selected from nitrogen, oxygen and sulphur, and optionally containing one or two dou- ble bonds, C' is a valence bond, C,-Cs-alkylene, -C;-Cg-alkyl-O-, -C4-Ce-alkyl-NH-, -NH-C;-Ce-alkyl, -NH-C(=0)-, -C(=0)-NH-, -O-C4-Ce¢-alkyl, -C(=0)-, or -C4-Cg-alkyl-C(=0)-N(RE)- wherein the alkyl moieties are optionally substituted by one or more R'F independently R'E and R'F are independently selected from C,-Ce-alkyl, ary! optionally substituted by one or more halogen, -COOH,;

AR? is e a valence bond « C,-Cs-alkylene, C-Cs-alkenylene , C,-Cg-alkynylene wherein the alkyl, alkenyl and alkynyl moieties are optionally substituted by one or more R? independently; « arylene, -aryloxy-, -aryloxy-carbonyl-, aryl-C,-Ce-alkyl, -aroyl-, aryl-C,-Ce-alkoxy-, aryl-C,-Ce-alkenyl-, aryl-C,-Cg-alkynyl-, heteroarylene, -heteroaryl-C,-Ce-alkyl-, -heteroaryl-C,-Ce¢-alkenyl-, -heteroaryl-C,-Cg-alkynyl- wherein the aryl and heteroaryl } moieties are optionally substituted by one or more R? independently;

R? is C,-Cg-alky!, Ci-Ce-alkoxy, aryl, aryloxy, aryl-C:-Ce-alkoxy, -C(=0)-NH-C,-Ce-alkyl-aryl, heteroaryl, heteroaryl-C;-Ce-alkoxy, -C,-Ce-alkyl-COOH, -O-C4-Cg-alkyl-COOH, -S(0),R%, -C,-Cs-alkenyl-COOH, -OR?, -NO,, halogen, -COOH, -CF3, -CN, -N(R*®R%®), wherein the aryl or heteroaryl moieties are optionally substituted by one or more C;-Cg-alkyl, C;- Ce-alkoxy, -C4-Cg-alkyl-COOH, -C,-Ce-alkenyl-COOH, -OR?, -NO,, halogen, -COOH, -CFs, -CN, or -N(R?R%) R% and R*® are independently selected from hydrogen and C;-Ce¢-alkyl

93. A zinc-binding ligand according to claim 92 wherein A’ is a valence bond, C;-Cs-alkylene, -NH-C(=0)-A2-, -C,-Cs-alkyl-S-, -C4-Cq-alkyl-O-, or -C(=0)-, wherein any C;-Cs-alkyl moiety is optionally substituted by R"

94. A zinc-binding ligand according to claim 93 wherein A’ is a valence bond, C,-Cs-alkylene, -NH-C(=0)-A?-, -C,-Cg-alkyl-S-, or -C,-Cs-alkyl-O, wherein any C;-Cg-alkyl moiety is option- ally substituted by R*

95. A zinc-binding ligand according to claim 94 wherein A’ is a valence bond, C,-Ce-alkylene, or -NH-C(=0)-A?, wherein any C;-Ce-alkyl moiety is optionally substituted by R™

96. A zinc-binding ligand according to claim 95 wherein A' is a valence bond or C;- Ce-alkylene, wherein any C,-Cq-alkyl moiety is optionally substituted by R™

97. A zinc-binding ligand according to claim 96 wherein A' is a vaience bond

98. A zinc-binding ligand according to any one of the claims 92 to 97 wherein A? is a valence bond or -C;-Cs-alkyl-O-

99. A zinc-binding ligand according to claim 98 wherein A is a valence bond

100. A zinc-binding ligand according to any one of the claims 92 to 99 wherein AR' is arylene or heteroarylene, wherein the arylene or heteroarylene moieties are optionally substituted by one or more R'® independently )

101. A zinc-binding ligand according to claim 100 wherein AR! is selected from the group of compounds consisting of phenylene, biphenylylene, naphthylene, anthracenylene, phenan- threnylene, fluorenylene, indenylene, azulenylene, furylene, thienylene, pyrrolylene, oxa- zolylene, 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, ben-

zisothiazolylene, benzoxazolylene, benzisoxazolylene, purinylene, quinazolinylene, quinoliz- ~ inylene, quinolinylene, isoquinolinylene, quinoxalinylene, naphthyridinylene, pteridinylene, carbazolylene, azepinylene, diazepinylene, or acridinylene, optionally substituted by one or more R'® independently

102. A zinc-binding ligand according to claim 101 wherein AR’ is selected from phenylene, biphenylylene, naphthylene, pyridinylene, fyrylene, indolylene, or carbazolylene, optionally substituted by one or more R*® independently

103. A zinc-binding ligand according to claim 102 wherein AR" is selected from the group of compounds consisting of phenylene, indolylene, or carbazolylene, optionally substituted by one or more R'® independently

104. A zinc-binding ligand according to claim 103 wherein AR’ is phenylene optionally substi- tuted by one or more R*® independently

105. A zinc-binding ligand according to claim 103 wherein AR’ is indolylene optionally substi- tuted by one or more R'® independently

106. A zinc-binding ligand according to claim 105 wherein AR’ is R'® oy \

107. A zinc-binding ligand according to claim 103 wherein AR’ is carbazolylene optionally substituted by one or more R'® independently

108. A zinc-binding ligand according to claim 107 wherein AR" is r'® = \

109. A zinc-binding ligand according to any one of the claims 92 to 108 wherein R"® is se- lected from « hydrogen, halogen, -CN, -CF;, -OCF;, -NO,, -OR'C, -NR'“R™, -SR'¢, -S(0).R'°, -NR'®C(O)R™, -OC;-Ce-alkyl-C(O)NR'R'P, —C,-C-alkenyl-C(=O)OR'®, -C(O)OR', =0, -NH-C(=0)-0-C,-Cs-alkyl, or -NH-C(=0)-C(=0)-O-C,-Cs-alkyl ¢ C4-Cs-alkyl or C,-Cs-alkenyl ]

which may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, OCF, -OR'C, and -NR'°R'® «aryl, aryloxy, aryl-Cs-Ce-alkoxy, aryl-C-Cs-alkyl, aryl-C,-Ce-alkenyl, heteroaryl, het- eroaryl-C;-Cg-alkyl, or heteroaryl-C,-Cg-alkenyl of which the cyclic moieties optionally may be substituted with one or more substituents se- lected from halogen, -C(O)OR'C, CN, -CFs, -OCF3, -NO,, -OR'®, -NR'“R'® and C;-Ce-alkyl

110. A zinc-binding ligand according to claim 109 wherein R'® is selected from « hydrogen, halogen, -CF3, -NO,, -OR'®, -NR'°R"®, -C(O)OR'®, =0, -NH-C(=0)-0-C+- Ce-alkyl, or -NH-C(=0)-C(=0)-O-C,-Cs-alkyl ¢ C,-Ces-alkyl

111. A zinc-binding ligand according to any one of the claims 92 to 110 wherein R' and R'™P independently are hydrogen, C,-Cs-alkyl, or aryl, wherein the aryl moieties may optionally be substituted by halogen or -COOH

112. A zinc-binding ligand according to claim 111 wherein R'® and R'® independently are hydrogen, methyl, ethyl, or phenyl, wherein the phenyl moieties may optionally be substituted by halogen or -COOH

113. A zinc-binding ligand according to any one of the claims 92 to 112 wherein C'isava- lence bond, C,-Ce-alkylene, -C;-Cg-alkyl-O-, -C4-Ce-alkyl-NH-, -NH-C,-Ce-alkyl, -NH-C(=0)-, -C(=0)-NH-, -O-C,4-Cs-alkyl, -C(=0)-, or -C,-Ce-alkyl-C(=0)-N(R'F)- wherein the alkyl moieties are optionally substituted by one or more R'F independently

114. A zinc-binding ligand according to claim 113 wherein C' is a valence bond, -CH,-, -CH,-CHy-, -CH-O-, -CH,-CH5-O-, -CHz-NH-, -CH-CH2-NH-, -NH-CHa-, -NH-CH,-CHo-, -NH-C(=0)-, -C(=0)-NH-, -O-CH-, -O-CH;-CH,-, or -C(=0)-

115. A zinc-binding ligand according to any one of the claims 92 to 114 wherein R'® and R'f are independently selected from C;-Cg-alkyl

116. A zinc-binding ligand according to any one of the claims 92 to 115 wherein AR? is «a valence bond « C;-Ce-alkylene, wherein the alkyl is optionally substituted by one or more R* inde- pendently earylene, aryl-C,-Cg-alkyl, heteroarylene, wherein the arylene and heteroarylene moieties are optionally substituted by one or more R? independently

117. A zinc-binding ligand according to claim 116 wherein AR? is a valence bond

* C4-Ce-alkylene, wherein the alkylene is optionally substituted by one or more R# independently » phenyl, phenyl-C,-Cg-alkyl, wherein the phenylene moieties are optionally substi- tuted by one or more R* independently

118. A zinc-binding ligand according to any one of the claims 92 to 117 wherein R* is Cy- Ce-alkyl, C,-Cg-alkoxy, aryl, aryloxy, heteroaryl, -C4-Cg-alkyl-COOH, -O-C,-Ce-alkyl-COOH, -S(0),R%, -C,-Cs-alkenyl-COOH, -OR?, -NO,, halogen, -COOH, -CF3, -CN, -N(R®R), wherein the aryl or heteroaryl moieties are optionally substituted by one or more C,-Cg-alkyl, C;-Ce-alkoxy, -C,-Cs-alkyl-COOH, -C,-Cs-alkenyl-COOH, -OR?*®, -NO,, halogen, -COOH, -CF3, -CN, or -N(R®®R%)

119. A zinc-binding ligand according to claim 118 wherein R* is C,-Cq-alkyl, C,-Cg-alkoxy, aryl, -OR%, -NO,, halogen, -COOH, -CF3, -CN, -N(R®R%), wherein the aryl is optionally sub- stituted by one or more C,-Ce-alkyl, C;-Cq-alkoxy, -OR?, -NO,, halogen, -COOH, -CF3, -CN, or -N(R®R%) ns

120. A zinc-binding ligand according to claim 119 wherein R* is C4-Ce-alkyl, C-Ce-alkoxy, aryl, halogen, -CF3, wherein the aryl is optionally substituted by one or more C,-Cg-alkyl, halogen, -COOH, -CF;, or -CN

121. A zinc-binding ligand according to claim 120 wherein R* is C;-Cg-alky!, C,-Ce-alkoxy, phenyl, halogen, -CF3, wherein the phenyl is optionally substituted by one or more C;- Cs-alkyl, halogen, -COOH, -CF,, or -CN

122. A zinc-binding ligand according to any of the claims 1 to 3 wherein A is . N=N Nags” S wherein AR? is C,-C¢-alkylene, arylene, heteroarylene, -aryl-C,.¢-alkyl- or -aryl-C,.¢-alkenyl-, wherein the alkylene or alkenylene is optionally substituted with one or more substituents in- dependently selected from halogen, -CN, -CF;, -OCF3, aryl, -COOH and —NH, and the ary- lene or heteroarylene is optionally substituted with one or more R* independently R* is independently selected from

« hydrogen, halogen, -CN, -CH,CN, -CHF;, -CF3, -OCF;, -OCHF;, -OCH,CF3, -OCF,CHF, -S(O),CFs, -OS(0),CFs, -SCF3, -NO,, -OR®, -NR®R*, -SR*, -NR®S(0),R*°, -S(0),NR®R?*®, -S(O)NR*®R*, -5(0)R®, -$(0),R*, -0S(0), rR, -C(O)NR®R¥, -OC(O)NR®R¥*, -NR*®C(0O)R¥, -CH,C(O)NR*R*", -OC+-Ce- alkyl-C(O)NR®ER®, -CH,OR®, -CH,0C(O)R®, -CH,NR®R*’, -OC(0)R*, -OC1-Ce- alkyl-C(O)OR®, -OC,-Cg-alkyl-OR®, -8C;-Cg-alkyl-C(O)OR™®, —C,-Ce-alkenyl- C(=0)OR®, -NR*®-C(=0)-C;-Cg-alkyl-C(=0)OR*, -NR*-C(=0)-C,-Ce- alkenyl-C(=0)OR® , -C(O)OR®, or —-C,-Cs-alkenyl-C(=0)R™, » C,-Cg-alkyl, Co-Ce-alkenyl or C,-Ce-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFs, -OCF;, -OR®, and ~NR*R* «aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C,-Ces-alkoxy, aryl-C,-Cg-alkyl, aryl-C,-Ce-alkenyl, aroyl-C,-Ce-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C,- Ce-alkyl, heteroaryl-C,-Ce-alkenyl or heteroaryl-C,-Ce-alkynyl, of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR®, -CH,C(O)OR®, -CH,OR®, -CN, -CF;, -OCF;, -NO,, -OR®, -NR*R* and C,-Ce-alkyl, R® and RC are independently hydrogen, OH, CF3, C,-Ciz-alkyl, aryl-C,-Ce-alkyl, -C(=0)-C,-Ce-alkyl or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OC,-Cg-alkyl, -C(O)OC4-Ce- alkyl, -C(=0)-R*®, -COOH and —NH,, and the aryl groups may optionally be substituted by halogen, -C(O)OC,-Cg-alkyl, -COOH, -CN, -CF3, -OCF3, -NO2, -OH, -OC,-Ce-alkyl, -NH, C(=0) or C,-Ce-alkyl; R*® and R3¢ 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 RP is C,-Cs-alkyl, aryl optionally substituted with one ‘or more halogen, or heteroaryl option- ally substituted with one or more C4-Ce-alkyl.

123. A zinc-binding ligand according to claim 122 wherein AR? is arylene, heteroarylene, or -aryl-C, ¢-alkyl-, wherein the alkyl is optionally substituted with one or more substituents in- dependently selected from halogen, -CN, -CF3, OCF, aryl, -COOH and —NH,, and the ary- lene or heteroarylene is optionally substituted with one or more R* independently ’ ‘5

124. A zinc-binding ligand according to claim 123 wherein AR? is arylene optionally substi- tuted with one or more R* independently

125. A zinc-binding ligand according to claim 124 wherein AR? is phenylene, naphthalene or anthranylene optionally substituted with one or more R* independently

126. A zinc-binding ligand according to claim 125 wherein AR? is phenylene optionally substi- tuted with one or more R* independently

127. A zinc-binding ligand according to any one of the claims 122 to 126 wherein Ris inde- pendently selected from «halogen, -CN, -CFs, -NO,, -OR®, -NR*R*, -SR®, -OC;-Cg-alkyl-C(O)OR™ or -C(O)OR™ « C,-Cg-alky! optionally substituted with one or more substituents selected from halo- gen, -CN, -CF3, -OCF;, -OR™, and -NR*R* e aryl, aryl-C,-C¢-alkyl, heteroaryl, or heteroaryl-C;-Ce-alkyl of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR®, -CN, -CF3, -OCF3, -NO,, -OR®, -NR3R3¢ and C,-Ce-alkyl

128. A zinc-binding ligand according to claim 127 wherein R* is independently selected from halogen, -OR%, -NR*®R, -C(O)OR®, -OC,-Ce-alkyl-C(O)OR™, or C,-Ce-alkyl

129. A zinc-binding ligand according to any one of the claims 122 to 128 wherein R*® and R* are independently hydrogen, CFs, Ci-C1z-alkyl, or -C(=0)-C-Cs-alkyl; R*® and R3¢ when at- tached to the same nitrogen atom may form a 3 to 8 membered heterocyclic ring with the said nitrogen atom

130. A zinc-binding ligand according to any of the claims 1 to 3 wherein A is "Nw NC DE \ “NAR N : \

wherein AR? is C-Cs-alkylene, arylene, heteroarylene, -aryl-C¢-alkyl- or -aryl-C,¢-alkenyl-, wherein the alkylene or alkenylene is optionally substituted with one or more substituents in- dependently selected from halogen, -CN, -CF3, -OCF3, aryl, -COOH and —NH,, and the ary- lene or heteroarylene is optionally substituted with one or more R* independently S R* is independently selected from «hydrogen, halogen, -CN, -CH,CN, -CHF;, -CF;, -OCF;, -OCHF,, -OCH.CF3, -OCF,CHF,, -S(O),CF3, -OS(0),CF3, -SCF3, -NO;, -OR™, -NR*R*, -SR*,

-NR*S(0),R, -S(0),NR®®R*C, -S(O)NR*®R*®, -S(O)R*, -S(0),R*, -0S(0). R*, -C(O)NR*ER*, -OC(O)NR*®R*, -NR*®C(O)R*’, -CH,C(O)NR**R*®, -OC,-Cs- alkyl-C(O)NR*8RC, -CH,OR*®, -CH,O0C(O)R*®, -CHNR**R, -OC(O)R*®, -OC;-Ce- alkyl-C(OYOR®, -OC;-Ce-alkyl-OR?®, -SC,-Ce-alkyl-C(O)OR*, —Co-Cs-alkenyl- C(=0)OR®, -NR*®-C(=0)-C1-Ce-alkyl-C(=0)OR®, -NR**-C(=0)-C1-Cs- alkenyl-C(=0)OR®® , -C(O)OR*, or -C,-Ce-alkenyl-C(=O)R*, C,-Cg-alkyl, C-Ce-alkenyl or C,-Ce-alkynyl, which may optionally be substituted with one or more substituents selected from halogen, -CN, -CFa, -OCF;, -OR*, and -NR*R* «aryl, aryloxy, aryloxycarbonyl, aroyl, arylsulfanyl, aryl-C,-Cg-alkoxy, aryl-C4-Ce-alkyl,

aryl-C»-Ce-alkenyl, aroyl-C,-Ce-alkenyl, aryl-C,-Ce-alkynyl, heteroaryl, heteroaryl-C;- : Ce-alkyl, heteroaryl-C,-Ce-alkeny! or heteroaryl-C,-Ce-alkynyl,

of which the cyclic moieties optionally may be substituted with one or more substitu- ents selected from halogen, -C(O)OR®, -CH,C(0)OR*®, -CH,OR*, -CN, -CF3, -OCF,, -NO,, -OR®, -NR*®R*® and C;-Cq-alkyl,

R® and R‘ are independently hydrogen, OH, CFs, C1-C,z-alkyl, aryl-C;-Ce-alkyl, -C(=O)-R*, or aryl, wherein the alkyl groups may optionally be substituted with one or more substituents selected from halogen, -CN, -CF3, -OCF3, -OC4-Ce-alkyl, -C(0)OC,-Ce-alkyl, -COOH and - NH,, and the aryl groups may optionally be substituted by halogen, -C(O)OC;-Ce-alkyl, - COOH, -CN, -CF3, -OCF3, -NO3, -OH, -OC;-Ce-alkyl, -NH,, C(=0) or C1-Ce-alkyl; R*® and R*¢ 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 heteroa- toms selected from nitrogen, oxygen and sulphur, and optionally containing one or two dou- ble bonds Ris C,-Cs-alkyl, aryl optionally substituted with one or more halogen, or heteroaryl option- ally substituted with one or more C4-Cg-alkyl.

131. A zinc-binding ligand according to claim 130 wherein AR‘ is arylene, heteroarylene or aryl-C,¢-alkyl-, wherein the alkyl is optionally substituted with one or more substituents inde- pendently selected from halogen, -CN, -CF3, -OCF3, aryl, -COOH and —NH, and the arylene or heteroaryl is optionally substituted with one or more R* independently

132. A zinc-binding ligand according to claim 131 wherein AR* is arylene or heteroarylene optionally substituted with one or more RA independently

133. A zinc-binding ligand according to claim 131 wherein AR* is phenylene, naphtylene, anthrylene, thienylene, pyridylene, or benzodioxylene optionally substituted with one or more R* independently “

134. A zinc-binding ligand according to claim 133 wherein AR* is phenylene optionally substi- tuted with one or more R** independently

135. A zinc-binding ligand according to any one of the claims 130 to 134 wherein R* is inde- pendently selected from hydrogen, halogen, -CFs, -OR*, -NR*®*R*°, C,-Ce-alkyl, aryl-C,-Ce-alkenyl or aryl optionally substituted with one or more substituents selected from halogen, -CF3, or -OR*®

136. A zinc-binding ligand according to any one of the claims 130 to 135 wherein R*® and R* are independently hydrogen, CFs, C4-Cq2-alkyl, -C(=0)-R*, or aryl

137. A zinc-binding ligand according to any one of the claims 130 to 136 wherein Ris C4-Cg-alkyl, pheny! optionally substituted with one or more halogen, or a heteroaryl selected from isoxazole and thiadiazole optionally substituted with one or more C,-Cq-alkyl

138. A zinc-binding ligand according to any one of the claims 1 to 137 wherein G® is of the formula -B'-B%-C(O)-, -B'-B%-SO;- or -B'-B>CH,-, wherein B' and B? are as defined in claim 1

139. A zinc-binding ligand according to any one of the claims 1 to 137 wherein G® is of the formula -B'-B2-C(O)-, -B'-B%-SO,- or -B'-B2-NH-, wherein B' and B? are as defined in claim 1

140. A zinc-binding ligand according to any one of the claims 1 to 137 wherein GP is of the formula -B'-B2-C(O)-, -B'-B%-CH;- or -B'-B%-NH-, wherein B' and B? are as defined in claim 1 .

141. A zinc-binding ligand according to any one of the claims 1 to 137 wherein GB is of the formula -B'-B2-CH,-, -B'-B%-S0,- or -B'-B%-NH-, wherein B' and B? are as defined in claim 1 -

142. A zinc-binding ligand according to any one of the claims 138 or 139 wherein G® is of the formula -B'-B2-C(O)- or -B'-B%-S0,-, wherein B' and B? are as defined in claim 1

143. A zinc-binding ligand according to any one of the claims 138 or 140 wherein G® is of the formula -B'-B2-C(O)- or -B'-B%-CHg-, wherein B' and B? are as defined in claim 1

144. A zinc-binding ligand according to any one of the claims 139 or 140 wherein GBis of the formula -B'-B%C(O)- or -B'-B2NH-, wherein B' and B? are as defined in claim 1

145. A zinc-binding ligand according to any one of the claims 138 or 141 wherein G® is of the formula -B'-B2-CH,- or -B'-B%-S0,- , wherein B' and Bare as defined in claim 1

146. A zinc-binding ligand according to any one of the claims 139 or 141 wherein G? is of the formula -B'-B2-NH- or -B'-B2SO,- , wherein B' and B? are as defined in claim 1

147. A zinc-binding ligand according to any one of the claims 140 or 141 wherein G® is of the formula -B'-B2%CH,- or -B'-B-NH- , wherein B' and B? are as defined in claim 1

148. A zinc-binding ligand according to any one of the claims 142, 143, or 144 wherein G® is of the formula -B'-B?-C(O)-

149. A zinc-binding ligand according to any one of the claims 143, 145 or 147 wherein GPis of the formula -B'-B*CH,-

150. A zinc-binding ligand according to any one of the claims 143, 145 or 146 wherein G® is of the formula -B'-B%-SO,-

151. A zinc-binding ligand according to any one of the claims 144, 146 or 147 wherein GPis of the formula -B'-B*NH-

152. A zinc-binding ligand according to any one of the claims 1 to 151 wherein B' is a va- lence bond, -O-, or =5-

153. A zinc-binding ligand according to any one of the claims 1 to 151 wherein B' is a va- lence bond, -O-, or -N(R®)-

154. A zinc-binding ligand according to any one of the claims 1 to 151 wherein B' is a va- lence bond, -S-, or -N(R°)-

155. A zinc-binding ligand according to any one of the claims 1 to 151 wherein B' is -O-, -S- or -N(R®)-

156. A zinc-binding ligand according to any one of the claims 152 or 153 wherein B' is a va- lence bond or —-O-

157. A zinc-binding ligand according to any one of the claims 152 or 154 wherein B' is a va- lence bond or —S-

158. A zinc-binding ligand according to any one of the claims 153 or 154 wherein B' is a va- lence bond or —N(R®)-

159. A zinc-binding ligand according to any one of the claims 152 or 155 wherein B'is -O-or -S-

160. A zinc-binding ligand according to any one of the claims 153 or 155 wherein B'is -O-or —N(R®)-

161. A zinc-binding ligand according to any one of the claims 154 or 155 wherein B'is -S-or N(R®)-

162. A zinc-binding ligand according to any one of the claims 156,157 or 158 wherein B'isa valence bond

163. A zinc-binding ligand according to any one of the claims 156, 159 or 160 wherein B'is -O-

164. A zinc-binding ligand according to any one of the claims 157, 159 or 161 wherein B'is -S-

165. A zinc-binding ligand according to any one of the claims 158, 160 or 161 wherein B'is -N(R®)-

166. A zinc-binding ligand according to any one of the claims 1 to 165 wherein Bis a va- lence bond, C;-Cis-alkylene, C,-Cqg-alkenylene, C,-Cis-alkynylene, arylene, heteroarylene, -C4-Cys-alkyl-aryl-, -C(=0)-C,-Cg-alkyl-C(=0)-, -C(=0)-C;-C1s-alkyl-O-C4-C,g-alkyl-C(=0)-, -C(=0)-C1-Crg-alkyl-S-C4-Cyg-alkyl-C(=0)-, -C(=0)-C4-C1g-alkyl-NR®-C,-Cs-alkyl-C(=0)-; and the alkylene and arylene moieties are optionally substituted as defined in claim 1

167. A zinc-binding ligand according to claim 166 wherein Bis a valence bond, C;-Cis- alkylene, C-Cis-alkenylene, C-Cys-alkynylene, arylene, heteroarylene, -C,-Cs-alkyl-aryl-, -C(=0)-C;-C1s-alkyl-C(=0)-, -C(=0)-C,-C,s-alkyl-O-C,-C1g-alkyl-C(=0)-, and the alkylene and arylene moieties are optionally substituted as defined in claim 1

168. A zinc-binding ligand according to claim 167 wherein B? is a valence bond, C;-Cis- alkylene, C,-Cqs-alkenylene, C,-Cqg-alkynylene, arylene, heteroarylene, -C,-C,g-alkyl-aryl-, -C(=0)-C,-C,s-alkyl-C(=0)-, and the alkylene and arylene moieties are optionally substituted as defined in claim 1

169. A zinc-binding ligand according to claim 168 wherein B? is a valence bond, C,-Cis- alkylene, arylene, heteroarylene, -C4-C4g-alkyl-aryl-, -C(=0)-C;-Cg-alkyl-C(=0)-, and the al- kylene and arylene moieties are optionally substituted as defined in claim 1

170. A zinc-binding ligand according to claim 169 wherein BZ? is a valence bond, C-Cs- alkylene, arylene, heteroarylene, -C,-Cyg-alkyl-aryl-, and the alkylene and arylene moieties are optionally substituted as defined in claim 1 ]

171. A zinc-binding ligand according to claim 170 wherein B? is a valence bond, C;-C1s- alkylene, arylene, -C4-Cg-alkyl-aryl-, and the alkylene and arylene moieties are optionally substituted as defined in claim 1

172. A zinc-binding ligand according to claim 171 wherein B? is a valence bond or -C4-C1s- alkylene, and the alkylene moieties are optionally substituted as defined in claim 1

173. A zinc-binding ligand according to any one of the claims 1 to 172 wherein C consists of 0 to 5 neutral amino acids independently selected from the group consisting of Abz, Gly, Ala, Thr, and Ser

174. A zinc-binding ligand according to claim 173 wherein C consists of 0 to 5 Gly

175. A zink-binding ligand according to claim 174 wherein C consists of 0 Gly

176. A zink-binding ligand according to claim 174 wherein C consists of 1 Gly

177. A zink-binding ligand according to claim 174 wherein C consists of 2 Gly

178. A zink-binding ligand according to claim 174 wherein C consists of 3 Gly

179. A zink-binding ligand according to claim 174 wherein C consists of 4 Gly

180. A zink-binding ligand according to claim 174 wherein C consists of 5 Gly

181. A zinc-binding ligand according to claim 173 wherein C is ~Abz-(Gly)o.4-

182. A zinc-binding ligand according to any one of the claims 1 to 181 wherein D comprises 1 to 16 positively charged groups

183. A zinc-binding ligand according to claim 182 wherein D comprises 1 to 12 positively charged groups

184. A zinc-binding ligand according to claim 183 wherein D comprises 1 to 10 positively charged groups

185. A zinc-binding ligand according to any one of the claims 1 to 184 wherein D is a frag- ment containing basic amino acids independently selected from the group consisting of Lys and Arg and D-isomers of these.

186. A zinc-binding ligand according to claim 185 wherein the basic amino acid is Arg

187. A zinc-binding ligand according to any one of the claims 1 to 186 wherein Xis -OHor — NH,

188. A zinc-binding ligand according to claim 187 wherein X is -NH;

189. 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.

190. An R-state insulin hexamer according to claim 189 wherein the insulin is selected from the group consisting of human insulin, an analogue thereof, a derivative thereof, and combinations of any of these :

191. An R-state insulin hexamer according to claim 190 wherein the insulin is an analogue of human insulin selected from the group consisting of v.An analogue wherein position B28 is Asp, Lys, Leu, Val, or Ala and position B29 is Lys or Pro; and vi.des(B28-B30), des(B27) or des(B30) human insulin.

192. An R-state insulin hexamer according to claim 191, wherein the insulin is an analogue of human insulin wherein position B28 is Asp or Lys, and position B29 is Lys or Pro.

193. An R-state insulin hexamer according to claim 191 wherein the insulin is des(B30) hu- man insulin.

194. An R-state insulin hexamer according to claim 190 wherein the insulin is a derivative of human insulin having one or more lipophilic substituents.

195. An R-state insulin hexamer according to claim 194 wherein the insulin derivative is se- lected from the group consisting of B29-N°-myristoyl-des(B30) human insulin, B29-N°- palmitoyl-des(B30) human insulin, B29-N°-myristoyl human insulin, B29-N*-palmitoyl human insulin, B28-N°-myristoyl Lys??® Pro®? human insulin, B28-N°-palmitoyl Lys®?® Pro? human insulin, B30-N*-myristoyl-Thr?®Lys®* human insulin, B30-N°-paimitoyl-Thr®2°Lys®*® human insulin, B29-N°-(N-paimitoyl-y-glutamyt)-des(B30) human insulin, B29-N°-(N-lithocholyl-y- glutamyl)-des(B30) human insulin, B29-N°*-(w-carboxyheptadecanoyl)-des(B30) human insu- lin and B29-N°-(w-carboxyheptadecanoyl) human insulin.

196. An R-state insulin hexamer according to claim 195 wherein the insulin derivative is B29- Ne-myristoyl-des(B30) human insulin.

197. An insulin hexamer according to any one of the claims 189 to 196 further comprising at least 3 phenolic molecules.

198. An aqueous insulin preparation comprising R-state insulin hexamers according to any of claims 189 to 197

199. Method of prolonging the action of an insulin preparation which comprises adding a zinc-binding ligand according to any of claims 1 to 188 to the insulin preparation.

200. Aqueous insulin preparation according to claim 198 wherein the ratio between precipi- tated insulin and dissolved insulin is in the range from 99:1 to 1:99.

201. Aqueous insulin preparation according to claim 200 wherein the ratio between precipi- tated insulin and dissolved insulin is in the range from 95:5 to 5:85

202. Aqueous insulin preparation according to claim 201 wherein the ratio between precipi- tated insulin and dissolved insulin is in the range from 80:20 to 20:80

203. Aqueous insulin preparation according to claim 202 wherein the ratio between precipi- tated insulin and dissolved insulin is in the range from 70:30 to 30:70

204. 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 His®°-Zn?" site « optionally attaching a fragment consisting of 0 to 5 neutral a- or B-amino acids « attaching a fragment comprising 1 to 20 positively charged groups independently se- : lected from amino or guanidino groups