ZA200605883B - Novel inhibitors of glutaminyl cyclase - Google Patents

Description

Novel Inhibitors of Glutaminyl Cyclase

Field of the invention

The invention relates to glutaminyl cyclase (QC, EC 2.3.2.5) that catalyzes the intramolecular cyclization of N-terminal glutamine residues into pyroglutamic acid (5- oxo-prolyl, pGlu*) under liberation of ammonia and the intramolecular cyclization of

N-terminal glutamate residues into pyroglutamic acid under liberation of water.

Background of the invention Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the intramolecular cyclization of N- terminal glutamine residues into pyroglutamic acid (pGlu®) liberating ammonia. A QC was first isolated by Messer from the latex of the tropical plant Carica papaya in 1963 (Messer, M. 1963 Nature 4874, 1299). 24 years later, a corresponding enzymatic activity was discovered in animal pituitary (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci U S A 84, 3628-3632). For the mammalian QC, the conversion of Gin into pGlu by QC could be shown for the precursors of TRH and GnRH (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci U S A 84, 3628-3632). In addition, initial localization experiments of QC revealed a co- localization with its putative products of catalysis in bovine pituitary, further improving the suggested function in peptide hormone synthesis (Bockers, T. M. et al. 1995 J

Neuroendocrinol 7, 445-453). In contrast, the physiological function of the plant QC is less clear. In the case of the enzyme from C. papaya, a role in the plant defense against pathogenic microorganisms was suggested (El Moussaoui, A. et al.2001 Cell

Mol Life Sci 58, 556-570). Putative QCs from other plants were identified by sequence comparisons recently (Dahl, S. W. et al.2000 Protein Expr Purif 20, 27-36).

The physiological function of these enzymes, however, is still ambiguous.

The QCs known from plants and animals show a strict specificity for L-Glutamine in the N-terminal position of the substrates and their kinetic behavior was found to obey the Michaelis-Menten equation (Pohl, T. et al. 1991 Proc Natl Acad Sci U S A 88, 10059-10063; Consalvo, A. P. et al. 1988 Anal Biochem 175, 131-138; Gololobov, M.

Y. et al. 1996 Biol Chem Hoppe Seyler 377, 395-398). A comparison of the primary structures of the QCs from C. papaya and that of the highly conserved QC from mammals, however, did not reveal any sequence homology (Dahl, S. W. et al. 2000

Protein Expr Purif 20, 27-36). Whereas the plant QCs appear to belong to a new enzyme family (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36), the mammalian

QCs were found to have a pronounced sequence homology to bacterial aminopeptidases (Bateman, R. C. et al 2001 Biochemistry 40, 11246-11250), leading to the conclusion that the QCs from plants and animals have different evolutionary origins.

Recently, it was shown that recombinant human QC as well as QC-activity from brain extracts catalyze both, the N-terminal glutaminyl as well as glutamate cyclization.

Most striking is the finding, that cyclase-catalyzed Glu;-conversion is favored around pH 6.0 while Gins-conversion to pGlu-derivatives occurs with a pH-optimum of around 8.0. Since the formation of pGlu-AB-related peptides can be suppressed by inhibition of recombinant human QC and QC-activity from pig pituitary extracts, the enzyme QC is a target in drug development for treatment of Alzheimer's disease.

EP 02 011 349.4 discloses polynucleotides encoding insect glutaminyl cyclase, as well as polypeptides encoded thereby. This application further provides host cells comprising expression vectors comprising polynucleotides of the invention. Isolated polypeptides and host cells comprising insect QC are useful in methods of screening for agents that reduce glutaminyl cyclase activity. Such agents are useful as pesticides.

Definitions

Enzyme inhibitors

Reversible enzyme inhibitors: comprise competitive inhibitors, non-competitive reversible inhibitors, slow-binding or tight-binding inhibitors, transition state analogs and multisubstrate analogs.

Competitive inhibitors show i) non-covalent interactions with the enzyme, ii) compete with substrate for the enzyme active site,

The principal mechanism of action of a reversible enzyme inhibitor and the definition of the dissociation constant can be visualized as follows:

Kon

E + 1 —_—— E-I

Kote

Ss

E-S m/——"= E~-Po——> E + P k

K =K =-% k,,

The formation of the enzyme-inhibitor [E-I] complex prevents binding of substrates, therefore the reaction cannot proceed to the normal physiological product, P. A larger inhibitor concentration [I] leads to larger [E-l], leaving less free enzyme to which the substrate can bind.

Non-competitive reversible inhibitors i) bind at a site other than active site (allosteric binding site) ii) cause a conformational change in the enzyme which decreases or stops catalytic activity.

Slow-binding or tight-binding inhibitors i) are competitive inhibitors where the equilibrium between inhibitor and enzyme is reached slowly, ii) (kon is slow), possibly due to conformational changes that must occur in the enzyme or inhibitor a) are often transition state analogs b) are effective at concentrations similar to the enzyme conc. (subnanomolar K; values) c) due to ken values being so low these types of inhibitors are "almost" irreversible

Transition state analogs are competitive inhibitors which mimic the transition state of an enzyme catalyzed reaction. Enzyme catalysis occurs due to a lowering of the energy of the transition state, therefore, transition state binding is favored over substrate binding.

Multisubstrate Analogs

For a reaction involving two or more substrates, a competitive inhibitor or transition state analog can be designed which contains structural characteristics resembling two or more of the substrates. irreversible enzyme inhibitors: drive the equilibrium between the unbound enzyme and inhibitor and enzyme inhibitor complex (E + | <—> E-I) all the way to the right with a covalent bond (~100 kcal/mole), making the inhibition irreversible.

Affinity labeling agents e Active-site directed irreversible inhibitors (competitive irreversible inhibitor) are recognized by the enzyme (reversible, specific binding) followed by covalent bond formation, and i) are structurally similar to substrate, transition state or product allowing for specific interaction between drug and target enzyme, ii) contain reactive functional group (e.g. a nucleophile, -COCH.Br) allowing for covalent bond formation

The reaction scheme below describes an active-site directed reagent with its target enzyme where K; is the dissociation constant and Kinactvation is the rate of covalent bond formation.

E+ 1< Xe spo tum pf

» Mechanism-based enzyme inactivators (also called suicide inhibitors) are active-site directed reagents (unreactive) which binds to the enzyme active site where it is transformed to a reactive form (activated) by the enzyme's catalytic capabilities. Once activated, a covalent bond between the inhibitor 5 and the enzyme is formed.

The reaction scheme below shows the mechanism of action of a mechanism based enzyme inactivator, where K; is the dissociation complex, k; is the rate of activation of the inhibitor once bound to the enzyme, k; is the rate of dissociation of the activated inhibitor, P, frorn the enzyme (product can still be reactive) from the enzyme and k, is the rate of covalent bond formation between the activated inhibitor and the enzyme.

SS. ENG. EIN - LE BN :

E+ P 16

Inactivation (covalent bond formation, ks) must occur prior to dissociation (ks) otherwise the now reactive inhibitor is released into the environment. Partition ratio, kJ/k,: ratio of released product to inactivation should be minimized for efficient inactivation of the system and minimal undesirable side reactions.

A large partition ratio (favors dissocation) leads to nonspecific reactions.

Uncompetitive enzyme inhibitors: From the definition of uncompetitive inhibitor (an inhibitor which binds only to ES complexes) the foll owing equilibria can be written:

Ks k2

E+S~——— ES—E+P +

ESI

The ES complex dissociates the subtrate with a dissociation constant equal to Ks, whereas the ESI complex does not dissociate it (i.e has a Ks value equal to zero).

The K's of Michaelis-Menten type enzymes are expected to be reduced. Increasing substrate concentration leads to increasing ES! concentration (a complex incapable of progressing to reaction products), therefore the inhibition can not be removed.

Preferred according to the present invention are competitive enzyme inhibitors.

Most preferred are competitive reversible enzyme inhibitors.

The terms “ki” or “K;" and “Kp" are binding constants, which describe the binding of an inhibitor to and the subsequent release from an enzyme. Another measure is the “ICs” value, which reflects the inhibitor concentration, which at a given substrate concentration results in 50 % enzyme activity.

The term “DP IV-inhibitor” or “dipeptidyl peptidase IV inhibitor” is generally known to a person skilled in the art and means enzyme inhibitors, which inhibit the catalytic activity of DP IV or DP IV-like enzymes. "DP IV-activity" is defined as the catalytic activityof dipeptidyl peptidase IV (DP IV) and DP IV-like enzymes. These enzymes are post-proline (to a lesser extent post- alanine, post-serine or post-glycine) cleaving serine proteases found in various tissues of the body of a mammal including kidney, liver, and intestine, where they remove dipeptides from the N-terminus of biologically active peptides with a high specificity when proline or alanine form the residues that are adjacent to the N- terminal amino acid in their sequence.

The term “PEP-inhibitor” or “prolyl endopeptidase inhibitor” is generally known to a person skilled in the art and means enzyme inhibitors, which inhibit the catalytic activity of prolyl endopeptidase (PEP, prolyl oligopeptidase, POP). "PEP-activity* is defined as the catalytic activity of an endoprotease that is capable to hydrolyze post proline bonds in peptides or proteins were the proline is in amino acid position 3 or higher counted from the N-terminus of a peptide or protein substrate.

The term “QC” as used herein comprises glutaminyl cyclase (QC) and QC-like enzymes. QC and QC-like enzymes have identical or similar enzymatic activity, further defined as QC activity. In this regard, QC-like enzymes can fundamentally differ in their molecular structure from QC.

The term “QC activity” as used herein is defined as intramolecular cyclization of N- terminal glutamine residues into pyroglutamic acid (pGlu®) or of N-terminal L- homoglutamine or L-B-homoglutamine to a cyclic pyro-homoglutamine derivative under liberation of ammonia. See therefore schemes 1 and 2.

Scheme 1: Cyclization of glutamine by QC peptide peptide

NH

HN

Hy o 0]

NH;

J NH

NH, Qc 0

Scheme 2: Cyclization of L-homoglutamine by QC peptide peptide

NH

HN

HN o 0

NH, — & 0 N

Qc [o}

NH,

The term “EC” as used herein comprises the side activity of QC and QC-like enzymes as glutamate cyclase (EC), further defined as EC activity.

The term “EC activity” as used herein is defined as intramolecular cyclization of N- terminal glutamate residues into pyroglutamic acid (pGlu*) by QC. See therefore scheme 3.

Scheme 3: N-terminal cyclization of uncharged glutamyi peptides by QC (EC) tide i . oo ee pers ° NH NH HN ne

HaN o Hs o o HO (0) (~5.0<pH<7.0) Q } (~7.0<pH<8.0) QC/EC 2 QC/EC NH 0 0” “OH HN O° o ©

The term “QC-inhibitor” “glutaminyl cyclase inhibitor” is generally known to a person skilled in the art and means enzyme inhibitors, which inhibit the catalytic activity of glutaminyl cyclase (QC) or its glutamyli cyclase (EC) activity.

Potency of QC inhibition in light of the correlation with QC inhibition, in preferred embodiments, the subject method and medical use utilize an agent with a Ki for QC inhibition of 10 pM or less, more preferably of 1 pM or less, even more preferably of 0.1 uM or less or 0.01 uM or less, or most preferably 0.01 uM or less. indeed, inhibitors with Ki values in the eee —

lower micromolar, preferably the nanomolar and even more preferably the picomolar range are contemplated. Thus, while the active agents are described herein, for convience, as “QC inhibitors”, it will be understood that such nomenclature is not intending to limit the subject of the invention to a particular mechanism of action.

Molecular weight of QC inhibitors

In general, the QC inhibitors of the subject method or medical use will be small molecules, e.g., with molecular weights of 1000 g/mole or less, 500 g/mole or less, preferably of 400 g/mole or less, and even more preferably of 350 g/mole or less and even of 300 g/mole or less.

The term “subject” as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.

The term “therapeutically effective amount” as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

As used herein, the term “pharmaceutically acceptable” embraces both human and veterinary use: for example the term “pharmaceutically acceptable” embraces a veterinarily acceptable compound or a compound acceptable in human medicine and health care.

Throughout the description and the claims the expression “acyl”, unless specifically limited, denotes a Ci.12 acyl residue, preferably a Cig acyl residue and especially preferred a Ci4 acyl residue. Examples of acyl include alkanoyl groups mentioned below and benzoyl. “Peptides” are selected from dipeptides to decapeptides, preferred are dipeptides, tripeptides, tetrapeptides and pentapeptides. The amino acids for the formation of the “peptides” can be selected from those listed below.

Throughout the description and the claims the expression "alkyl", unless specifically limited, denotes a Ci.12 alkyl group, preferably a C1 alkyl group. Alkyl groups may be straight chain or branched. Suitable alkyl groups include, for example, methyl, ethyl, propyl (e.g. n-propyl and isopropyl), (n-butyl, tert-butyl and sec-butyl), pentyl, hexyl, heptyl (e.g. n-heptyl) and octyl (e.g. n-octyl). The expression "alk", for example in the expression "alkoxy", and the expression "alkan”, for example in the expression "alkanoyl’, should be interpreted in accordance with the definition of "alkyl". Exemplary alkoxy groups include methoxy, ethoxy, butoxy (e.g. n-butoxy), heptyloxy (e.g. n-heptyloxy) and octyloxy (e.g. n-octyloxy). Exemplary alkanoy! (i.e. acyl groups) include ethanoyl (i.e. acetyl), propionyl and butyryl.

The expression "alkenyl", unless specifically limited, denotes a C,.12 alkenyl group, preferably a C.¢ alkenyl group, which contains at least one double bond at any desired location. Alkenyl groups may be straight chain or branched. Exemplary alkenyl groups include ethenyl, propenyl and butenyl.

The expression "alkynyl", unless specifically limited, denotes a Ca.12 alkynyl group, preferably a Cz. alkynyl group, which contains at least one triple bond at any desired location. Alkynyl groups may be straight chain or branched. Exemplary alkenyl groups include ethynyl, propynyl and butynyl.

The expression “cycloalkyl”, unless specifically limited, denotes a Ca42 cycloalkyl group, preferably a Cas cycloalkyl group. Exemplary cycloalkyl groups include cylcopropyl, cyclobutyl, cyclopropyl, cyclohexyl, cycloheptyl and cyclooctyl.

Cycloalkyl groups may be branched in which case the number of carbons indicates the total number of carbons in the moiety.

The expression “heterocyclic”, unless specifically limited, denotes a cycloalkyl residue, wherein one or more (e.g. 1, 2 or 3) ring atoms are replaced by heteroatoms selected from N, S or O. Exemplary heterocyclic groups containing one hetero atom include pyrrolidine, tetrahydrofuran and piperidine. Such groups may be optionally substituted eg by alkyl, oxo or hydroxyl.

Concrete examples of a heterocyclic group comprise a substituted or unsubstituted oxirano, aziridino, oxacyclopropyl, azacyclopropyl, thiirano, oxetano, thietano, pyrrolidino, tetrahydrofurano, thiolano, 1,1-dioxo-thiolano, 1,3-dioxolano, thiazolidino, imidazolidino, oxazolidino, pyrazolidino, tetrahydropyrano, piperidino, urotropino, piperazino, N-methyl-piperazino, (2-(N-methyl)-N’-piperazinyl)-ethyl, (4N-(2'- hydroxyethyl)-1N-piperazinyl), (2-(4N-(2"-hydroxyethyl)-1 N-piperazinyl)-ethyloxy), morpholino, 2-(N-morpholino)-ethyl group, as well as lactams, lactones, cyclic imides and cyclic anhydrides.

The expression “carbocylic”, unless specifically limited, denotes a carbocylic group containing between 3 and 12 carbon atoms, more preferably between 3 and 8 carbon atoms. A carboyclic group, as used herein, refers to a group other than aryl or cycloalkyl which comprises at least one ring of carbon atoms without heteroatoms.

Examples of carbocylic groups include bridged ring systems (e.g. bicyclo[2.2.1]heptenyl) and partially unsaturated ring systems.

The expression “aryl”, unless specifically limited, denotes a Ce412 aryl group, preferably a Ces aryl group. Aryl groups will contain at least one aromatic ring (e.g. one, two or three rings), but may also comprise partially or fully unsaturated rings.

An example of an aryl group with one aromatic ring is phenyl. Examples of aromatic groups with two aromatic rings include naphyl. Examples of aryl groups which contain partially or fully unsaturated rings include pentalene and indene. As noted below, aryl groups may optionally be substituted. Further examples for aryl groups are 4-fluoro-phenyl, 3-fluoro-phenyl, pentafiuoro-phenyl, 4-hydroxyphenyl-, 3-nitro- phenyl-, 4-(trifluoromethyl)-phenyl-, 4-anilinyl-, 2-biphenylyl-, 3-biphenylyl-, 4- biphenylyl-, indenyl-, 1-naphthyl-, or 2-naphthyl-, 1-anthracenyl-, 2-anthracenyl-, 3- anthracenyl- groups.

Examples of -alkylaryl include phenylmethyl- (benzyl) and phenylethyl, 2-phenyleth-1- yl, p-tolyl-methyl-, p-tolyl-ethyl-, m-tolyl-methyl-, m-tolyl-ethyl-, o-tolyl-methyl-, o-tolyi- ethyl, 2-(4-ethyl-phenyl)-eth-1-yl-, 2,3-dimethyl-phenyl-methyl-, 2,4-dimethyi-phenyl- methyl, 2,5-dimethyl-phenyl-methyl-, 2,6-dimethyl-phenyl-methyl-, 3,4-dimethyl- phenyl-methyl-, 3,5-dimethyl-phenyl-methyl-, 2,4,6-trimethyl-phenyl-methyl-, 2 3-

dimethyl-phenyl-ethyl-, 2 4-dimethyl-phenyl-ethyl-, 2,5-dimethyl-phenyl-ethyl-, 2,6- dimethyl-phenyl-ethyl-, 3,4-dimethyl-phenyl-ethyl-, 3,5-dimethyl-phenyl-ethyl-, 2,4,6- trimethyl-phenyl-ethyl-, benzhydryl (= diphenyl-methyl), benzhydryl (= diphenyl-ethyl), trityl (= triphenyl-methyl), trityl (= triphenyl-ethyl), a-styryl, B-styryl, cumyl, 2-ethyl- phenyl-methyl-, 3-ethyl-phenyl-methyl-, 4-ethyl-phenyl-methyl-, 2-ethyl-phenyl-ethyl-, 3-ethyl-phenyl-ethyl-, 4-ethyl-phenyl-ethyl-, 2-fluoro-benzyl, 1-methyl-2-fluoro-phen-6- yl-methyl-, 1-methyl-2-fluoro-phen-4-yl-methyl-, 1-methyl-2-fluoro-phen-6-yl-ethyl-, 1- methyl-2-fluoro-phen-4-yl-ethyl-, 1H-indenyl-methyl-, 2H-indenyl-methyl-, 1H-indenyl- ethyl-, 2H-indenyl-ethyl-, indanyl-methyl-, indan-1-on-2-yl-methyl-, indan-1-on-2-yl- ethyl-, tetralinyl-methyl-, tetralinyl-ethyl-, fluorenyl-methyl-, fluorenyl-ethyl-, (3- phenyl)-cyclopent-1-yi ?, dihydronaphthalinyl-methyl-, dihydronaphthalinyl-ethyl-, or (4-cyciohexyl)-phenyl-methyl-, (4-cyclohexyl)-phenyl-ethyl-.

The expression “heteroaryl”, unless specifically limited, denotes as an aryl residue, wherein one or more (e.g. 1, 2, 3, or 4, preferably 1, 2 or 3) ring atoms are replaced by heteroatoms selected from N, S and O or else a 5-membered aromatic ring containing one or more (e.g. 1, 2, 3, or 4, preferably 1, 2 or 3) ring atoms selected from N, S and O. As noted below, heteroaryl groups may optionally be substituted.

Exemplary heteroaryl groups include, pyridine (eg 2, 3 or 4-pyridine), pyrimidine, quinoline, pyrrole, furan, thiophene, oxazole, pyrazole, benzodioxolane, benzodioxane, benzothiophene, benzodioxepine, and thiazolyl, 1-imidazolyl, 2- imidazolyl, 4-imidazolyl, 3-phenyl-1-pyrrolyl, isoxazolyl, isothiazolyl, 3-pyrazolyl, 1,2,3-triazolyl, 1,2,4- triazolyl, tetrazolyl, pyridazinyl, pyrazinyl, indazolyl, 6-indolyl, benzimidazolyl, isochinolinyl, purinyl, carbazolinyl, acridinyl, and 2,3 -bifuryl groups.

Examples of -alkyiheteroaryl include pyridinylmethyl-, N-methyl-pyrrol-2-methyl- N- methyl-pyrrol-2-ethyl-, N-methyl-pyrrol-3-methyl-, N-methyl-pyrrol-3-ethyl-, 2-methyl- pyrrol-1-methyl-, 2-methyl-pyrrol-1-ethyl-, 3-methyl-pyrrol-1-methyl-, 3-methyl-pyrrol- 1-ethyl-, 4-pyridino-methyl-, 4-pyridino-ethyl-, 2-(thiazol-2-yl)-ethyi-, tetrahydroisochinolinyl-methyl-, tetrahydroisochinolinyl-ethyl-, 2-ethyl-indol-1-methyl-, 2-ethyl-indol-1-ethyl-, 3-ethyl-indol-1-methyl-, 3-ethyl-indol-1-ethyl-, 4-methyl-pyridin- 2-methyl-, 4-methyl-pyridin-2-yl-ethyl-, 4-methyl-pyridin-3-methyl, 4-methyi-pyridin-3- ethyl.

The aforementioned aryl and heteroaryl groups may, where appropriate, optionally be substituted.

The expression “substitution” or “substituted” includes the substitution by one or more (e.g. 1, 2 or 3, preferably 1 or 2) monovalent or multivalent functional groups.

Suitable substituent groups include alkyl, cycloalkyl, aryl (eg phenyl), heteroaryl (eg furyl), carbocylic, heterocyclic, alkoxy, cycloalkoxy, aryloxy, heteroaryloxy, carbocyclicoxy, hetercyclicoxy, alkenyloxy, alkynyloxy, alkenyl, alkynyl, acyl, alkanoyl, alkoxyalkanoyl, alkoxyalkyl, heteroarylalkyl, arylalkyl, arylalkyloxy, heteroarylalkyloxy, nitro, -S-alkyl (e.g. methyithio) halo (e.g. fluoro, chloro, bromo and iodo), cyano, hydroxyl, -SQO.alkyl, -SOjaryl, -SOheteroaryl, -SO.cycloalkyl -

SOzheterocyclic, -CO.H, -COaalkyl, -NH2, -NHalkyl, -N(alkyl). (e.g. dimethylamino), -CO-N(alkyl), and -CO-NH(alkyl).

Alkyl groups including derivatives such as alkoxy together with alkenyl, alkynyl and cycloalkyl groups may optionally be halogen substituted e.g. substituted by fluoro.

For example, halo substituted alkyl groups include trifluromethyl and halo substituted alkoxy groups include trifluoromethoxy.

The term "halogen" comprises fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (1), respectively.

Amino acids which can be used in the present invention are L and D-amino acids, N- alkylated amino acids, N-methyl-amino acids, aza-amino acids; allo- and threo-forms of lle and Thr, which can, e.g. be a-, - or e-amino acids, whereof a-amino acids are preferred.

Examples of amino acids are: aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), lysine (Lys), histidine (His), glycine (Gly), serine (Ser), cysteine (Cys), threonine (Thr), asparagine (Asn), glutamine (Gin), tyrosine (Tyr), alanine (Ala), proline (Pro), valine (Val), isoleucine (lle), leucine (Leu), methionine (Met), phenylalanine (Phe), tryptophan (Trp), hydroxyproline (Hyp), beta-alanine (beta-Ala), 2-aminooctanoic acid (Aoa), acetidine- (2)-carboxylic acid (Ace), pipecolic acid (Pip), 3-aminopropionic acid, 4-aminobutyric acid and so forth, alpha-aminoisobutyric acid (Ab), sarcosine (Sar), ornithine (Om), citrulline (Cit), homoarginine (Har), t-butylalanine (f-butyi-Ala), t-butylglycine (f-butyl-

Gly), N-methylisoleucine (N-Melle), phenylglycine (Phg), cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) and methionine sulfoxide (MSO), acetyl-Lys, modified amino acids such as phosphoryl-serine (Ser(P)), benzyl-serine (Ser(Bz!)) and phosphoryl-tyrosine (Tyr(P)), 2-aminobutyric acid (Abu), aminoethyicysteine (AECys), carboxymethylcysteine (Cmc), dehydroalanine (Dha), dehydroamino-2- butyric acid (Dhb), carboxyglutaminic acid (Gla), homoserine (Hse), hydroxylysine (Hyl), cis-hydroxyproline (cisHyp), trans-hydroxyproline (transHyp), isovaline (Iva), pyroglutamic acid (Pyr), norvaline (Nva), 2-aminobenzoic acid (2-Abz), 3- aminobenzoic acid (3-Abz), 4- aminobenzoic acid (4-Abz), 4-(aminomethyl)benzoic acid (Amb), 4-(aminomethyl)cyciohexanecarboxylic acid (4-Amc), Penicillamine (Pen), 2-amino-4-cyanobutyric acid (Cba), cycloalkane-carboxylic aicds. Examples of w-amino acids are e.g.: 5-Ara (aminoraleric acid), 6-Ahx (aminohexanoic acid), 8-Aoc 16 (aminooctanoic aicd), 9-Anc (aminovanoic aicd), 10-Adc (aminodecanoic acid), 11-

Aun (aminoundecanoic acid), 12-Ado (aminododecanoic acid). Further amino acids are: indanylglycine (Igl), indoline-2-carboxylic acid (ldc), octahydroindole-2-carboxylic acid (Oic), diaminopropionic acid (Dpr), diaminobutyric acid (Dbu), naphtylalanine (1-

Nal) and (2-Nal), 4-aminophenylalanine (Phe(4-NH)), 4-benzoylphenylalanine (Bpa), diphenylalanine (Dip), 4-bromophenylalanine (Phe(4-Br)), 2-chlorophenylalanine (Phe(2-Cl)), 3-chlorophenylalanine (Phe(3-Cl)), 4-chlorophenylalanine (Phe(4-Cl)), 3,4-chlorophenylalanine (Phe (3,4-Cl)), 3-fluorophenylalanine (Phe(3-F)), 4- fluorophenylalanine (Phe(4-F)), 3.4-fluorophenylalanine (Phe(3,4-F5)), pentafluorophenylalanine (Phe(Fs)), 4-guanidinophenylalanine (Phe(4-guanidino)), homophenylalanine (hPhe), 3-jodophenylalanine (Phe(3-J)), 4-jodophenylalanine (Phe(4-J)), 4-methylphenylalanine (Phe(4-Me)), 4-nitrophenylalanine (Phe-4-NOy)), biphenylalanine (Bip), 4-phosphonomethylphenylalanine (Pmp), cyclohexylglycine (Ghg), 3-pyridinylalanine (3-Pal), 4-pyridinylalanine (4-Pal), 3,4-dehydroproline (A-

Pro), 4-ketoproline (Pro(4-keto)), thioproline (Thz), isonipecotic acid (inp), 1,234,.- tetrahydroisoquinolin-3-carboxylic acid (Tic), propargyiglycine (Pra), 6- hydroxynorieucine (NU(6-OH)), homotyrosine (hTyr), 3-jodotyrosine (Tyr(3-J)), 3,5- dijodotyrosine (Tyr(3,5-J2)), methyityrosine (Tyr(Me)), 2',6 -dimethyltyrosine (Dmt), 3-

NOo-tyrosine (Tyr(3-NOp)), phosphotyrosine (Tyr(PO3H2)), alkylglycine, 1- aminoindane-1-carboxylic acid, 2-aminoindane-2-carboxylic acid (Aic), 4-amino-

methylpyrrol-2-carboxylic acid (Py), 4-amino-pyrrolidine-2-carboxylic acid (Abpc), 2- aminotetraline-2-carboxylic acid (Atc), diaminoacetic acid (Gly(NHy)), diaminobutyric acid (Dab), 1,3-dihydro-2H-isoinole-carboxylic acid (Disc), homocylcohexylalanine (hCha), homophenylalanine (hPhe or Hof), trans-3-phenyl-azetidine-2-carboxylic acid, 4-phenyl-pyrrolidine-2-carboxylic acid, 5-phenyl-pyrrolidine-2-carboxylic acid, 3- pyridylalanine (3-Pya), 4-pyridylalanine (4-Pya), styrylalanine, tetrahydroisoquinoline- 1-carboxylic acid (Tig), 1,2,3,4-tetrahydronorharmane-3-carboxylic acid (Tpi), B-(2- thienryl)-alanine (Tha). “Peptides” are selected from dipeptides to decapeptides, preferred are dipeptides, tripeptides, tetrapeptides and pentapeptides. The amino acids for the formation of the “peptides” can be selected from those listed above.

An “aza-amino acid” is defined as an amino acid where the chiral a-CH group is replaced by a nitrogen atom, whereas an “aza-peptide” is defined as a peptide, in which the chiral a-CH group of one or more amino acid residues in the peptide chain is replaced by a nitrogen atom.

Other amino acid substitutions for those encoded in the genetic code can also be included in peptide compounds within the scope of the invention and can be classified within this general scheme. Proteinogenic amino acids are defined as natural protein-derived a-amino acids. Non-proteinogenic amino acids are defined as all other amino acids, which are not building blocks of common natural proteins. “Peptide mimetics” per se are known to a person skilled in the art. They are preferably defined as compounds which have a secondary structure like a peptide and optionally further structural characteristics; their mode of action is largely similar or identical to the mode of action of the native peptide; however, their activity (e.g. as an antagonist or inhibitor) can be modified as compared with the native peptide, especially vis a vis receptors or enzymes. Moreover, they can imitate the effect of the native peptide (agonist). Examples of peptide mimetics are scaffold mimetics, non- peptidic mimetics, peptoides, peptide nucleic acids, oligopyrrolinones, vinylogpeptides and oligocarbamates. For the definitions of these peptide mimetics see Lexikon der Chemie, Spektrum Akademischer Verlag Heidelberg, Berlin, 1999.

Claims (57)

Claims

1. A compound of the formula 1 including pharmaceutically acceptable salts thereof, including all stereoisomers and polymorphs: =\ LL N-A-8 = formula 1 wherein: Ais either: an alkyl chain, alkenyl chain or alkynyl chain; or A is a group selected from: n RS n RS . "(a n R? R? R® R® R? 10 10 R8 n' R8 R RS R 0) 0) (ty gia f (iV) V) wherein: RS, R’, R® R® and R' are independently H or an alkyl chain, alkenyl chain, alkynyl chain, cycloalkyl, a carbocycle, aryl, heteroaryl, or a heterocycle; n and n' are independently 1 - 5; mis 1-5; ois 0-4; and B is a group selected from:

X X X .D D H H H vi (Via) (Vib) Y X X bp ~Joe JE —N N Z N Z H R17 R8 4 Ltrs (vi X R16 (Vin) (1X) 1" n R ( R12 J YS ~N SN R13 H R14 11 (0) RM 0 R11 n R R12 R12 R12 x? N N EN R13 NE R13 “NN R13 H R14 H H Rw H H Rw“ (XI) (XN) (Xr) x2 RM R12 —N WN R13 H R14 (XIV) wherein: D and E independently represent an alkyl chain, alkenyl chain, alkynyl chain, a cycloalkyl, carbocycle, aryl, -alkylaryl, heteroaryl, -alkylheteroaryl, acyl or a heterocycle.

ZisCHorN;

X represents CRZRY, 0, S, NR, with the proviso for formulas (Vill) and (IX) that, if Z= CH, Xis OorS; R' is selected from the group consisting of H, alkyl, cycloalkyl, aryl, heteroaryl, - oxyalkyl, -oxyaryl, carbonyl, amido, hydroxy, NO, NH,, CN; R? and R?' are independently selected from H, alkyl, cycloalkyl, heterocycle, aryl heteroaryl, -oxyalkyl, -oxyaryl, carbonyl, amido, NO; , NH, CN, CFs;

X'. X2 and X° are independently O or S provided that X* and X® are not both O; Y is O or S, with the proviso that Y may not by O, when the carbocycle formed by R'” and R'® has 3 members in the ring; 16 ZisCHorN; R'" R®, R" and R" can be independently selected from H, an alkyl chain, an alkenyl chain, an alkynyl chain, cycloalkyl, carbocycle, aryl, heteroaryl, a heterocycle, halo, alkoxy-, -thioalkyl, carboxyl, carboxylic acid ester, carbonyl, carbamide, carbimide, thiocarbamide or thiocarbonyl, NH, NO2; R® and R'® are independently of each other H or a branched or unbranched alkyl chain, or a branched or unbranched alkenyl chain; RY and R'® are independently selected from H or an alkyl chain, alkenyl chain, a alkynyl chain, a carbocycle, aryl, heteroaryl, heteroalkyl, or can be connected to form a carbocycle with up to 6 ring atoms; nisOor1,; with the proviso that the following compounds:

S I NTN ~n [ H H N @ and s N [IN meh Sy S 0) NF and 0) iN NW / CH, (c) and X R 0 R CH. 4-F = Mx CH. 3-Cl rN N CH: 4-CHa WL) CH. H (d) are excluded from formula 1.

2. The compound according to claim 1, wherein A is an unbranched Cs; alkyl chain.

3. The compound according to claim 1, wherein A is group (1), (11) or (lll), with n and n' are each equal to 1.

4, The compound according to any one of claims 1 to 3, wherein B is group (VI).

5. The compound according to any one of claims 1 to 3, wherein B is group (Via).

6. The compound according to either claim 4 or 5, wherein X represents S.

7. The compound according to either claim 4 or 5, wherein X represents NR’.

8. The compound according to either claim 4 or 5, wherein X represents O.

9. The compound according to any one of claims 1 to 3, wherein B is group (VII).

10. The compound according to claim 9, wherein Y represents S.

11. The compound according to either claim 9 or 10, wherein one of R"” and R'® is H and the other is Me.

12. The compound according to either of claim 9 or 10, wherein one of R'” and R'® is H and the other is phenyl.

13. The compound according to either of claim 9 or 10, wherein R'” and R'® are connected to form a carbocycle with up to 6 ring atoms.

14. The compound according to any one of claims 4 to 13, wherein D represents substituted phenyl.

15. The compound according to claim 11, wherein D represents 3,4- dimethoxyphenyl.

16. The compound according to any one of claims 1 to 3, wherein B is selected from (X) to (XIV) and R'' and R'* are H.

17. The compound according to any one of claims 1 to 3, wherein B is selected from (X) to (XIV) and at least one of R" and R'® is H.

18. The compound according to any one of claims 1 to 3, wherein B is selected from (X) to (XIV) and R'® and R'® are both H.

19. A compound according to claim 1 corresponding to any one of Examples 1-5, 7,8, 10-28, 38-59, 61-85, 94, 96-99, 101, 102, 119, 136 and 140 or a pharmaceutical salt, stereoisomers or polymorph thereof.

20. A compound according to claim 1 corresponding to any one of Examples 29, 30, 34-37, 86-93, 95, 100, 106-113, 115-118, 120, 123-127, 130, 132, and 134, or a pharmaceutical salt, stereoisomers or polymorph thereof.

21. A compound according to claim 1 corresponding to any one of Examples 6, 9, 103-105, 121-122 and 137 or a pharmaceutical salt, stereoisomers or polymorph thereof.

22. A compound according to claim 1 corresponding to any one of Examples 31 - 33, 60, 114, 128, 129, 131, 133, 135, 138 and 139 or a pharmaceutical salt, stereoisomers or polymorph thereof.

23. A compound according to claim 1 corresponding to Example 13 or a pharmaceutical salt, stereoisomers or polymorph thereof.

24 A compound according to claim 1 corresponding to Example 119 or a pharmaceutical salt, stereoisomers or polymorph thereof.

25. A compound according to claim 1 corresponding to Example 125 or a pharmaceutical salt, stereoisomers or polymorph thereof.

26. The compound according to any one of the claims 1 to 23 for use as a pharmaceutical.

27. A pharmaceutical composition comprising at least one compound according to any one of the claims 1 to 26 optionally in combination with a therapeutically acceptable carrier and/or excipient.

28. A pharmaceutical composition according to claim 27 for parenteral, enteral or oral administration,

29. The pharmaceutical composition of claim 27 or 28 without the proviso excluding compounds (a) - (d) or the proviso that X* and X* are not both O or the proviso that Y may not be O when the carbocycle formed by R' and R'™ has 3 members in the ring, which comprises additionally at least one compound , selected from the group consisting of PEP-inhibitors, LiCl, inhibitors of dipeptidyl aminopeptidases, preferably inhibitors of DP IV or DP IV-like enzymes, NPY-receptor ligands, NPY agonists, ACE inhibitors, PIMT enhancers, inhibitors of beta secretases, inhibitors of gamma secretases, inhibitors of neutral endopeptidase, PDE-4 inhibitors, MAO inhibitors, TNFalpha inhibitors, amyloid protein or amyloid peptide deposition inhibitors, sigma-1 receptor inhibitors and histamine H3 antagonists.

30. The pharmaceutical composition according to claim 29, wherein said inhibitor of DP IV/DP IV-like enzymes is selected from the group consisting of L-threo-isoleucyl pyrrolidide, L-allo-isoleucyl thiazolidide, L-allo-isoleucyl pyrrolidide; and salts thereof or valine pyrrolidide, BMS-477118, CP-867534-01, LAF-237, PHX-1004, SSR- 162369, SYR-322, TSL-225, FE-999011 GW-229A, 815541, K-579, MK-431, PT-100 or one of ) ZZ N rd >=o ~ 0S N )

ye WT / NY N n_/ and

F. ., N “Arh n° N

31. The pharmaceutical composition according to claim 29, wherein said NPY antagonist is selected from 3a,4,5,9b-tetrahydro-1h-benzle]indol-2-yl amine, BIBP3226 and, (R)-N2-(diphenylacetyl)-(R)-N-[1-(4-hydroxy- phenyl) ethyl] arginine amide.

32. The pharmaceutical composition according to claim 29, wherein said PEP- inhibitor is selected from the group consisting of chemical derivatives of proline or small peptides containing terminal prolines, e.g. benzyloxycarbonyl-prolyl-prolinal, N- terminal substituted L-proline or L-prolylpyrmolidine, substituted N-benzyloxycarbonyl (2) dipeptides containing prolinal at the carboxy terminus, substituted thioprolines, substituted thiazolidines, substituted oxopyrrolidines, carboxy terminal modified prolines including fluorinated ketone derivatives, chloromethyl ketone derivatives of acyl-proline or acylpeptide-proline (Z-Gly-Pro-CH;Cl) and 2-acylpyrrolidine derivatives.

33. The pharmaceutical composition according to claim 29, wherein said PEP- inhibitor is selected from the group consisting of Fmoc-Ala-Pymrr-CN, Z-321, ONO- 1603, JTP-4819 and S-17092.

34. The pharmaceutical composition according to claim 29, wherein said PEP- inhibitor is qo 0 0 C1) PALS O g

35. The phamaceutical composition according to claim 29, wherein said ACE- inhibitor is SDZ ENA 713 (rivastigmine (+)-(S)-N-ethyi-3-[(1-dimethylamino)ethyl}-N- methylphenylcarbamate hydrogen tartrate.

36. The pharmaceutical composition according to claim 29, wherein said PDE4 inhibitor is selectede from the group consisting of Rolipram, CC-002, L-826141, Sch- 351591 (D-4396), 0S-0217, 1BFB-130011, IBFB-150007, IBFB-130020, IBFB- 140301, 1C-485, VMX-554, VMX-565, MEM-1414, MEM-1018, MEM-1091, MEM- 1145, CI-1044, BHN, ZK-117137 and SB-207499 or analogs thereof.

37. The pharmaceutical composition according to claim 29, wherein said PIMT enhancer is a 10-aminoaliphatyl-dibenz[b, f] oxepine of the general formula -R ak R= 0 , , wherein alk is a divalent aliphatic radical, R is an amino group that is unsubstituted or mono- or di-substituted by monovalent aliphatic and/or araliphatic radicals or disubstituted by divalent aliphatic radicals, and Rs, Rz, Rs and Rs are each, “independently of the others, hydrogen, lower alkyl, lower alkoxy, halogen or trifluoromethyl.

38. The pharmaceutical composition according to claim 29, wherein said gamma secretase inhibitor is Ph OH i Bu-i Ph on Ss “Rr R S +-Bu0 NH 0 NH, T Tove 0 0

39. The pharmaceutical composition according to claim 29, wherein said beta secretase inhibitor is

F. F 0 oO [o} JY ZN N Y Y N H FE H P oH OH

40. The pharmaceutical composition according to claim 29, wherein said MAO inhibitor is ladostigil of the formula O ~~ NZ

41. The pharmaceutical composition according to claim 29, wherein said histamine H3 antagonist is a compound selected from the group consisting of A-331440, A- 349821, 3874-H1, UCL-2173, UCL-1470, DWP-302, GSK-189254A, GSK-207040A,

cipralisant, GT-2203, 1S,2S)-2-(2-Aminoethyl)-1-(1 H-imidazol-4-yl)cyclopropane, JNJ-5207852, NNC-0038-0000-1049, dual H1/H3, Sch-79687 or one of 0] O eee N 0 H-0 yoo H oO and Z °N N rs ae XN ZZ x Cl

42. The use of the pharmaceutical compound according to any of claims 1 to 26 or a composition according to any of the claims 27 to 41 and without the proviso excluding compounds (a) - (d) or the proviso that X? and X3 are not both O or the proviso that Y may not be O when the carbocycle formed by R'” and R'® has 3 members in the ring, in the manufacture of a medicament for the treatment of neuronal disorders, especially Alzheimer's disease, Down Syndrome, Parkinson disease, Chorea Huntington, pathogenic psychotic conditions, schizophrenia, impaired food intake, sleep-wakefulness, impaired homeostatic regulation of energy metabolism, impaired autonomic function, impaired hormonal balance, impaired regulation, body fluids, hypertension, fever, sleep dysregulation, anorexia, anxiety related disorders including depression, seizures including epilepsy, drug withdrawal and alcoholism, neurodegenerative disorders including cognitive dysfunction and dementia.

43. A method of treatment of neuronal disorders, especially Alzheimer's disease, Down Syndrome, Parkinson disease, Chorea Huntington, pathogenic psychotic conditions, schizophrenia, impaired food intake, sleep-wakefulness, impaired homeostatic regulation of energy metabolism, impaired autonomic function, impaired hormonal balance, impaired regulation, body fluids, hypertension, fever, sleep dysregulation, anorexia, anxiety related disorders including depression, seizures including epilepsy, drug withdrawal and alcoholism, neurodegenerative disorders including cognitive dysfunction and dementia which comprises administering to a mammal an effective amount of a compound of formula (1) as defined in any of claims 1 to 25 and without the proviso excluding compounds (a) - (d) or the proviso that X2 and X® are not both O or the proviso that Y may not be O when the carbocycle formed by R' and R'® has 3 members in the ring.

44. The use or method according to one of claims 42 or 43 for the treatment of a neuronal disease selected from the group consisting of Alzheimer's disease, Down Syndrome, Parkinson disease and Chorea Huntington.

45. The use of inhibitors of glutaminyl cyclase for the preparation of a medicament for the treatment of treatment of neuronal disorders, especially Alzheimer's disease, Down Syndrome, Parkinson disease, Chorea Huntington, pathogenic psychotic conditions, schizophrenia, impaired food intake, sleep-wakefulness, impaired homeostatic regulation of energy metabolism, impaired autonomic function, impaired hormonal balance, impaired regulation, body fluids, hypertension, fever, sleep dysregulation, anorexia, anxiety related disorders including depression, seizures including epilepsy, drug withdrawal and alcoholism, neurodegenerative disorders including cognitive dysfunction and dementia, wherein said inhibitor inhibits glutaminyl cyclase with a K; of 10 pM or less.

46. A method of treatment of neuronal disorders, especially Alzheimer's disease, Down Syndrome, Parkinson disease, Chorea Huntington, pathogenic psychotic conditions, schizophrenia, impaired food intake, sleep-wakefulness, impaired homeostatic regulation of energy metabolism, impaired autonomic function, impaired hormonal balance, impaired regulation, body fluids, hypertension, fever, sleep dysregulation, anorexia, anxiety related disorders including depression, seizures including epilepsy, drug withdrawal and alcoholism, neurodegenerative disorders including cognitive dysfunction and dementia, which comprises administering to a

120 ] i mammal an effective amount of an inhibitor of glutaminyl cyclase, wherein said inhibitor inhibits glutaminyl cyclase with a K; of 10 uM or less.

47. The use or method according to any one of claims 42 to 46, wherein said inhibitor inhibits glutaminyl cyclase with a K; of 1 uM or less.

48. The use or method according to any one of claims 42 to 46, wherein said inhibitor inhibits glutaminyl cyclase with a K; of 0.1 uM or less.

49. The use or method according to any one of claims 42 to 46, wherein said inhibitor inhibits glutaminyl cyclase with a K; of 0.01 uM or less.

50. The use or method according to any one of claims 42 to 49, wherein said glutaminyl cyclase inhibitor has a molecular weight of 1000 g/mole or less.

51. The use or method according to any one of claims 42 to 49, wherein said glutaminyl cyclase inhibitor has a molecular weight of 500 g/mole or less.

52. The use or method according to any one of claims 42 to 49, wherein said glutaminyl cyclase inhibitor has a molecular weight of 400 g/mole or less.

53. The use or method according to any one of claims 42 to 49, wherein said glutaminyl cyclase inhibitor has a molecular weight of 350 g/mole or less.

54. The use or method according to any one of claims 42 to 53, wherein said glutaminyl cyclase inhibitor is a competitive inhibitor.

55. The use or method according to any one of claims 42 to 54, wherein said glutaminyl cyclase inhibitor is a competitive reversible inhibitor.

56. The use or method according to any one of claims 42 to 55, wherein said glutaminyl cyclase inhibitor is selected from a compound of any one of claims 1 to 25.

57. The use or method according to claim 56, wherein said glutaminyl cyclase inhibitor is administered to a mammal optionally in combination with a compound selected from the group consisting of PEP-inhibitors, LiCl, inhibitors of dipeptidy! aminopeptidases, preferably inhibitors of DP IV or DP IV-like enzymes, NPY-receptor ligands, NPY agonists, ACE inhibitors, PIMT enhancers, inhibitors of beta secretases, inhibitors of gamma secretases, inhibitors of neutral endopeptidase, PDE-4 inhibitors, MAO inhibitors, TNFalpha inhibitors, amyloid protein or amyloid peptide deposition inhibitors, sigma-1 receptor inhibitors and histamine H3 antagonists. :