EP3349744A1

EP3349744A1 - Sulfolipids as new glutaminyl cyclase inhibitors

Info

Publication number: EP3349744A1
Application number: EP16769934.7A
Authority: EP
Inventors: Stephanie Hielscher-Michael; Carola Griehl; Hans-Ulrich Demuth; Stephan Schilling; Ludger Wessjohann; Norbert Arnold
Original assignee: Hochschule Anhalt; Leibniz-Institut fur Pflanzenbiochemie (ipb); Leibniz Institut fuer Pflanzenbiochemie
Current assignee: Hochschule Anhalt; Leibniz-Institut fur Pflanzenbiochemie (ipb); Leibniz Institut fuer Pflanzenbiochemie
Priority date: 2015-09-16
Filing date: 2016-09-15
Publication date: 2018-07-25
Also published as: WO2017046256A1; US20180228825A1; DE102015011780A1

Abstract

The invention relates to the use of a compound of formula (I) or a composition containing said compound as a glutaminyl cyclase (QC) inhibitor as well as to production methods.

Description

SULFOLIPIDS AS NEW GLUTAMI NYLCYCLASE INHIBITORS

Field of the invention

The invention relates to the use of a compound or compound containing the compound as

Glutaminyl cyclase (QC) inhibitor and method of preparation.

State of the art

The glutaminylcyclases (QCs) (EC 2.3.2.5) belong to the class of acyltransferases (EC 2.3.2). QC catalyzes the

intramolecular cyclization of N-terminal L-glutamine residues of peptides and proteins to pyroglutamic acid (pGlu)

Release of ammonia and the intramolecular cyclization of N-terminal glutamate to pyroglutamic acid under

Release of water. QC is at a variety of

involved physiological processes and it has been shown that QC pathophysiologically at different

Diseases is involved. Accordingly, QC sets

pharmacologically interesting target, since the inhibition of QC is considered as a promising possibility for the treatment of these QC-associated diseases / conditions.

Various QC inhibitors are already known.

For example, thiourea derivatives and imidazole-propyl-thioamides of Buchholz et al. (J Med Chem (52), 7069-7080; 2006) as effective QC inhibitors.

Further QC inhibitors are described, for example, in WO 2010/026212, WO 2011/029920; WO 2011/101433; WO 2011/107530; and where Described 2011/110613. QC inhibitors from natural

Sources are not known yet.

Sulfolipids are known as biologically active natural products. The bioactivities so far described for sulfolipids include: immunosuppressive effects, antiviral effects (e.g., against HIV), antineoplastic effects, a

inhibiting effect of the enzymatic activities of the DNA polymerases pol α and pol ß, the α-glucosidase and the

Caspase, anti-inflammatory activities (e.g., an effect in inflammatory skin diseases or diseases such as psoriasis in particular) and anti-proliferative effects on various human cancer cell lines, a prophylactic action against a Mycobacterium tuberculosum infection, and an anti-protozoal activity.

A common problem of inhibitors is their side effects, such as cytotoxicity. Also the increasing

Occurrence of incompatibilities with certain

Active ingredients or molecular components provides a great

Problem. Accordingly, there is a great need for inhibitors without cytotoxic effect.

Object of the present invention was to provide new QC inhibitors derived from natural sources, have no cytotoxic effects and are usually well tolerated.

Description of the invention

Glutaminyl cyclase (QC) inhibitor and method of preparation. For this purpose, the present invention provides a compound of the formula (I):

or a salt thereof, ready for use as a glutaminyl cyclase (QC) inhibitor, wherein

R ^{1 is} hydrogen, -C (= O) Ci-C ₂ 6alkyl or -C (= O) C ₂ -C ₂ 6alkenyl; and

R ^{2 is} a hydrogen atom, -C (= O) Ci-Czealkyl or -C (= 0) C ₂ - C26alkenyl.

The term "C ₁ -C ₂ 6 alkyl" as used herein means an alkyl group and denotes a saturated,

straight-chain or branched, optionally substituted,

Hydrocarbon group containing 1 to 26 carbon atoms

having. Representative "Ci-C26alkyl" radicals are, for example, the methyl, ethyl, propyl, butyl, pentyl (or valeryl), hexyl (or capro), heptyl (or Önanthyl-), octyl ( or caprylic), nonyl (or pelargonyl), decyl (or caprine), undecyl; Dodecyl (or lauryl), tridecyl, tetradecyl (or myristyl), pentadecyl, hexadecyl (or cetyl), heptadecyl, octadecyl (or stearyl); Nonadecyl, eicosyl, heneicosyl, doeicosyl, tricosyl, tetracosyl, pentacosyl, Hexacosyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl and the 2-methylhexyl group.

The term "-C 2 -C ₂₆ alkenyl" as used herein means an alkenyl group and denotes an at least partially unsaturated, straight-chain or branched, optionally substituted hydrocarbon group containing 2 to 26

Having carbon atoms. Representative "-C 2 -C ₆ alkenyl" radicals are, for example, the vinyl, allyl, 1-butenyl, 2-butenyl, - isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl , 2-methyl-2-butenyl, 2, 3-dimethyl-2-butenyl-ethenyl, hex-2-enyl, 9-hexadecenyl (or palmitoleyl), 9-octadecenyl (oleyl), II-octadecenyl, 9,12-octadecadienyl (or

Linoleyl), 6, 9, 12-octadecatrienyl (or linolenyl),

5, 8, 11, 14-eicosatetraenyl or 5, 8, 11, 14, 17-eicosapentaenyl. The alkenyl groups contain one or more

Double bond (s), preferably 1, 2, 3, 4 or 5

Double bond (s).

The term "optionally substituted" as used herein refers to groups in which one or more hydrogen atom (s) is represented by one or more fluorine, chlorine, bromine or iodine atom (s), OH, = 0, SH, SS, NH ₂ , NHNH, NO ₂ , -C (O) R ', -O C (O) R', - C (O) OR ', -C (O) NH ₂ , -C (O) NHR *, -C (O) N (R ') 2, -NHC (O) R', -S (O) ₂ R ', -S (O) R', -N3, -NH (R '), -N (R ') 2 or -CN is / are replaced, preferably by one or more fluorine or chlorine atom (s), OH, C (O) R', -O C (O) R ', or -C ( 0) 0R ', more preferably by one or more fluorine, or chlorine atom (s), or OH, wherein each R' is independently selected from unsubstituted C ₁ -C ₃ alkyl.

Depending on its structure, the compound of the formula (I) can be used in stereoisomeric forms (enantiomers, diastereomers) exist. The invention therefore includes the enantiomers or diastereomers as well as mixtures thereof. From such mixtures of enantiomers and / or diastereomers, the stereoisomerically uniform constituents can be isolated in a known manner, for example by means of column chromatography.

If the compound of the formula (I) can occur in tautomeric form, the present invention covers all tautomeric forms.

As salts are within the scope of the present invention

physiologically acceptable salts of the compound of the formula (I) are preferred. But are also included salts for

physiological applications themselves are not suitable, however, for example as a research tool, in cosmetic

Applications, or for the isolation or cleaning of the

Compound or other molecules can be used.

Physiologically acceptable salts of the compound of formula (I) may include acid addition salts of mineral acids, carboxylic acids and sulfonic acids, e.g. Salts of hydrochloric acid,

Hydrobromic acid, sulfuric acid, phosphoric acid,

Methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,

Benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid,

Trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fumaric acid, maleic acid and

Be benzoic acid. Further, physiologically acceptable salts of the compound of formula (I) may also include salts of conventional bases, such as, for example, alkali metal salts (e.g., sodium and potassium salts); Alkaline earth salts (e.g., calcium and

Magnesium salts); and ammonium salts derived from ammonia or organic amines having 1 to 16 C atoms, such as

for example, ethylamine, diethylamine, triethylamine, Ethyldiisopropylamine, monoethanolamine, diethanolamine,

Triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine, dibenzylamine, N-ethylmorpholine, arginine, lysine, ethylenediamine and N-ethylpiperidine.

Preferred is a compound of formula (I) or a salt thereof, for use as a QC inhibitor, wherein

R ^{1 is} hydrogen, -C (= O) Ci 3 -C ₂ oalkyl or -C (= O) Ci 3 -C ₂ alkenyl; and

R ^{2 is} a hydrogen atom, -C (= O) Ci3-C ₂ ialkyl or -C (= 0) Ci ₃ - C2ialkenyl.

Further preferred is a compound of the formula (I) or a salt thereof, for use as a QC inhibitor, wherein

R ^{1 is} a hydrogen atom, a palmitoyl, palmitoleoyl, oleoyl, linoleoyl, linolenoyl, eicosatetraenoyl or eicosapentaenoyl group; and

R ^{2 is} a hydrogen atom, a palmitoyl, palmitoleoyl, oleoyl, linoleoyl, linolenoyl, eicosatetraenoyl or eicosapentaenoyl group.

According to the invention, the compound of the formula (I) or a salt thereof may be selected for use as QC inhibitor from the group: 1,2-di-O-palmitoyl -3-0- (β'-deoxy-6 '- sulfo-D-glycopyranosyl, 1-0-palmitoyl-2-0-linolenyl-3-0- (6'-deoxy-6 'sulfo-D-glycopyranosyl, 10-linolyl-2-0-palmitoyl-3-0 - (6 ^'-deoxy-6' -sulfo-D-glycopyranosyl, 1-0- palmitoyl-3-0 (6 '-sulpho- -quinovopyranosyl) -glycerol and 1-0- (6-deoxy-6-sulphoglucopyranosyl) glycerol. Also according to the invention is the use of the compound according to the formula (I) or a salt thereof, as QC inhibitor, wherein R ^{1 is} a hydrogen atom, -C (= O) Ci-C26 alkyl; or -C (= O) C2-C26alkenyl; and R ^{2 is} a hydrogen atom, -C (= O) Ci-C26alkyl; or -C (= O) C 2 -C ₂₆ alkenyl. In this case, preferably a

Compound of the formula (I) or a salt thereof, wherein R ¹ is a hydrogen atom, -C (= 0) Cl 3-C ₂ Oalkyl, or -C (= 0) Ci3- C2ialkenyl; in particular a hydrogen atom, a palmitoyl, palmitoleoyl, oleoyl, linoleoyl, linolenoyl,

Eicosatetraenoyl, or an eicosapentaenoyl group; and R ^{2 is} a hydrogen atom, -C (= O) C 13 -C 20 -alkyl, or -C (= O) C 13 -C 20 -alkenyl; in particular a hydrogen atom, a palmitoyl, palmitoleoyl, oleoyl, linoleoyl, linolenoyl,

Eicosatetraenoyl, or an eicosapentaenoyl group; used as QC inhibitor. Specifically, l, 2-di-0- palmitoyl -3-0- (6 ^{^'-deoxy-6'} -sulfo-D-glycopyranosyl, 1-0- palmitoyl-2-0-1inoleny1-3-0- (6 ^' -deoxy-6 ^{'-sulfo-D-glycopyranosyl,} l-0-linolyl-2-0-palmitoyl-3-0- ^(6' -deoxy-6 ^'- sulfo-D-glycopyranosyl, lO-palmitoyl-3-0 (6 ^* -sulpho-a-quinovopyranosyl) -glycerol, or 1-0- (6-deoxy-6-sulphoglucopyranosyl) -glycerol may be used as the QC inhibitor The use may be in vitro or in vivo

Use in vivo, for example, refers to the compound of formula (I) or a salt thereof for use in the prophylaxis or treatment of a QC-associated disease. However, when used in vivo, methods for the surgical or therapeutic treatment of the

human or animal body and

Diagnostic procedures that are performed on the human or animal body are excluded. On the other hand, it includes the use of the compound of the formula (I) or a salt thereof for the manufacture of a medicament for a Use for the prophylaxis or treatment of a QC-associated disease.

Also, a composition comprising any of the aforementioned compounds of the formula (I) or a salt thereof for use as a QC inhibitor is the subject of the present invention. Preferably, the composition comprising a compound of the formula (I) or a salt

the same, includes, being an extract of a microalgae. For example, microalgae of the species are suitable for obtaining the extract: Scenedesmus sp., E.g. B. Scenedesmus producto capitatus, Scenedesmus rubescens, Scenedesmus acuminatus

Scenedesmus vacuolatus and Scenedesmus pectinatus as well

Tetradesmus wisconsinensis; Eustigmatos sp., Z. B. Eustigmatos magnus, Cyclotella sp., Odontella sp., Synechocystis s. , Spirulina sp., E.g. Spirulina maxima and Spirulina platensis; Prochlorococcus sp., Synechococcus sp., Trichodesmium erythraeum, Arthrospira platensis, Scytonema hofmanni, Nostoc punctiforme, Microcystis aeruginosa, Stephanodiscus sp. , Skeletonema costatum, Phaeodactylum tricornutum, Heterosigma carterae, Nannochloropsis sp., E.g. Nannochloropsis oculata, Porphyridium sp., E.g. Porphyridium cruentum and Porphyridium purpureum; Chilomonas paramecium, Bumilleriopsis filiformis, Emiliana huxleyi, Chlorella sp. , z. Chlorella vulgaris, Chlorella sorokiniana, Chlorella variegate, Chlorella

kessleri, Chlorella minutissima and Muriella zofingiensis;

Chloroidium saccharophilum, Parietochloris incisa, Prototheca portoricensis, Dictyochloris fragrans, Chlamydomonas

reinhardtii, Dunaliella parva, Tetraselmis chuii and Deasonia sp. e.g. Deasonia punctata and Deasonia granata; and particularly preferred examples of the microalgae extract include

Extracts from a species that is selected from Scenedesmus acuminatus, Scenedesmus pectinatus, Chlorella vulgaris and Spirulina platensis.

The composition or microalgae extract for use as a QC inhibitor may further contain a solvent. The solvent may for example be selected from:

Methanol, ethanol, isopropanol, water, chloroform,

Ethyl acetate, dichloromethane, acetone, diethyl ether, hexane, and mixtures thereof; preferably methanol.

The composition or microalgae extract for use as QC inhibitor may further comprise two, three or more than three compounds according to formula (I) or salt thereof

include.

The composition or microalgae extract for use as a QC inhibitor may also be another therapeutic agent

Agent which is selected from the group comprising

Acetylcholinesterase inhibitors (e.g., Aricept®, Exelon®

and Reminyl®), NMDA receptor antagonists (eg, memantine), neuroprotectors, anti-Parkinson's drugs, antidepressants, anxiolytic drugs, antipsychotic drugs, drugs for multiple sclerosis, ACE inhibitors (eg, Enap, Enalapril, Capoten, Renitec, Prestarium (Berlipril , Diroton, Capoten, Quadropril, Monopril, Renitec, Prestarium, Noliprel-Forte, Enap-N), Diuretics (eg Furosemide,

Veroshpiron, Hypothiazide, Arifon Retard, Indapamide,

Hypothiazide, Diuver, Indap, Indapamide), β-receptor blockers, nitrates, cardiac glycosides, calcium antagonists (eg (normodipine, Cordäflex, Amlovas, amlodipine, Amlovas, amlotope, cardilopin, Corda lex, lipid-lowering drugs (eg (Vasilip, Liprimar, Liptonorm, Simvahexal, Simvastol, Simvacard, Simgal, Tulip),

Inhibitors of aggregation (eg (acetylsalicylic acid, CardiASK, Cardiomagnyl, Thrombo ASS), antihypoxemic, anticoagulant (eg thrombomodulin, warfarin), cytostatics and antibiotics is selected.

The composition or microalgae extract for use as a QC inhibitor may also contain a carrier,

preferably a pharmacologically inert, inorganic or organic excipient substance, e.g. mannitol,

Lactose, sucrose, glucose, gelatin, malt, silica gel, starch or derivatives thereof, talc, stearic acid or theirs

Salts, dry skimmed milk, vegetable oils, petroleum,

animal or synthetic oils, waxes, fats, polyols.

Furthermore, the composition or the microalgae extract for use as QC inhibitor also additives to

Preservation, stabilization, emulsifiers, sweeteners,

Flavorings, salts for changing the osmotic pressure, buffers, coating additives and antioxidants.

Moreover, the invention also relates to a process for the preparation of a compound of formula (I) according to the

above, or a salt thereof, comprising:

(a) culturing at least one microalgae;

(b) recovery of algal biomass and digestion of the algae biomass

Algal cells;

(c) Extraction of digested algal cells from step (b) with methanol comprising the suspension of the

digested algae cells from step (b) in methanol, stirring the suspension, and separating the liquid

Methanol extract phase from the cell mass;

(dl) isolating a compound of formula (I), or a salt thereof, from that obtained in step (c)

Extraction product. In the process for producing a compound of the formula (I), as microalgae, any microalgae which produces compounds of the formula (I) as a secondary metabolite or secondary metabolites, or a mixture of such microalgae, for example, a microalgae of Phyla Bacillariophyta, Chlorophyta, Ochrophyta and cyanobacteria, and preferably the species: Scenedesmus sp. , z. B.

Scenedesmus producto-capitatus, Scenedesmus rubescens,

Scenedesmus acuminatus Scenedesmus vacuolatus and Scenedesmus pectinatus as well as Tetradesmus wisconsinensis; Eustigmatos sp. , z. B. Eustigmatos magnus, Cyclotella sp. , Odontella sp.,

Synechocystis sp., Spirulina sp., E.g. Spirulina maxima and Spirulina platensis; Prochlorococcus sp., Synechococcus sp., Trichodesmium erythraeum, Arthrospira platensis, Scytonema hofmanni, Nostoc punctiforme, Microcystis aeruginosa,

Stephanodiscus sp., Skeletonema costatum, Phaeodactylum tricornutum, Heterosigma carterae, Nannochloropsis sp., E.g. Nannochloropsis oculata, Porphyridium sp., E.g. Porphyridium cruentum and Porphyridium purpureum; Chilomonas paramecium, Bumilleriopsis filiformes, Emiliana huxleyi, Chlorella sp. , z. Chlorella vulgaris, Chlorella sorokiniana, Chlorella variegate, Chlorella kessleri, Chlorella minutissima and

Muriella zofingiensis; Chloroidium saccharophilum,

Parietochloris incisa, Prototheca portoricensis, Dictyochloris fragrans, Chlamydomonas reinhardtii, Dunaliella parva,

Tetraselmis chuii and Deasonia sp. e.g. Deasonia punctata and Deasonia granata; and most preferably scenedesmus

acuminatus, Scenedesmus pectinatus, Chlorella vulgaris and Spirulina platensis.

The cultivation of the microalgae (s) according to step (a) can be carried out under known conditions, for example in one Photobioreactor, pH 5-8 (preferably pH 7), and a

Temperature of 10-45 ° C, preferably 20-40 ° C, stronger

preferably 23-35 ° C, and more preferably 25-31 ° C. When

Medium suitable for the cultivation of the microalgae are known media, such as, for example, Setlik medium (KNO3 (2020.00 mg / l), H2PO4 (340.00 mg / l), MgSO ₄ .7H ₂ O (990.00 mg / l)). L), Fe-EDTA (18.50 mg / L), Ca (N0 ₃₎ 2 * 4 H ₂ 0 (10, 00 mg / L), H3BO3 (3.09 mg / L), MnS0 ₄ * 4 H ₂ 0 (1.20 mg / L), CoSO ₄ (1.40 mg / L), CuSO ₄ * 5 H ₂ O (1.24 mg / L), ZnSO ₄ (1, 43 mg / L), ( ΝΗ) ₆ Μο ₇ θ 2 ₄ * 4 H ₂ 0 (1.84 mg / L)).

Recovery of the algal biomass in step (b) may be accomplished by any one of numerous known physical or chemical methods for separating biomass from a

Culture medium, for example, be obtained by centrifuging off the medium for cultivation and / or lyophilization. The digestion of the algal cells in step (b) may be conveniently carried out using one of the

Numerous known physical or chemical methods for the cell disruption of microorganisms take place, e.g. in Ullmann's Encyclopedia of Industrial Chemistry, 2003, Vol. 5, Biochemical Separations, Table 3. One possibility is, for example, grinding, e.g. with mortar and pestle using sea sand (ratio

Seaweed biomass: sea sand 1: 2) and solvent (e.g., water). The cell disruption may be at any suitable

Temperature, e.g. Room temperature, done.

For the extraction of the digested algae cells with

Methanol according to step (c) is preferably a solid-liquid extraction is applied, wherein the extraction is carried out with stirring and each 100 ml of methanol / l g of the

disrupted algae cells are used. The separation the liquid methanol extract phase from the biomass may be by centrifugation, eg 1000-5000 rpm, preferably 1500-3000 rpm, more preferably 2000-2500 rpm, and especially at about 2000 rpm. Preferably, the biomass can be extracted three times each with methanol. The liquid methanol extract phases of the extractions of the biomass can be combined. The combined liquid methanol extract phases can be concentrated to dryness in vacuo, for example using a vacuum rotary evaporator.

The isolation of a compound of formula (I) from the extraction product obtained in step (d) in step (d1) can be carried out by one of the numerous known physical or chemical processes for the isolation of compounds from an extract, for example

Solid Phase Extraction (SPE). There are many for the SPE

Sorbents known, for example, an aminopropyl-modified silica gel phase can be used. When

Solvents for elution can be known in the SPE

suitable solvents are used, such as, for example, methanol, ethanol, isopropanol, water, chloroform,

Ethyl acetate, dichloromethane, acetone, diethyl ether, hexane, and mixtures thereof; preferably methanol. To remove non-lipid contaminants, the product of the SPE can be further purified, for example by means of

Chromatography or other known purification methods; Preferably, the product of SPE can be prepared according to the method of Folch et al. (J. Biol. Chem., 226, 497-509 (1957)).

The invention also provides a process for the production of an extract from a microalgae, which has a

Compound of the formula (I) according to the above statements, or a salt thereof, the process comprising:

(a) cultivation of the microalgae;

(b) recovery of algal biomass and digestion of the algae biomass

Algal cells;

(c) Extraction of digested algal cells from step

(b) with methanol comprising the suspension of

Methanol extract phase from the cell mass;

(d2) Removal of chlorophyll from the extraction product obtained in step (c).

The steps (a), (b) and (c) of the process for preparing an extract from a microalgae can be carried out analogously to the steps (a), (b) and (c) of the above-described

Process for the preparation of a compound of formula (I) are carried out.

The removal of chlorophyll from the recovered

Extraction product can be prepared using a known

Procedure, for example by

Ion exchange chromatography, e.g. under use

commercially available columns for the exchange of cations. As solvents for ion exchange chromatography, known suitable solvents can be used,

for example, methanol. For further purification of the extract, a solid phase extraction can be carried out. Numerous sorbents are known for solid phase extraction, for example, an octadecyl-modified silica gel phase can be used. As the solvent for elution, suitable solvents known in solid phase extraction may be used, preferably methanol. Brief description of the illustrations

Figure 1: Schematic representation of the method according to the invention for the preparation of a compound of formula (I) having QC-inhibiting activity or for producing an extract from a microalgae containing at least one compound of formula (I) with QC-inhibiting activity.

Figure 2: QC-inhibiting effect of compositions of the invention. Concentration-activity diagram for the activity of extracts of Eustig atos magnus, Tetradesmus wisconsinensis _f Scenedesmus pectinatus, Scenedesmus

acuminatus, Scenedesmus rubescens, and Scenedesmus producto capitatus. As can be seen from Fig. 2, the extracts inhibiting microalgae in the exponential

Growth phase were obtained, QC stronger than the extracts, which were obtained from microalgae in the stationary growth phase. Furthermore, it can be seen from Fig. 2 that the QC inhibition is specific and concentration-dependent.

FIG. 3: Base-peak chromatogram and MS ¹ mass spectrum of the compounds of the formula (I) according to the invention with QC-inhibiting action. These

Compounds are also in the he indungsgemäßen

Contain compositions or microalgae extracts.

The MS ¹ mass spectrum shows a molecular ion [MH] ^~ at m / z 555 for the retention time of 6.9 min, which can be assigned to a SQMG. The peak at the retention time of 9.9 - 10 min shows in the MS ¹ mass spectrum the molecular ions [MH] - at m / z 794 and 815. It is thus the peak the SQDG. As the main compounds are thus the sulfolipids 1,2-di-O-palmitoyl -3-0- (6 ^'-deoxy-6' -sulfo-D-glycopyranosyl) with [MH] ^~ at m / z 793 and l-0- palmitoyl-2-0-linolenyl-3-0- (6 ^'-deoxy-6' -sulfo-D-glycopyranosyl) included with [MH] ^"at m / z 815th Furthermore, it could as by connecting the SQMG 1-0- palmitoyl -3-0 (6 ^* -sulpho -quinovopyranosyl) -glycerol

be detected.

Hereinafter, the present invention will be described with reference to FIG

Examples explained.

Examples

Obtaining the AIgenbiomasse

Microalgae of the species Eustigmatos magnus, Tetradesmus wisconsinensis, Scenedesmus pectinatus, Scenedesmus

acuminatus, Scenedesmus rubescens, and Scenedesmus producto capitatus were cultivated to obtain sufficient biomass in a tubular photobioreactor PBR 100 GS / PL from IGV GmbH with a reactor volume of 100 L. The

Pump speed, temperature and pH were controlled by means of a Biostat control unit from Braun, with the following parameters: temperature 28 ° C +/- 3 ° C; pH = 7, pump speed 1750 rpm were set. The reactor system has a flow cell in which both a

Temperature and pH probe and a probe for measuring the optical density (OD) and a p02 probe were integrated. An inline measurement of the following parameters was carried out: temperature, pH value, OD and pump speed. These were recorded using the MFSC win software over the entire cultivation period. Cultures in the 100 L PBR were also carried out at a temperature of 28 ° C +/- 3 ° C. It was with a light intensity of 60 μπιο1 / ιτι ² * 3 at OD <20 and a Light intensity of 150 μπιο1 / Γη ² * 3 of the light module at OD> 20 cultivated. The cultivation was carried out in a modified fed-batch process.

For the cultivation of the microalgae Setlik medium (Table 1) was used.

Tab. 1: Composition of the Setlik medium

Nutrient salt Amount used [mg / L]

KN0 ₃ 2020, 00

KH2PO4 340, 00

MgS0 * 7H ₂ 0 990, 00

Fe-EDTA 18.50

Ca (NO ₃ ) ₂ * 4 H ₂ O 10, 00

H3BO3 3.09

MnS0 ₄ * 4H ₂ 0 1,20

C0SO4 1.40

CuSO ₄ * 5H ₂ O 1.24

ZnS0 ₄ 1.43

(NH ₄ ) 6M07O24 * 4H ₂ O 1.84

The nutrient salts were dissolved in 1 L of distilled water and autoclaved at 121 ° C for 20 min.

The biomasses of the exponential and stationary growth phase of the mentioned microalgae were investigated.

Extraction of algae biomass - methanol extract

The lyophilized algal biomasses of the exponential and stationary growth phases of each microalgae studied were digested with sea-sand in a 1: 2 ratio with mortar and pestle using solvent. Of the

Cell disruption was carried out at RT for 10 min. For extraction, a one-step extraction method based on solid-liquid extraction was used. In each case, 10 g algae biomass after cell disruption with the

Solvent methanol extracted. The extraction was carried out in three steps with 100 mL each of Lsm. / 1 g BM with stirring on the magnetic stirrer at RT for 1 h. The separation of the liquid extract phase from the biomass was carried out by means of

Centrifugation at 2000 rpm and RT, so that the biomass in the 2nd and 3rd extraction step could be extracted analogously. The extracts from the three extraction steps were combined and concentrated to absolute dryness using Büchi vacuum rotary evaporator.

Chlorophyll elimination

The elimination of the dye chlorophyll was carried out using CHROMABOND ^® SA (SCX) of the company

Macherey & Nagel from the methanol extracts. The SA cartridges were coated with 0.5 g of a2SC> 4 and conditioned with 1 v / v dichloromethane / acetone (3: 1, v / v). Subsequently, the task of the methanol extract was carried out in each case. The

chlorophyllf fluat was collected in glass test tubes.

Recovery microalgae extract with compound of formula (I)

The chlorophyll-free extracts were then transferred over

Chromabond® C18ec cartridge (500 mg / 3 mL or 1000 mg / 6 mL, Macherey & Nagel). Elution was carried out with 100% MeOH.

QC inhibitory effect of microalgae extracts

The chlorophyll-free microalgae extracts purified on Chromabond® C18ec cartridges were tested for their QC inhibitory activity was investigated. For this purpose, known detection methods of QC catalysis can be used.

For example, the Schilling et al. (Anal. Biochem., 303, 49-56 (2002); Biol. Chem. 384, 1583-1592 (2003)) developed a continuous testing method based on the conversion of free ammonia by the cyclization of the N-terminal glutamine residues (loc. cit., 2003) and, on the other hand, the pyroglutamate peptide formed is reacted with pyroglutaminylaminopeptidase (pGAP) (loc.cit., 2002), see reaction scheme 1 below. In this method, fluorogenic groups are cleaved off by the auxiliary enzyme pGAP, which leads to an increase in fluorescence.

Reaction Scheme 1

The QC-inhibiting activity of the invention

Chlorophyll-free via Chromabond® C18ec cartridges

Purified microalgae extracts were treated with the above

Test method of Schilling et al. in two concentrations

(2 mg / mL and 0.2 mg / mL). The assay was performed in 96-well transparent microtiter plates (NUNC, Costar Corning Incorporated, Acton, MA, USA) as a triplicate. The measurements showed a total volume of 250 pL per well. These consisted of 100 μΐ, 0.25 mM substrate, 50 pL 0.2 mg / mL sample and 25 pL auxiliary enzymes pGAP together. Since QC activity is pH dependent, 50 μl Tris buffer (0.1 M, pH 8) was added to each assay. The start of the reaction was carried out after 10 minutes incubation at 30 ° C by the addition of 25 pL QC. The measurements or the

Increase in free AMC has been continuous over one

Period of 12 min (~ 27 cycles) at a temperature of 30 ° C on a FluoStar Multiplate Reader from BMG Labtech measured.

The results of the measurement are shown in Fig. 2. How out

Fig. 2 can be seen, the inventive inhibited

Microalgae extracts the activity of QC specific and concentration dependent.

Identification of QC inhibitors in the microalgae extratens

To identify the QC inhibitors, the microalgae extracts according to the invention were analyzed by mass spectrometry.

The mass spectrometric analyzes were carried out using an API-150EX mass spectrometer with a "Turbo Ion Spray" ion source from Applied Biosystems.The sample injection (10 μΐι) was carried out via direct inlet into a continuous flow of a MeOH / H 2 O mixture (6: 4; v) at a flow rate of 400 pL / min.

Furthermore, the microalgae extracts according to the invention were purified by means of UPLC-ESI ion trap MS ⁿ on a Waters Acquity UPLC system using an RP18 column (Acquity UPLC HSS T3 1.8 μπι, 1x100mm, Waters) examined. The samples were analyzed by negative ionization using electrospray ionization (ESI) at a spray voltage of 4 kV, a capillary temperature of 275 ° C and a capillary voltage of 27V using nitrogen as a shielding gas (flow rate 35-40 arb. Units).

The mass spectrometric analyzes of the extracts investigated showed that the compounds of the formula (I) are responsible for the QC-inhibiting action. In the mass spectrometry-identified compounds it was the SQDG ^'s 1,2-di-O-palmitoyl -3-0- (6' -deoxy-6 ^'- sulfo-D-glycopyranosyl with [MH] ^~ at m / z 793, 1-O-palmitoyl 2-0-linolenyl-3-0- (6 ^'-deoxy-6' -sulfo-D-glycopyranosyl with [M- H] - at m / z 815 and lO-linolyl-2 -0- palmitoyl-3-Ο- (6 '-deoxy- ^6' -sulfo-D-glycopyranosyl with [MH] - at m / z 817 and around the SQMG lO-palmitoyl-3-0 (6 ^* -sulpho- quinovopyranosyl) glycerol with [MH] ^" at m / z 555 and 1-0- (6-deoxy-6-sulpho-glucopyranosyl) -glycerol with [MH] - at m / z 317.

Recovery of compounds of formula (I)

To obtain the QC-inhibiting compounds from the methanol extracts obtained in the above-described extraction from the algal biomass, a solid-phase extraction followed by "folch wash" was used.

For the solid phase extraction of methanol extracts, aminopropyl-modified silica gel cartridges (NH2 cartridge) from Macherey & Nagel (3 mL / 500 mg or 6 mL / 1000 mg) were used. The conditioning of the NH2 cartridges was carried out in several steps using 2 mL MeOH, 2 mL dist. H ₂ 0, 4 mL 0.1M HCl (for 1h on column), 2 mL dist. H ₂ 0, 2 mL MeOH, 2 mL DCM / I isopropanol / MeOH (15:30:50 v / v / v). Each sample of NH 2 was sampled with 20 mg of methanolic extract dissolved in DCM / MeOH (1: 1, v / v). When 6 mL / 1000 mg NH2 cartridges were used, the volumes of solvents used and the sample applied were doubled.

The elution was done in 2 steps. In step 1, the uncharged substance was first eluted with 9 ml DCM / isopropanol / MeOH 15:30:50 (v / v / v). In step 2, the compounds according to formula (I) were mixed with 5 mL DCM / ACN / isoprop / MeOH / 0.1M NH ₄ Ac (10: 10: 10: 50: 15; v / v / v / v / v). eluted; Fraction 2.

The fraction 2 was removed by means of a vacuum rotary evaporator, the solvent and then with the addition of DCM / MeOH / 0.1 NaAc buffer pH = 4.0 (8: 4: 3, v / v / v) by vigorous shaking for several minutes back , After overnight incubation at 4 ° C, the compounds of formula (I) accumulated in the lower lipophilic phase. The lipophilic phase with the compounds of formula (I) was removed with a Pasteur pipette, transferred to a round bottom flask and concentrated to dryness by means of a vacuum rotary evaporator.

The yields of the compounds of the formula (I) varied depending on the biomass used (algae species and growth phase) between 3 - 26%. For example, from 25 mg of the methanolic extract from Scenedesmus acuminatus (stationary phase) compounds according to formula (I) could be obtained with a yield of 26%, ie, it could be isolated 6.5 mg. Identification of the compounds obtained

For identification, the isolated compounds were analyzed by mass spectrometry.

The mass spectrometric investigations of the isolated compounds were carried out as in the microalgae extracts according to the invention by means of ESI-API-MS and UPLC-MS in the negative ion mode. The base-peak chromatogram and the associated MS ¹ spectrum are shown in Fig. 3 with the peaks characteristic of compounds according to formula (I) at 9, 9 - 10.0 min and at 6.9 min.

The isolated compounds are compounds according to formula (I), namely the SQDG- sulfolipids 1,2-di-O-palmitoyl -3-0- (6 ^'-deoxy-6' -sulfo-D-glycopyranosyl ) with [MH] - for m / z 793 and lO-palmitoyl-2-Ο-linolenyl-3-Ο- (6'-deoxy-6'-sulfo-D-glycopyranosyl) with [MH] - at m / z 815, as well as the SQMG sulfolipid 10-palmitoyl-3-0 (6 "-sulpho-a-quinovopyranosyl) -glycerol with [MH] ^" at m / z 555.

QC-inhibiting effect

To confirm that sulfolipids isolated from the microalgae, Scenedesmus acuminatus, inhibit QC, they were assayed in two concentrations (0.25 mg / mL and 0.025 mg / mL, respectively) by the Schilling et al. examined. It was found that the sulfolipids isolated from the microalgae inhibit QC with 81% (0.25 mg / mL) and 76% (0.025 mg / mL), respectively. As a positive control for the QC-inhibiting effect of the compounds identified as activity-relevant according to the formula (I), a SQDG standard from the company Lipid Products in the concentrations 0.25 mg / mL and 0.025 mg / mL in the test method of Schilling et al. examined. The SQDG standard inhibited QC by 77% (0.25 mg / mL) or 76%

(0.025 mg / mL). The IC ₅ o value determined for the SQDG standard was 10.9 μΜ, which is even below the range

(greater effectiveness) of the known from US 7,304,086 B2 QC inhibitors with IC 50 values of 0.22 μΜ - 14 μΜ.

Since the SQDG standard as the reference substance for the compounds according to the formula (I) inhibits the QC, it is clearly demonstrated that compounds according to the formula (I) are QC inhibitors.

Claims

Godparent sayings

1. Use of a compound of the formula

, or a salt thereof, as a glutaminyl cyclase (QC) inhibitor, wherein

R ^{1 is} a hydrogen atom, -C (= O) Ci-C ₂ 6alkyl or -C (= 0) C ₂ -

C ₂₆ alkenyl; and

R ^{2 is} a hydrogen atom, -C (= O) Ci-C ₂₆ alkyl or -C (= 0) C ₂ - C26alkenyl.

2. The use of the compound of formula (I) or a salt thereof according to claim 1, wherein

R ^{1 is} a hydrogen atom, -C (= O) Ci ₃ -C ₂ oalkyl or -C (= O) Ci ₃ - C2ialkenyl; and

R ^{2 is} a hydrogen atom, -C (= 0) Ci3-C ₂ ialkyl or -C (= 0) Ci ₃ - C ₂ ialkenyl.

3. The use of the compound of formula (I) or a salt thereof according to claim 1 or 2, wherein R ^{1 is} a hydrogen atom, a palmitoyl, palmitoleoyl, oleoyl, linoleoyl, linolenoyl, eicosatetraenoyl or eicosapentaenoyl group; and

4. The use of the compound of the formula (I) or a salt thereof according to any one of claims 1 to 3, wherein the compound is selected from the group: l, 2-di-0-palmitoyl -3-0- (6 ^' - deoxy-6 ^{'-sulfo-D-glycopyranosyl,} lO-palmitoyl-2-0- linolenyl-3-0- (6' -deoxy-6 ^{'-sulfo-D-glycopyranosyl,} 1-0 linol l-2-0 -palmitoyl-3-0- (6 ^'-deoxy-6' -sulfo-D-glycopyranosyl, 1-0 palmito 1-3-0 (6 "-sulpho-a-quinovopyranosyl) -glycerol and 1-0- ( 6-deoxy-6-sulphoglucopyranosyl) glycerol.

5. Use of a composition comprising a

Compound of the formula (I):

or a salt thereof, as a QC inhibitor, wherein

R ¹ is a hydrogen atom, -C (= 0) _Ci-C2 6alkyl or -C (= 0) C ₂ - C26alke yl; and R ^{2 is} a hydrogen atom, -C (= O) Ci-C ₂ 6alkyl or -C (= 0) C ₂ -C ₂ alkenyl.

The use of the composition of claim 5, wherein the composition is an extract of a microalgae.

The use of the composition of claim 5 or 6, wherein the composition further contains a solvent.

8. The use of the composition according to claim 7, wherein the solvent is selected from: methanol, ethanol,

Isopropanol, water, chloroform, ethyl acetate, dichloromethane, acetone, diethyl ether, hexane and mixtures thereof.

9. The use of the composition according to one of

Claims 5 to 8, wherein the composition comprises two, three or more than three compounds according to formula (I) or salts thereof.

10. The use of the composition according to one of

Claims 5 to 9, wherein the composition is another therapeutic agent selected from the group comprising acetylcholinesterase inhibitors, NMDA receptor antagonists,

Neuroprotectors, antiparkinsonian drugs, antidepressants, anxiolytic drugs, antipsychotic drugs, antidiabetic drugs, ACE inhibitors,

Diuretics, ß-receptor blockers, nitrates, cardiac glycosides,

Calcium antagonists, lipid lowering agents, anti-aggregation agents,

Antihypoxema, anticoagulants, cytostatics and antibiotics is selected.

A process for producing a compound of the formula (I) or a salt thereof described in claims 1 to 4, comprising:

(a) culturing at least one microalgae;

(b) recovery of algal biomass and digestion of the algae biomass

Algal cells;

Methanol extract phase from the cell mass;

Extraction product.

12. A process for producing an extract from a

Microalgae containing at least one compound of the formula (I) described in claims 1 to 4 or a salt

the same, the method comprising:

(a) cultivation of the microalgae;

(b) recovery of algal biomass and digestion of the algae biomass

Algal cells;

Methanol extract phase from the cell mass;

(d2) removal of chlorophyll from that in step (c)

recovered extraction product.