WO1997031901A1

WO1997031901A1 - Hydroxyhexahydropyridazines

Info

Publication number: WO1997031901A1
Application number: PCT/DK1997/000090
Authority: WO
Inventors: Mikael Bols
Original assignee: Mikael Bols
Priority date: 1996-02-29
Filing date: 1997-02-28
Publication date: 1997-09-04
Also published as: AU2090897A

Abstract

The present invention relates to hydroxyhexahydropyridazines of general formula (I). These compounds are found to be inhibitors of glycoside cleaving enzymes and it is therefore envisaged that such compound may be used for treatment of or for controlling diabetes, cancer, or AIDS caused by human immunodeficiency virus. Furthermore, such compounds may be used in plant protection. The mechanism for the action of the compound is that they, in protonated form, resemble the transition state of natural substrates for the glucosidases in question.

Description

HYDROXYHEXAHYD OPYRIDAZINES

FIELD OF THE INVENTION

The invention relates to a new group of chemical compounds, hydroxyhexahydropyridazines (formula I herein), and their biological application and use. It has been found that the compounds according to the invention inhibit glycoside cleaving enzymes such as glycosidases and glycosyl phosphorylases. The invention also relates to such compounds for use as a medicament, e.g. in the treatment of the diseases: diabetes, cancer and ALDS, as well as the use of these compounds in crop protection.

BACKGROUND OF THE INVENTION

In the group of enzymes that convert carbohydrates, some of the most important are those which hydrolyse or phosphorylate glycosidic linkages to mono- and oligosaccharides or saccharide phosphates, respectively. These enzymes, which are called glycosidases or glycoside phosphorylases, are important for all growth and development of living organisms. They participate in a wide range of important biological events such as digestion of carbohydrate- foodstuffs, glycoprotein modification in eukariotes, and degradation of polysaccharides and glycoconjugates.

Chemical compounds that inhibit glycoside-cleaving enzymes can be used to block certain biochemical processes, and consequently such compounds may be applied for treatment of diseases and crop protection.

Diabetes may, e.g., be treated by controlling or reducing the addition of glucose to the blood. This can either be done by inhibition of the α-glucosidase catalysed degradation of foodstuff- carbohydrates to glucose or by inhibition of the glycogen phosphorylase catalysed degradation of the carbohydrate storage.

Cellulose cannot be degraded to glucose by humans, but can on the other hand be degraded by micro-organisms, fungi and insects (crop pests), living of plant material. Cellulase and β- glucosidase inhibitors may, thus, block the crop pests metabolism in a for humans harmless way, and thus be applied for crop protection

In recent years, the interest for development and synthesis of inhibitors of glycosidases have grown tremendously The most effective glycosidase inhibitors are polyhydroxylated piperidines such as nojirimycin and isofagomine, 5-amino-5-deoxy-gluconolactam, poly¬ hydroxylated pyrrolidines, the indolizidine alkaloids swainsonin and castanospermin, the polyhydroxylated aminocyclopentanes and the polyhydroxylated aminocyclohexene acarbose

As mentioned above, inhibitors of α-glucosidases can be used for treatment of diabetes The strongest known α-glucosidase inhibitors are acarbose and castanospermin Acarbose is used as a pharmaceutical for treatment of diabetes, but its effect is limited, and it has side effects There is thus a need for alternative α-glucosidase inhibitors

As mentioned above, diabetes may alternatively be treated with a glycogen phosphoryiase inhibitor A known strong glycogen phosphoryiase inhibitor is isofagomine (WO 95/24391) Isofagomine is however difficult to prepare There is thus a need for more readily available strong glycogen phosphoryiase inhibitors

Also as mentioned above, inhibitors of β-glucosidase may be used for crop protection and the strongest known β-glucosidase inhibitor is isofagomine (Jespersen et al Angew. Chem. Int. Ed. 33 (1994) 1778-9 ) Isofagomine is however difficult to prepare and thus unsuitable for crop protection There is thus a need for more readily available strong β-glucosidase inhibitors

Furthermore, inhibitors of α-mannosidase, α- and β-fucosidase and α-glucosidase can potentially be used in the treatment of AIDS There is thus a need for new strong α-manno¬ sidase, α- and β-fucosidase and α-glucosidase inhibitors

Furthermore, the inhibitors of β-glucuronidase and other glycosidases can potentially be applied to reducing cancer metastases There is thus a need for new strong glycosidase inhibitors Thus, the aim of the present invention is to provide novel glycosidase inhibitors. The inventor has surprisingly found that compound of the hydroxyhexahydropyridazine type possess glycosidase inhibitor properties as well glycogen phosphoryiase inhibitor properties.

Some other hydroxyhexahydropyridazines are known in the literature. Paulsen and Steinert {Chem. Ber. 1970 103 1834-45) made 3-methyl-di- and trihydroxyhexahydropyridazines more specifically (3R, 4S, 5R, 6S)-6-methyl-3,4,5-trihydroxy-l,2-diazinane, (3S, 4S, 5R, 6S)-6- methyl-3,4,5-trihydroxy-l,2-diazinane, (3R, 4S, 5R, 6S)-l,6-dimethyl-3,4,5-trihydroxy-l,2- diazinane, (3S, 4S, 5R, 6S)-l,6-dimethyI-3,4,5-trihydroxy-l,2-diazinane, (3S, 4R, 5R)-3- methyl-4,5-dihydroxy-l,2-diazinane, (3S, 4R, 5R)-l-acetyl-3-methyl-4,5-dihydroxy-l,2- diazinane and (3S, 4R, 5R)-l-acetyl-4,5-diacetoxy-3-methyl-l,2-diazinane. These compounds have a stereochemistry unlike naturally occurring sugars. Various monohydroxy-hexahydro- pyridazine-3-carboxylic acids (specifically l-/er -butoxycarbonyl-3-ethoxycarbonyl-4-hydroxy- 1,2-diazinane and l-ter/-butoxycarbonyl-3-ethoxycarbonyl-4-acetoxy-l,2-diazinane) and derivatives have been described (1)J. Chem. Soc. Perkin. Trans. 1 1976 22 2390-4. 2) J.

Chem. Soc. D. 1969 18 1079-80. 3) Heterocycles 1977 7 119-22. 4) Experientia 1970 26 122- 3. 5) J. Chem. Soc. C 1971 3 522-6. 6) J Chem. Soc. Chem. Commun. 1994 1867-8 ). No biological activity was described in connection with any of these compounds.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to novel compounds of the general formula I

R₂

^R'-N-^N^"'

OL, CH R^ CH" ^R₄

R<

wherein

Ri designates hydrogen, hydroxy, halogen, optionally substituted Cι.₇-alkyl, optionally substi¬ tuted C].₇-alkylcarbonyl, optionally substituted Cι_-7-alkoxycarbonyl, optionally substituted aryl(Cι.₇-alkoxy)carbonyl, aminocarbonyl, optionally substituted C_]-7-all ylaminocarbonyl, di(optionally substituted Cι- -alkyl)aminocarbonyl, optionally substituted C_2- -alkenyloxy, optionally substituted Cι-7-alkylcarbonyloxy or -CH₂-O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; or Ri designates two Cι_-7-alkyl groups thereby leading to a quaternarisation of the nitrogen atom to which Ri (the two Cι- -alkyl groups) is/are attached; and

R₂ designates hydrogen, hydroxy, halogen, optionally substituted C_]-7-alkyl, optionally substi¬ tuted C_2-7-alkenyloxy, optionally substituted C_)-7-alkylcarbonyloxy or -CH₂-O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide;

each of R3, R4, R5, and Re independently designates hydrogen, hydroxy, halogen, optionally substituted C_]-7-alkyl, amino, optionally substituted Cι-7-alkylamino, di(optionally substituted Cι.7-alkyl)amino, tri(optionally substituted Cι.7-alkyl)ammonium, carboxy, carboxyamino, optionally substituted Cι.₇-alkylcarbonylamino, optionally substituted arylcarbonylamino, nitro, sulphanyl, Cι_-7-alkylthio, cyano, azido, optionally substituted C_2-7-alkenyl, optionally substi¬ tuted C_2-7-alkynyl, optionally substituted aryl, optionally substituted Cι-7-alkylcarbonyl, optio¬ nally substituted Cι- -alkoxycarbonyl, aminocarbonyl, optionally substituted C_]-7-alkylamino- carbonyl, di(optionally substituted Cι.₇-alkyl)aminocarbonyl, optionally substituted C_2- - alkenyloxy, optionally substituted Cι.₇-alkylcarbonyloxy, or -CH2-O-X or -O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide;

or a salt thereof;

with the provisos that (a) at least one of R₃, R₄, R5 or Ro designates optionally substituted Cι_-7-alkyl, carboxy, cyano, optionally substituted C_2-7-alkenyl, optionally substituted C₂.₇-alkynyl, optionally substituted aryl, optionally substituted Cι_-7-alkylcarbonyl, optionally substituted Cι_-7-alkoxycarbonyl, aminocarbonyl, optionally substituted Cι_-7-alkylaminocarbonyl, di(optionally substituted C_1- - alkyl)aminocarbonyl, or -CH₂-O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; and (b) at least one of R , R₄, R₅ or Re designates hydroxy, optionally substituted Cι_-7-alkoxy, optionally substituted C₂.₇-alkenyloxy, optionally substituted Cι_-7-alkylcarbonyloxy, or -O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; and

(c) when R₃ designate (3S)-carboxy and R4 and Re designate hydrogen, then R₅ designates a group different from (5S)-hydroxy; and

(d) that said compound is not selected from l-ter/-butoxycarbonyl-3-ethoxycarbonyl-4- hydroxy-l,2-diazinane, l-tert-butoxycarbonyl-3-ethoxycarbonyl-4-acetoxy-l,2-diazinane, (3R, 4S, 5R, 6S)-6-methyl-3,4,5-trihydroxy-l,2-diazinane, (3S, 4S, 5R, 6S)-6-methyl-3,4,5- trihydroxy-l ,2-diazinane, (3R, 4S, 5R, 6S)-l,6-dimethyl-3,4,5-trihydroxy-l,2-diazinane, (3S, 4S, 5R, 6S)-l,6-dimethyl-3,4,5-trihydroxy-l,2-diazinane, (3S, 4R, 5R)-3-methyl-4,5- dihydroxy-l,2-diazinane, (3S, 4R, 5R)-l-acetyl-3-methyl-4,5-dihydroxy-l,2-diazinane, (3S, 4R, 5R)- 1 -acetyl-4, 5-diacetoxy-3-methyl- 1 ,2-diazinane.

The compounds according to the invention have glucosidase inhibitor activity and may, thus, be used for the treatment of and for controlling diabetes, AIDS, and cancer as well as for crop protection. These compounds are, as described below, easier to prepare than most known inhibitors of glycoside cleaving enzymes.

DETAILED DESCRIPTION OF THE INVENTION

Glycoside-cleaving enzymes work by catalysing the cleavage of the glycosidic linkage in a glycoside to a unstable high energy intermediate, a transition state, by stabilising this in its catalytic pocket (see Fig. 1). The transition state is a carbocation, which is in resonance equilibrium with an oxocarbenium ion. Chemical compounds that electronically and sterically resemble transition state to a high extent, transition state analogues, are expected to bind strongly to the enzyme's catalytic pocket and thereby prevent, i.e. inhibit, its action. The compounds according to the present invention, hydroxyhexahydropyridazines, as per formula I, e.g. compound 1, are basic and will be protonated under neutral conditions. Thereby two forms of the protonated hydrazine are formed (see Fig 1). These are in equilibria and will each represent a transitionstate-analogues of respectively the carbocation and the oxocarbenium ion ofglycoside-cleavage. Hydroxyhexahydropyridazines, as 1 and derivatives, are therefore potentially strong inhibitors of glycoside-cleaving enzymes e.g. glycosidases and glycoside phosphorylases.

In the present context, the term "Cι- -alkyl" is intended to mean a linear, cyclic or branched hydrocarbon group having 1 to 7 carbon atoms, such as methyl, ethyl, propyl, iso-propyl, cyclopropyl, cyclopropylmethyl, butyl, tert-butyl, iso-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl. Preferred examples of "Cι.₇-alkyl" are methyl, ethyl, propyl, iso- propyl, butyl, fert-butyl, iso-butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, in particular methyl, ethyl, propyl, iso-propyl, tert-butyl, iso-butyl and cyclohexyl.

The term "alkoxy" is intended to mean alkyl-oxy.

Similarly, the term "C2-7-alkenyl", is intended to mean a linear, cyclic or branched hydrocarbon group having 2 to 7 carbon atoms and comprising one unsaturated bond. Examples of alkenyl groups are vinyl, allyl, butenyl, pentenyl, hexenyl, and heptenyl. Preferred examples of alkenyl are vinyl, allyl, butenyl, especially allyl.

Similarly, the term "C _-7-alkynyl" is intended to mean a linear or branched hydrocarbon group having 2 to 7 carbon atoms and comprising a triple bond. Examples hereof are ethynyl, propynyl, butynyl, octynyl, and dodecanyl.

In connection with the terms "alkyl", "alkoxy", "alkenyl", and "alkynyl", the term "optionally substituted" is intended to mean that the alkyl group, the alkoxy group, the alkenyl group, or the alkynyl group, respectively, in question may be substituted one or several times, preferably 1-3 times, with group(s) selected from hydroxy, C].₇-alkoxy, carboxy, Cι_-7-alkoxycarbonyl, Cι-₇-alkylcarbonyl, formyl, optionally substituted aryl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyl, amino, mono- and di(Cι.7-alkyl)amino; carbamoyl, mono- and di(Cι-₇-alkyl)aminocarbonyl, amino-Cι_-7-alkyl-aminocarbonyl, mono- and di(Cι_-7-alkyl)- amino-Cι-7-alkyl-aminocarbonyl, Cι-7-alkylcarbonylamino, guanidino, carbamido, Cι.₇-alkyl- carbonyloxy, sulphono, nitro, sulphanyl, Cι.7-alkylthio, trihalogenalkyl, halogen such as fluoro, chloro, bro o or iodo. Preferably, the substituents are selected from hydroxy, Cι.₇-alkoxy, carboxy, Cι_-7-alkoxycarbonyl, Cι.₇-alkylcarbonyl, formyl, optionally substituted aryl, optionally substituted aryloxycarbonyl, optionally substituted arylcarbonyl, amino, mono- and di(Cι.₇- alkyl)amino, carbamoyl, mono- and di(Cι-7-alkyl)aminocarbonyl, amino-Cι-7-alkyl-aminocar- bonyl, mono- and di(Cι_-7-alkyl)amino-Cι-7-alkyl-aminocarbonyl, Cι_-7-alkylcarbonylamino, guanidino, carbamido, trihalogenalkyl, halogen such as fluoro, chloro, bromo or iodo, where aryl and heteroaryl may be substituted with methyl, nitro or halogen. Especially preferred examples of substituents are hydroxy, Cι_-7-alkoxy, carboxy, optionally substituted aryl, amino, mono- and di(Cι_-7-alkyl)amino, mono- and di(C].₇-alkyl)amino, and halogen such as fluoro, chloro, bromo or iodo.

In the present context the term "aryl" is intended to mean an aromatic carbocyclic ring or ring system, such as phenyl, naphthyl, anthracyl, phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, among which phenyl is a preferred example. Furthermore, "aryl" is also inten¬ ded to mean such groups where one or more of the carbon atoms have been replaced with heteroatoms, e.g. nitrogen, sulphur, and/or oxygen atoms. Examples of such further groups are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, indolyl, benzopyrazolyl, phenoxazonyl, among which pyridinyl, benzopyrazolyl, and imidazolyl are preferred examples.

In the present context, i.e. in connection with the term "aryl", the term "optionally substituted" is intended to mean that the group in question may be substituted one or several times, prefera- bly 1-5 times, in particular 1-3 times, with group(s) selected from hydroxy (which when pre¬ sent in an enol system may be represented in the tautomeric keto form), Cι. -alkoxy, carboxy, Cι.₇-alkoxycarbonyl, Cι_-7-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono- and di(Cι_-7-alkyl)amino; carbamoyl, mono- and di(Cι_-7-alkyl)aminocarbonyl, amino-Cι- -alkyl-aminocarbonyl, mono- and di(Cι_-7-alkyl)amino-Cι_-7-alkyl-aminocarbonyl, C1.7-alkylcarbonylarr.ino, guanidino, carbamido, C_1-7-alkanoyloxy, sulphono, Cι_-7-aikylsulpho- nyloxy, nitro, sulphanyl, trihalogenalkyl, halogen such as fluoro, chloro, bromo or iodo. Pre¬ ferred examples are hydroxy, Cι_-7-alkoxy, carboxy, Cι-7-alkoxycarbonyl, C_]-7-alkylcarbonyl, aryl, amino, mono- and di(Cι_-7-alkyl)amino, aryl and halogen such as fluoro, chloro, bromo or iodo.

In the present context, the term glycosyl group (of a mono-, di- or trisaccharide) is intended to mean a hexopyranosyl group, a (O-hexopyranosyl)-hexopyranosyl group or a ((O-hexopyrano- sy!)-0-hexopyranosyl)-hexopyranosyl group. The individual hexopyranose groups are typically selected from glucose, mannose, galactose, fucose, glucosamine, galactosamine, N-acetyl- glucosamine, N-acetylgalactosamine, and rhamnose.

In preferred compounds of the general formula I, Ri designates hydrogen, optionally substitu- ted Cι.7-alkyl, optionally substituted Cι_-7-alkylcarbonyl, aryl(Cι_-7-alkoxy)carbonyl, aminocar¬ bonyl, optionally substituted Cι-7-alkylaminocarbonyl, di(optionally substituted Cι_-7-alkyl)- aminocarbonyl, or optionally substituted C_1-7-alkylcarbonyloxy; or Ri designates two C₁.₇-alkyl groups; and R₂ designates hydrogen or optionally substituted d-7-alkyl. More preferably, each of Ri and R₂ independently designates hydrogen or optionally substituted Cι-₇-alkyl, and in particular each of Ri and R₂ independently designates hydrogen or Cι_-7-alkyl.

With respect to the further substituents, preferred compounds are those where each of R₃, R₄, R₅, and Re independently designates hydrogen, hydroxy, halogen, optionally substituted Cι_-7- alkyl, amino, optionally substituted Cι.₇-alkylamino, di(optionally substituted Cι_-7-alkyI)amino, tri(optionally substituted Cι-7-alkyl)ammonium, carboxy, carboxyamino, optionally substituted Cι-7-alkylcarbonylamino, optionally substituted arylcarbonylamino, sulphanyl, Cι.₇-alkylthio, cyano, azido, optionally substituted aryl, optionally substituted Cι_-7-alkoxycarbonyl, amino¬ carbonyl, optionally substituted Ci_-7-alkylaminocarbonyl, di(optionally substituted Cι_-7-alkyl)- aminocarbonyl, optionally substituted C_2-7-alkenyloxy, optionally substituted C_t-7-alkylcarbo- nyloxy, or -CH₂-O-X or -O-X, where X designates a glycosyl group of a mono-, di- or tri¬ saccharide. More preferred are those where and each of R3 and Re independently designates hydrogen, hydroxy, halogen, optionally substituted Cι_-7-alkyl, carboxy, cyano, azido, optionally substituted aryl, optionally substituted Cι.₇-alkoxycarbonyl, aminocarbonyl, optionally sub¬ stituted Cι.₇-alkylaminocarbonyl, di(optionally substituted Cι-7-alkyl)aminocarbonyl, or -CH₂- O-X or -O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; and each of R and R₅ independently designates hydrogen, hydroxy, halogen, optionally substituted C₁-7- alkyl, amino, optionally substituted Cι-7-alkylamino, di(optionally substituted Cι-7-alkyl)amino, carboxy, carboxyamino, optionally substituted Cι.7-alkylcarbonylamino, optionally substituted arylcarbonylamino, sulphanyl, Ci.₇-alkylthio, cyano, azido, optionally substituted aryl, optio- nally substituted C].₇-alkoxycarbonyl, aminocarbonyl, optionally substituted C1.7-alkylam.no- carbonyl, di(optionally substituted Cι.₇-alkyl)aminocarbonyl, optionally substituted C₂.₇-alke- nyloxy, optionally substituted Cι.₇-alkylcarbonyloxy, or -CH₂-O-X or -O-X, where X desig¬ nates a glycosyl group of a mono-, di- or trisaccharide. Particularly preferred are those where each of R₃ and R* independently designates hydrogen, hydroxy, Cι_-7-alkyl, carboxy, cyano, azido, Cι- -alkoxycarbonyl, aminocarbonyl, Cι- -alkylaminocarbonyl, or di(Cι. -alkyl)amino- carbonyl; and each of R and R₅ independently designates hydrogen, hydroxy, C1.7-a.kyl, amino, Cι-₇-alkylamino, di(Cι-7-alkyl)amino, carboxy, carboxyamino, Cι-7-alkylcarbonylamino, cyano, azido, optionally substituted aryl, Cι_-7-alkoxycarbonyl, aminocarbonyl, Cι.₇-alkylamino- carbonyl, di(Cι.₇-alkyl)aminocarbonyl, or Cι.₇-alkylcarbonyloxy.

Furthermore, the invention covers salts of the compounds according to the invention. The preparation of pharmaceutical salts of the compounds according to the invention is well known to a professional in the field, and will not be described in detail. Examples of salt, which furthermore are pharmaceutically acceptable, includes, but is not limited to, organic carboxylic acids such as acetic acid, lactic acid, tartaric acid, maleic acid, isothionic acid, lactobionic acid, and succinic acid; organic sulfonic acids such as methane sulfonic acid, ethane sulfonic acid, benzene sulfonic acid and toluene sulfonic acid, and inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfaminic acid. Also included are pharmaceutical acceptable salts of basic salts of the compounds, made with a suitable base such as alkalimetal (e.g. sodium, potassium), earth alkalimetal (e.g. magnesium), ammonium, and NW_nH_m bases where n and m are from 0 to 4, and n+m are 4, and where W is a Ci.ig-alkyl group.

Preferred specific compound are:

4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine such as

{4S, 5R, 6R)~4, 5-dihydroxy-6-hydroxymethyl-hexahydropyridazine,

(^R,JιS^,,6^'5)-4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine,

(4R, 5S, 6R)-4, 5-dihydroxy-6-hydroxymethyl-hexahydropyridazine, {4S,5R, 6S)-4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine,

{4R,5R, 67?)-4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine, and

{4S, 5S, 6S)-4, 5-dihydroxy-6-hydroxymethyl-hexahydropyridazine,

4,5-dihydroxy-6-methyl-hexahydropyridazine such as

{4S, 5R, 6S)-4, 5 -dihydroxy-6-methyl-hexahydropyridazine, {4R, 5S, 6R)-4, 5-dihydroxy-6-methyl-hexahydropyridazine,

{4R,5S, 6S)-4,5-dihydroxy-6-rnethyl-hexahydropyridazine, and

(4S, 5R, 6R)-4, 5-dihydroxy-6-methyl-hexahydropyridazine, 4,5-dihydroxy-6-fluoromethyl-hexahydropyridazine,

4-fluoro-5-hydroxy-6-hydroxymethyl-hexahydropyridazine, l-N-(2-hydroxyethyl)-4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine,

2-N-(2-hydroxyethyl)-4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine,

4,5-dihydroxy-hexahydropyridazine-6-carboxylic acid, and salt of these compounds.

The compounds according to the invention are expected to resemble the transitionstate of natural substrates so much that they will inhibit glycoside cleaving enzymes

It is, thus, believed that the most interesting compounds are those which mimic the stereo- chemisty of glucose, mannose, galactose, glucosamine, galactosamine, rhamnose or fucose because such compounds will better fit into the active site of a glycosidase. Furthermore the substituents R_?-Re of the compound I will then have a spatial position similar to the hydroxy groups of the natural substrate saccharide, and, thus, the substituents are more likely to fit into the corresponding pockets of the glucosidase in question. Also, if one or more of the substi¬ tuents are hydroxy groups, or, alternatively, derivatives thereof, such substituents may par¬ ticipate in hydrogen bonding similar to the hydroxy groups of the natural substrate. This will increase the binding and thereby the inhibition, and is also likely to increase the specificity of the compound

Thus, a preferred embodiment of the present invention relates to compounds I having any of the general formulae II, III, IV or V

π m

rv v

In compounds I where the substituents R₃ and R4, or R₃ and R₅, respectively, are substituents different from hydrogen, Formula II represents compounds that have a stereochemistry resem¬ bling D-glucose, D-mannose and D-glucosamine; Formula III represents compounds that have a stereochemistry resembling L-rhamnose; Formula IV represents compounds that have a stereochemistry resembling D-glucose, D-mannose, D-galactose, D-galactosamine and D- glucosamine; Formula V represents compounds that have a stereochemistry resembling L- fucose.

Interesting examples are compound which fulfils the criteria set forth in both of Formulae II and IV, thus having, at least partially, a stereochemistry resembling D-glucose, D-glucosamine, or D-mannose.

The present invention is also related to a pharmaceutical formulation comprising a compound I for the treatment of or for controlling diabetes, cancer or AIDS caused by human immuno¬ deficiency virus which consist of at least one compound of the invention as described above, mixtures hereof and/or pharmaceutical salts hereof; and a pharmaceutical acceptable carrier material. Such formulations are made after the established pharmaceutical procedures, e.g. as described in Remington 's Pharmaceutical Sciences, 17. edition, ed. Alfonso R. Gennaro,

Mack Publishing Company, Easton, PA (1985). More generally, the present invention relates to a compound I as a medicament, e g for the treatment of or for controlling diseases such as diabetes or cancer, or for fighting or controlling human immunodeficiency virus

Furthermore, the invention covers a method for protecting a crop characterised by treatment of the crop with one or more compounds of the invention in an amount necessary to protect the crop

Still further, the invention covers a method for inhibiting cellulase activity characterised by treatment of one or more compounds of the invention in an amount necessary to inhibit cellulase activity

The invention is also intended to cover the known compound 4,5-dihydroxy-tetrahydropyrida- zine (formula I where Ri = R₂ = R₃ = e = H and R4 = Rs = OH) as inhibitor of glycoside cleaving enzymes for treatment of diabetes, cancer, AIDS and for crop protection

SYNTHESIS

The novel compounds of the invention can be made by the synthesis described in the reaction scheme in Fig 2 The synthetic method includes a sequence of steps known to a professional in the field, thus, the synthetic method includes one or more of the following steps

1) Diels- Alder reaction between a diene and a azadienophile

This reaction is as generally described in Forrest, A K., Schmidt, R R , Huttner, G , Jibril, I

De novo Synthesis of Carbohydrates Part 14 Preparation of 4- Amino-lyxose Derivatives X- ray Molecular Structure of Ethyl 6-Ethoxycarbonylamino-8-hydroxymethyl-3,3-dimethyl-2,4- dioxa-7-azabicyclo(3 3 0)octane-7-carboxylate J Chem Soc Perkin Trans 1 1984 1981-7 or in Batchelor, M J , Mellor, J M The Use of Dichloromaleic and Bromomaleic Anhydrides in the Synthesis of Lactones by the Intramolecular Diels-Alder Reaction J Chem Soc Perkin Trans 1 1989 985-95 The diene can be any known 1,3 -butadiene optionally substituted in the 1, 2, 3 and/or 4 position with any available substituent Such substituents may correspond to the substituents in the compound I, or may be substituents which may be converted to a substituent in the compound I E g , when a R₃ substituent is present in compound I, such a substituent is normally introduced via the 1 -position of the diene, analogously, when R», R₅ and/or R_o substituent are present in the compound I, those substituents may directly be introduced via the 2, 3 and/or 4-position of the diene, respectively However, the substituents R₄ and R₅ may also conveniently be introduced in a separate step, see below Furthermore, all the substituents of the diene can normally be modified at a later stage The diene may, e g , be butadiene, 2,3-dimethylbutadiene, phenylbutadiene, Danishefskis diene, hexadienes, penta- diene, pentadienol, pentadienoic acid, methyl pentadienoate, ethyl pentadienoate or sorbyl alcohol As explained such dienes may carry suitable substituents The azadienophile is typical¬ ly either 4-phenyl-l,2,4-triazol-3,5-dione or a dialkyl azodicarboxylate The Diels-Alder reaction is carried out by mixing the diene and the dienophile in an organic solvent, e g selec- ted from ethyl acetate, ethanol, methanol, dichloromethane, chloroform, toluene, dioxane, tetrahydrofuran, benzene, acetone, acetonitrile, dimethyl sulphoxide or dimethyl formamide The reaction is carried out at a temperature between -50 and 200°C depending on the reacti¬ vity of the reagents, typically at a temperature between 0 and 50°C, such as around 25 °C

2) Addition to the double bond of the Diels-Alder adduct

This reaction step can be used to introduce the substituents R4 and/or R₅ when such substitu¬ ents are absent in the diene, or when such substituents are present in the diene to reduce the partially unsaturated ring (the Diels-Alder adduct) to a fully saturated ring by the addition of hydrogen atoms (hydrogenation) The following illustrative examples of reaction conditions may be used, of course depending on the nature of the substituents R₄ and R₅

a) Addition of hydrogen to the double bond, typically when R4 and R₅ designate hydrogen, can be carried out by reaction of the Diels-Alder adduct with hydrogen, as generally described in J March Advanced Organic Chemistry, 3 ed p 745-758, at a pressure of 1 to 100 atmospheres, typically 1-3 atmospheres, in the presence of a metal catalyst such as palladium, rhodium, Raney nickel or platinum, typically palladium, in a suitable organic solvent such as ethyl acetate, ethanol, methanol, dioxane, tetrahydrofuran, toluene or benzene

b) Dihydroxylation of the double bond (R4 and R5 designate hydroxy or a derivative thereof) can be earned out with osmium tetroxide with or without N-methyl-N-morpholine as co-oxi- dant, as generally described in J March Advanced Organic Chemistry, 3 ed pp 732-734, at 0-60°C, typically at around 25°C, for lh to 7 days, typically V2-I day, in a suitable solvent such as ethyl acetate, ethanol, methanol, dichloromethane, chloroform, toluene, dioxane, tetrahydro¬ furan, benzene, acetone, acetonitrile, dimethyl sulphoxide, water or dimethyl formamide Alter¬ natively dihydroxylation can be carried out by epoxidation followed by acidic hydrolysis This is done by first reaction of the Diels-Alder adduct with an oxidant, as generally described in J March Advanced Organic Chemistry, 3 ed pp 735-737, such as 3-chloroperbenzoic acid or a dialkyldioxirane, typically trifluoromethylmethyl dioxirane, in a solvent such as ethyl acetate, ethanol, methanol, dichloromethane, dichloroethane, chloroform, toluene, dioxane, tetrahydro¬ furan, benzene, acetone, acetonitrile, dimethyl sulphoxide or dimethyl formamide, at 0-150°C, typically at around 25°C, for lh to 7 days, typically Vι-\ day, to give an epoxide Secondly the epoxide is reacted with aqueous acid, as generally described in J March Advanced Organic Chemistry, 3 ed p 332, e g aqueous perchloric acid 0 1-5% at 25-100°C for 1-100 h, typi¬ cally around 5 h The resulting hydroxy groups may subsequently or intermittently be conver¬ ted to a derivative thereof, e g by acetylation or alkylation or glycosidation (step 5), thereby leading to other variants of the substituents R4 and R₅

c) Monohydroxylation can be made by hydroboration/oxidation, as generally described in J March Advanced Organic Chemistry, 3 ed pp 702-707, 550 It is carried out by reaction of the Diels-Alder adduct with borane or a alkyl- or dialkylborane in an aprotic organic solvent, typically tetrahydrofuran or dioxane, at -78-100°C for 1-24 hours, then the product is oxidised with a suitable oxidant such as hydrogen peroxide As above, the resulting hydroxy group may be converted to a derivative thereof

d) Halogen is added (as R4 and/or R5), as generally described in J March Advanced Organic Chemistry, 3 ed pp 724-726, by treatment of the Diels-Alder adduct with halogen such as bromine, chlorine or iodine in an inert solvent typically dichloromethane for 1-18 hours at 25- 100°C

e) Halogen and hydroxy groups (one or R4 and R5 is halogen and the other is hydroxy or a derivative thereof) are added by treatment, as generally described in J March Advanced Organic Chemistry, 3 ed pp 726-728, with halogen in water This is carried out by treatment of the Diels-Alder adduct with halogen in water for 1-18 hours at 25-100°C As above, the resulting hydroxy group may be converted to a derivative thereof f) A hydroxy group and another substituent (one of R4 and R₅ is halogen, alkylthio, cyano, hydrogen, optionally substituted alkyl, amino, thio, optionally substituted alkoxy or optionally substituted alkylamino and the other is hydroxy or a derivative thereof) may be introduced by first reaction of the Diels-Alder adduct with an oxidant, as generally described above. Secondly the epoxide is reacted with a nucleophile, as generally described in J. March Advanced Organic Chemistry, 3. ed. pp. 255-446. The nucleophile may, e.g., be a halide, cyanide, a sulphide, a xanthogenate, an amine, a carbanion, an alkyl group form a organometalic reagent, azide and hydride. With a halide nucleophile a halogen substituent is obtained; with a cyanide nucleophile a cyano substituent is obtained; with a sulphide nucleophile an alkylthio substituent is obtained; with a xanthogenate nucleophile a thiol substituent is obtained after treatment with sodium methoxide in methanol; with an amine nucleophile an amino, alkylamino or dialkylamino substituent is obtained; with a carbanion nucleophile an optionally substituted alkyl substituent is obtained.; with an organometallic reagent an alkyl substituent is obtained; with an azide nucleophile an azido substituent is obtained; and, finally, with a hydride nucleophile, from e.g. lithium aluminium hydride, a hydrogen "substituent" is obtained. As a reagent, the pure nucleophile, the metal, typically potassium, salt of the nucleophile, the corresponding acid form, H-Nucleophile, of the nucleophile or the trimethylsilyl derivative of the nucleophile (Me₃Si-Nucleophile) may be used. The solvent is typically ethyl acetate, ethanol, methanol, dichloromethane, dichloroethane, chloroform, toluene, dioxane, tetrahydrofuran, benzene, acetone, acetonitrile, dimethyl sulphoxide or dimethyl formamide. The reaction temperature is often between 25-180°C, typically 25-60°C. In some cases a Lewis acid catalysts such as borontrifluoride, tin tetrachloride or trimethylsilyl triflate is added.

3) Hydrazinolysis of phenylurazole or hydrolysis of dialkyl azodicarboxylates.

Hydrazinolysis of a phenylurazole is, e.g., carried out by mixing the compound with hydrazine- hydrate at 25-100°C for lh to 2 days without a solvent. Hydrolysis of dialkyl azodicarboxy- lates is typically carried out by treatment of the dialkyl azodicarboxylate with aqueous acid such as aqueous perchloric acid 0.1-5% at 25-100°C for 1-100 h. In some cases the product will be the final compound, i.e. the introduction of any substituents R₄ and R₅ has been per¬ formed and Ri and R₂ designate hydrogen. Alternatively, the product is subjected to the reac¬ tion steps 4) and/or 5) below, and optionally also to reaction step 2) above. 4) Substitution on nitrogen followed by separation of the products

This step is used when Ri and/or R₂ substituents are desired Reaction with an alkyl halide will give a N-alkyl substituent, while reaction with an acid chloride will result in an acyl substituent Other reactive nitrogen atoms (other substituents) are optionally protected by using protection- /deprotection schemes known to the person skilled in the art.

a) The hydrazine derivative (the adduct from step 3) is dissolved in a suitable solvent such as ethyl acetate, ethanol, methanol, dichloromethane, dichloroethane, chloroform, toluene, dioxane, tetrahydrofuran, benzene, acetone, acetonitrile, dimethyl sulphoxide or dimethyl formamide, and treated with an alkyl halide, which can be any commercially available alkyl halide, e g but not limited to methyl iodide, ethyl iodide, 2-bromoethanol, benzyl bromide, allyl bromide or propyl bromide, in excess The reaction is carried out at 0-200°C typically at 25°C for 0 1 hour to 7 days depending on whether both of Ri and R₂ should be alkylated or not The reaction is stopped and the product is separated In some cases the product will be the final compound, in some cases the product is subjected to reactions described under 4b) and/or 5) below and/or 2) above

b) The hydrazine derivative (optionally treated as described under 4b) is dissolved in a suitable solvent such as ethyl acetate, ethanol, methanol, dichloromethane, dichloroethane, chloroform, toluene, dioxane, tetrahydrofuran, benzene, acetone, acetonitrile, dimethyl sulphoxide or dimethyl formamide, and treated with an acylhalide or anhydride, which can be any commer¬ cially available acyl halide or anhydride, e.g.,. but not limited to acetic anhydride, benzoyl chloride, pivaloyl chloride, propanoyl chloride, butanoyl chloride or crotonyl chloride, in excess The reaction is carried out at 0-200°C typically at 25°C for 0.1 hour to 7 days. The reaction is stopped and the product is separated In some cases the product will be the desired compound I, and in some cases the product is further subjected to reaction step 5) below or reaction step 2) above.

5) Glycosidation of hydroxy groups

The compound is typically reacted with a the trichloroacetimidate of a sugar as generally described in Schmidt, R R Angew Chem 1986, 98, 213-236 The compound is dissolved in a suitable solvent such as ethyl acetate, dichloromethane, dichloroethane, chloroform, toluene, dioxane, tetrahydrofuran, benzene, acetone, acetonitrile or dimethyl formamide, and a perbenzyl glycosyl trichloroacetimidate, such as tetra-O-benzyl glucosyl trichloroacetimidate, tetra-O-benzyl mannosyl trichloroacetimidate, tetra-O-benzyl galactosyl trichloroacetimidate, tetra-O-benzyl fucosyl trichloroacetimidate or tetra-O-benzyl rhamnosyl trichloroacetimidate, is added together with a Lewis acid catalyst, such as borontrifluoride, tin tetrachloride or trimethylsilyl triflate, and the mixture is reacted at -78 to 25°C for 0.1 to 24 hours. After this reaction the benzyl groups are removed by dissolving the product in ethyl acetate adding palladium on carbon and reacting with hydrogen at 1 to 50 atmospheres at 25°C for 1 to 24 hours The product will typically be the final compound.

For therapeutic use in a method to treat or control diabetes, cancer or human immuno¬ deficiency virus can one of the compounds in this invention or its salt be given in the form of a pharmaceutical formulation consisting of at least one compound of the invention, and/or pharmaceutical salts hereof; and a pharmaceutical acceptable carrier material. Suitable carriers are known to a professional and can vary with form and the method used for treatment in the pharmaceutical formulation.

EXAMPLES

The following examples illustrate the invention further, and how, e.g., 1 can be made from commercially available materials (see Fig. 3) and penta-2,4-dien-l-ol (2), which can be made as described in J. Chem. Soc. 1990, p 1640., and te -butylhypochlorite, prepared as described by M. J. Mintz et al. Organic Synthesis Coll. Vol 5, John Wiley & sons New York 1973, ed. H. E. Baumgarten, p 184-7.

Example 1

⁽6^'R/6^'S⁾-2.4-dioxo-6-hydroxymethyl-3-phenyl-1.3.5-triaza-f4.3.0]-bicvclonon-7-ene (4r 4- Phenylurazol (3, 20 g, 113 mmol) were dissolved in EtOAc (60 ml) at 0°C and tert- butylhypochlorite (12.4 g) was added, where after a red, homogeneous solution was obtained. After 5 min. penta-2,4-dien-l-ol (2, 10.0 g, 119 mmol) was added, and the solution was stirred while being heated over 30 minutes to room temperature. After filtration and concentration, chloroform (200 ml) was added to the residue, and the mixture was filtered again The filtrate was concentrated to a crystalline residue of 4 (17.2 g, 59%) Mp 150 °C Η-NMR (CDC1₃) δ 7 4-7 5 (m, 5H), 6 1 (m, IH), 5 85 (m, IH), 4 6 (ddd, IH, J = 8 3, 6 2 and 3 3 Hz), 4.25 (m, IH), 4 15 (m, IH), 3 95 (dd, IH, J = 12 4 and 3 3 Hz), 3 9 (dd, IH, J = 12 4 and 6 2 Hz)

Example 2 (^R.7R._fy '/<?S.7 '._f RV2.4-dioxo-7.8-epoxy-6-hvdroxymethyl-3-phenyl-1.3.5-triaza-r4.3.01- bicyclononane (5) Alcohol 4 (1 00 g, 3 86 mmol) was dissolved in MeCN (30 ml) and water (20 ml) in a flask with an addition funnel, and a dry ice/acetone condenser The solution was cooled to 0°C with an ice bath, and 1 , 1 , 1 -trifluoroacetone (4 ml) and NaHCO₃ (2 6 g) followed by oxone (12 3 g) was added in small portions over 5 min The mixture was stirred at room temperature for 18 hours More NaHCO₃ (1 3 g) and oxone (6 15 g) was added, and after 2 hours the reaction was stopped by addition of water (200 ml) followed by extraction with CHC1₃ (8 x 100 ml) The combined organic layers were dried (MgSO4) and concentrated to a solid mixture of trans and cis epoxides (1 09 g) in ratio 3 1 By addition of CHC1₃ (40 ml) the pure transepoxide 5 crystallised (635 mg, 60%) Mp 182-4 ¹³C-NMR (CDC1 ) δ 128 3, 127 5, 124 6, 61 1 , 54 2, 50 2, 47 6, 41 0

Example 3 (t5R.7R.^R/6S.7,S^,.^y>-7.8-dihvdroxy-2.4-dioxo-6-hvdroxymethyl-3-phenyl-1.3.5-triaza-r4.3.0 bicyclononane (6) Epoxide 5 (500 mg) was dissolved under heating in water (50 ml) and 70% HCIO₄ (1 25 ml) was added The solution was heated to 100°C in 5 hours, and then neutralised with potassium carbonate The solution was concentrated, and the residue was flash-chromato- graphed in ethyl acetate Thereby the triol 6 was obtained (388 mg, 73%) ¹³C-NMR (D₂O) δ 136 6, 133 8, 66 5, 66 2, 61 2, 58 5, 46 1

Example 4

(4R.5R.6RI4S.5S.6SV4.5-dihvdroxy-6-hvdroxymethyl-hexahvdropyridazine (1 . Triol 6 (256 mg) was dissolved in hydrazine hydrate (5 ml) and heated to 100°C for 18 hours After evaporation, the residue was flash-chromatographed in EtOH-25% NH4OH 10 1, giving the title compound 1 (109 mg, 84%) as a syrup ¹³C-NMR (D₂O) δ 73 2, 72 7, 64 3, 60 9, 53 0 Example 5 rf5R/<5S)-2.4-dioxo-3-phenyl-1.3.5-triaza-r4.3.01-bicvclonon-7-ene-6-carboxylic acid (7). 4- Phenylurazole (3, 1.1 g) was dissolved in EtOAc (3 ml) at 0°C, and tert-butylhypochlorite (0.7 g) was added, where after a red, homogenous solution was obtained. After 5 min. was added penta-2,4-dienoic acid (0.7 g), and the solution was stirred while being heated over 30 minutes to room temperature. After filtration, the filtrand was washed with pentane (15 ml). Yield of 7: 1.33 g (79%). ^]H-NMR (CD₃OD): δ 7.25 (m, 5H, Ph), 5.95 (s, 2H, H-7 & H-8), 4.95 (bs, IH, H-6),.4.15 (bd, IH, J 16.7 Hz, H-9a), 3.85 (bd, IH, J 16.7 Hz, H-9b).

Example 6

(6R/6^' ₁S^-2,4-dioxo-3-phenyl-1.3.5-triaza-r4,3.01-bicvclonon-7-ene-6-carboxylic acid methylester (8). 4-Phenylurazole (3, 2.2 g) was dissolved in EtOAc (6 ml) at 0°C, and tert- butylhypochlorite (1.1 g) was added where after a red, homogeneous solution was obtained After 5 min. was added penta-l,3-dienenoic acid methylester (1.5 g), and the solution was stirred while being heated over 30 minutes to room temperature. After filtration, the filtrand was recrystallised from chloroform. Yield of 8: 3.45 g (97%).1H-NMR (CDC1₃): δ 7.4-7.5 (m, 5H, Ph), 6.1 (s, 2H, H-7 & H-8), 5.1 (bs, IH, H-6),.4.4 (bd, IH, J 16.6 Hz, H-9a), 4.0 (bd, IH, J 16.6 Hz, H-9b), 3.8 (s, 3H, Me).

Example 7

(6R/6^' _tSV2.4-dioxo-6-acetoxymethyl-3 -phenyl- 1.3.5 -triaza-f 4.3.0] -bicyclonon-7-ene (9) . The alcohol 4 (518 mg) was dissolved in CH₂C1₂ (10 ml), pyridine (1 ml) and acetic anhydride (1 ml) was added, and the solution was kept at 25°C for 18 hours. The solution was washed with water, HC1 and NaHCO₃-solutions and dried with MgSO₄. After filtration, the filtrate was concentrated to a crystalline residue of 9 (563 mg, 94%). Mp 1 18-124°C. ¹ C-NMR (CDC1₃). δ 125-8 (3s, Ph), 123.1 (C-8), 121.5 (C-7), 61.8 (C-6'), 52.6 (C-6), 43.7 (C-9), 20.6 (Ac).

Example 8

(67?/<5^-2.4-dioxo-6-f2.2.2-trimethylacetoxy)methyl-3-phenyl-I.3.5-triaza-r4.3.0]-bicvclonon- 7-ene flOY Alcohol 4 (1.04 g) was dissolved in CH₂C1₂ (20 ml), pyridine (2 ml), 4-dimethyl- aminopyridine (10 mg) and pivaloyl chloride (0.97 g) was added, and the solution was kept at 25°C for 3 days. The solution was washed with water, HC1 and NaHCO₃-solution and was dried with MgSO₄ After filtration, the filtrate was concentrated to a residue of 10 which crystallised from ether (1 11 g, 81%) Mp 97-8°C

Example 9 r6R.7S._fyR/6S.7R.<yS)-2.4-dioxo-7.8-epoxy-3-phenyl-1.3.S-triaza-[4.3.01-bicvclononan-6- carboxylic acid methylester (11) Ester 8 (1 12 mg) was dissolved in CH C1₂ (1 ml), and a solution of dimethyldioxirane in acetone (0 092 M, 18 5 ml, 1 7 mmol, prepared as described by McMurry et al J. Org. Chem. 1985 50 2847-53 ), was added The solution was kept at 25°C for 3 days and then concentrated to a solid mixture of trans and cis epoxides (113 mg) in ratio 3 1 Flash-chromatography in EtOAc/pentane 1 1 gave the pure trans epoxide 11 (78 mg, 66%) ¹³C-NMR (CDC1₃) δ 166 7 (C=O), 129 9, 129 1, 126 4 (Ph), 55 4 (C-7), 53 9 (C-8), 50.8 (C-6), 49 5 (OMe), 43 6 (C-9)

Example 10 (6R.7R.gS/6S.7S.^RV6-acetoxymethyl-2.4-dioxo-7.8-epoxy-3-phenyl-1.3.5-triaza-[^"4.3.01- bicyclononane (12) Acetate 9 (100 mg) was dissolved in (CH₂C1)₂ (3 ml), and m-chloro- perbenzoic acid (168 mg) was added The solution was kept at 80°C for 3 hours and cooled After filtration, the filtrand was washed with CH₂C1₂ (10 ml), and the filtrates was washed with Na₂CO₃-solution, was dried with MgSO₄, filtered and concentrated to a solid mixture (102 mg, 96%) of 12 and the cis epoxide in ratio 2 1 ¹³C-NMR (CDC1₃) δ 130 0, 129 1, 126 3 (Ph), 61 9 (C-6'), 52 5 (C-7), 50 7 (C-8), 48 5 (C-6), 42 7 (C-9), 21 4 (Ac)

Example 11

(<5R.7R.gS/6S.7S.gR)-2.4-dioxo-7.8-epoxy-3-phenyl-6-(2.2.2-trimethylacetoxy)methyl-1.3.5- triaza-f4.3.0]-bicvclononane (13) Pivalate 10 (79 mg) was dissolved in (CH₂C1) (3 ml), and m-chlorperbenzoic acid (200 mg) was added The solution was kept at 80°C for 18 hours, and was then cooled After filtration, the filter was washed with CH₂C1₂ (10 ml), and the filtrates was washed with Na CO₃-solution, was dried with MgSO₄, filtered and concentrated to a residue (98 mg) This was purified by flash-chromatography in EtOAc to a syrup (35 mg, 42%) of 13 and the cis epoxide in ratio 3 2 1H-NMR (CDC1₃) δ 7 4-5 (m, 5H, Ph), 4 78 (m, IH, H-6), 4 69 (dd, IH, J 12 2 & 4 3 Hz, H-6a), 4 34 (dd, IH, J 12 2 & 3 8 Hz, H-6b), 4 12 (d, IH, J 14 1 Hz, H-9a), 3 99 (dd, IH, J 14 1 & 3 8 Hz, H-9b), 3 59 (m, IH, H-7), 3 5 (m, IH, H-8), 1 25 (s, 9H, Me's)

Example 12 (<5R/6S)-2.4-dioxo-6-methyl-3-Dhenyl-1.3.5-triaza-[4.3.01-bicvclonon-7-ene (14) 4-Phenyl- urazole (3, 1 77 g) was dissolved in EtOAc (5 ml) at 0°C, and tert-butylhypochlorite (1 1 g) was added, where after a red, homogeneous solution was obtained After 5 min was added penta- 1,3 -diene (1 5 g), and the solution was stirred while being heated over 30 minutes to room temperature After filtration, the filtrand was washed with chloroform (10 ml), and the filtrates was concentrated to a crystalline residue of 14 (2 22 g, 91%) Mp 122-5 °C ¹³C-NMR (CDC1₃) δ 129 6, 128 6, 127 9, 125 9, 120 0 (Ph, CH=CH), 50 8 (C-6), 44 0 (C-9), 18 0 (Me)

Example 13 (<?R.7R.^S/5S.7S.( R)-7.8-dihvdroxy-2.4-dioxo-6-methyl-3-phenyl-1.3.5-triaza-|^"4.3.01- bicvclononane (15) Alkene 14 (564 mg) was dissolved in water (1 ml) and acetone (1 ml), and N-methylmorpholin-N-oxide (400 mg) was added Finally was added 1 ml of a solution of OsO₄ in butanol (10 g/1), and the solution was kept at 25°C for 4 days A solution of 0 5 g Na₂S₂O₅ in 30 ml water was added, and extraction with ethyl acetate (5x 20 ml) was performed The combined organic layers were dried with MgSO₄, filtered and concentrated From the residue was crystallised diol 15 (431 mg, 67%) with EtO Ac-ether ¹³C-NMR (CD₃CN/D₂O) δ 128 3, 127 5, 125 8 (Ph), 69 3 (C-7), 63 0 (C-8), 54 3 (C-6), 43 2 (C-9), 17 8 (C-6')

Example 14

(4R.5S.6S/4S.5R.6R)-4.5-dihvdroxy-6-methyl-hexahvdropyridazine ( 16) Diol 15 (176 mg) was dissolved in hydrazinhydrate (10 ml) and warmed to 100 °C for 18 hours Then it was concentrated, and the residue was flash-chromatographed in EtOH-25% NH₄OH 50 1, where after the compound 16 (76 mg, 90%) was obtained as a syrup ¹³C-NMR (D₂O) δ 75 7 (C-5), 69 0 (C-4), 55 2 (C-6), 54 3 (C-3), 17 8 (C-6') Example 15

⁽67?.7R.^S/<?S.7S.<gR⁾-7.8-dihvdroxy-2.4-dιoxo-6-hvdroxymethyl-3-phenyl-1.3.5-tnaza-r4.3.01- bicyclononane (17) Alkene 4 (601 mg) was dissolved in water (1 ml) and acetone (1 ml), and N-methylmorphohne-N-oxide (400 mg) was added Finally was added 1 ml of a solution of OsO₄ in butanol (10 g/1), and the solution was kept at 25°C for 5 days 0 5 g of Na₂S₂O₅ was added, and the mixture was concentrated The residue was soxlet-extracted with acetone (100 ml) The organic layers were concentrated From the residue crystallised from acetone the triol 17 (537 mg, 79%) ¹³C-NMR (CD₃CN/D₂O) δ 151 3, 150 6 (CO), 128 6, 127 6, 127 3, 124 8 (Ph), 63 6 (C-7), 61 9 (C-8), 58 2 (C-6'), 56 3 (C-6), 42 5 (C-9)

Example 16

(4R.5S.6SI4S.5R. <5R)-4.5-dιhvdroxy-6-hvdroxymethyl-hexahvdropyridazιne (18) Triol 17 (180 mg) was dissolved in hydrazinhydrate (10 ml) and warmed to 100 °C for 24 hours Then the solution was concentrated, and the residue was flash-chromatographed in EtOH-25% NH₄OH 20 1, giving the compound 18 as a syrup (85 mg, 93%) ^I3C-NMR (D₂O) δ 70 4 (C-5), 68 9 (C-4), 63 0 (C-6'), 60 7 (C-6), 54 1 (C-3)

Example 17 Enzyme assays Enzymes and substrates were obtained from Sigma As substrate was employed p-rutrophenyl-α-glucopyranoside for α-glucosidase and p-nitrophenyl-β-gluco- pyranoside for β-glucosidase The expeπments were performed in 0 05 M phosphate buffer at 22°C Enzyme assays were performed as described by H Halvorson Methods Enzym. 8 (1966) 559-62

Enzyme α-glucosidase β-glucosidase

Kι (μM, pH= 7 5) 1 02

Ki (μM, pH= 6 8) 3 72 0 61

Ki (μM, pH= 5 0) 0 72 Table 1 Inhibitor constants for 1 Example 18

Glycogen phosphoryiase inhibition The assay which was described by Johnson et al {Biochemistry 1991 30 10101-16 ) was used Glycogen phosphoryiase A was bought from Sigma As substrate was used α-D-glucopyranose 1 -phosphate (0 1 M) and a 4% glycogen- solution The experiments were performed in 0 1 M NaF/HCl buffer, pH 6 2 at temperature 26°C The reaction was followed in direction of glycogen synthesis, as phosphate development was measured as described by Palmgren et al The Plant Cell 1995 7 1655-6

[compound] μM % inhibition

3 7 16

37 66

3700 78

Table 2 Inhibition constants for 1

The results in table 2 can be extrapolated to a IC₅₀ value of 13 5 μM

The chemical compound 1 and related compounds are strong inhibitors of glycoside-cleaving enzymes This is illustrated in table 1 where it can be seen that 1 show potent inhibition of α- glucosidase from bakers yeast and β-glucosidase from almonds

1. A compound of the general formula I

R₂

^Ri\ /^N- ^ NT CH^

CH. CH

R^ OT ^R,

R<

wherein

Ri designates hydrogen, hydroxy, halogen, optionally substituted Cι.₇-alkyl, optionally substituted Cι-7-alkylcarbonyl, optionally substituted Cι_-7-alkoxycarbonyl, optionally substituted aryl(C_]-7-alkoxy)carbonyl, aminocarbonyl, optionally substituted Cι- -alkylamino- carbonyl, di(optionally substituted Cι.7-alkyl)aminocarbonyl, optionally substituted C₂-₇- alkenyloxy, optionally substituted Cι.₇-alkylcarbonyloxy or -CH₂-O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; or Ri designates two C].₇-alkyl groups thereby leading to a quaternarisation of the nitrogen atom to which Ri (the two Cι-7-alkyl groups) is/are attached; and

R₂ designates hydrogen, hydroxy, halogen, optionally substituted Cι-₇-alkyl, optionally substituted C₂.7-alkenyloxy, optionally substituted Cι_-7-alkylcarbonyloxy or -CH₂-O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide;

each of R₃, R₄, R₅, and R« independently designates hydrogen, hydroxy, halogen, optionally substituted Cι.₇-alkyl, amino, optionally substituted Cι_-7-alkylamino, di(optionally substituted Cι.₇-alkyl)amino, tri(optionally substituted C_]- -alkyl)ammonium, carboxy, carboxyamino, optionally substituted Cι.₇-alkylcarbonylamino, optionally substituted arylcarbonylamino, nitro, sulphanyl, Cι.₇-alkylthio, cyano, azido, optionally substituted C₂.7-alkenyl, optionally substituted C₂.₇-alkynyl, optionally substituted aryl, optionally substituted Cι.₇-alkylcarbonyl, optionally substituted Cι_-7-alkoxycarbonyl, aminocarbonyl, optionally substituted Cj.₇- alkylaminocarbonyl, di(optionally substituted Cι.₇-alkyl)aminocarbonyl, optionally substituted C_2-7-alkenyloxy, optionally substituted Cι.₇-alkylcarbonyloxy, or -CH₂-O-X or -O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide;

or a salt thereof;

with the provisos that

(a) at least one of R₃, Ri, R5 or R„ designates optionally substituted Cι-7-alkyl, carboxy, cyano, optionally substituted C_2-7-alkenyl, optionally substituted C_2-7-alkynyl, optionally substituted aryl, optionally substituted G-7-alkylcarbonyl, optionally substituted C].₇-alkoxycarbonyl, aminocarbonyl, optionally substituted Cι-7-alkylaminocarbonyl, di(optionally substituted Cι_-7- alkyl)aminocarbonyl, or -CH₂-O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; and

(b) at least one of R₃, R4, R5 or R designates hydroxy, optionally substituted Cι- -alkoxy, optionally substituted C_2-7-alkenyloxy, optionally substituted Cι.₇-alkylcarbonyloxy, or -O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; and

(d) that said compound is not selected from l-tørt-butoxycarbonyl-3-ethoxycarbonyl-4- hydroxy- 1 ,2-diazinane, 1 -tert-butoxycarbonyl-3-ethoxycarbonyl-4-acetoxy- 1 ,2-diazinane, (3R, 4S, 5R, 6S)-6-methyl-3,4,5-trihydroxy-l,2-diazinane, (3S, 4S, 5R, 6S)-6-methyl-3,4,5- trihydroxy-l,2-diazinane, (3R, 4S, 5R, 6S)-l,6-dimethyl-3,4,5-trihydroxy-l,2-diazinane, (3S, 4S, 5R, 6S)-l,6-dimethyl-3,4,5-trihydroxy-l,2-diazinane, (3S, 4R, 5R)-3-methyl-4,5- dihydroxy-l,2-diazinane, (3S, 4R, 5R)-l-acetyl-3-methyl-4,5-dihydroxy-l,2-diazinane, (3S, 4R, 5R)- 1 -acetyl-4, 5 -diacetoxy-3 -methyl- 1,2-diazinane.

2. A compound according to claim 1, wherein Ri designates hydrogen, optionally substituted Cι-7-alkyl, optionally substituted Cι.7-alkylcarbonyl, aryl(Cι_-7-alkoxy)carbonyl, aminocarbonyl, optionally substituted Cι-7-alkylaminocarbonyl, di(optiona!ly substituted Cι_-7-alkyl)amino- carbonyl, or optionally substituted G.₇-alkylcarbonyloxy; or Ri designates two Cι.₇-alkyl groups; and R₂ designates hydrogen or optionally substituted C].₇-alkyl; and each of R₃, R,, R₅, and Rg independently designates hydrogen, hydroxy, halogen, optionally substituted Cι.₇-alkyl, amino, optionally substituted Cι_-7-alkylamino, di(optionally substituted Cι.₇-alkyl)amino, O 97/31901 26 PC17DK97/00090 tri(optionally substituted Cι.7-alkyl)ammonium, carboxy, carboxyamino, optionally substituted Cι.7-alkylcarbonylamino, optionally substituted arylcarbonylamino, sulphanyl, Cι.₇-alkylthio, cyano, azido, optionally substituted aryl, optionally substituted Cι.7-alkoxycarbonyl, amino¬ carbonyl, optionally substituted G-7-alkylaminocarbonyl, di(optionally substituted Cι-₇-alkyl)- aminocarbonyl, optionally substituted C_2-7-alkenyloxy, optionally substituted C₁-₇- alkylcarbonyloxy, or -CH₂-O-X or -O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide: or a salt thereof.

3. A compound according to claim 1 or 2, wherein each of Ri and R₂ independently designates hydrogen or optionally substituted C1.7-a.kyl; and each of R₃ and Re independently designates hydrogen, hydroxy, halogen, optionally substituted G-7-alkyl, carboxy, cyano, azido, optionally substituted aryl. optionally substituted Cι-7-alkoxycarbonyl, aminocarbonyl, optionally substituted Cι.7-alkylaminocarbonyl, di(optionally substituted Cι.₇-alkyl)aminocarbonyl, or -CH₂-O-X or -O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; each of R4 and R$ independently designates hydrogen, hydroxy, halogen, optionally substituted Cι_-7- alkyl, amino, optionally substituted Cι-7-alkylamino, di(optionaIly substituted Cι.₇-alkyl)amino, carboxy, carboxyamino, optionally substituted Cι-7-alkylcarbonylamino, optionally substituted arylcarbonylamino, sulphanyl, Cι-7-alkylthio, cyano, azido, optionally substituted aryl, optio¬ nally substituted G-₇-alkoxycarbonyl, aminocarbonyl, optionally substituted Cι-7-alkyl- aminocarbonyl, di(optionally substituted Cι_-7-alkyl)aminocarbonyl, optionally substituted C₂.₇- alkenyloxy, optionally substituted Cι.₇-alkylcarbonyloxy, or -CH₂-O-X or -O-X, where X designates a glycosyl group of a mono-, di- or trisaccharide; or a salt thereof.

4. A compound according to any of the claims 1-3, wherein each of Ri and R₂ independently designates hydrogen or G.7-alkyl; and each of R₃ and Re independently designates hydrogen, hydroxy, G.₇-alkyl, carboxy, cyano, azido, C].₇-alkoxycarbonyl, aminocarbonyl, C₁.₇- alkylaminocarbonyl, or di(Cι.7-alkyl)aminocarbonyl; each of R4 and R5 independently designates hydrogen, hydroxy, Cι.₇-alkyl, amino, G-7-alkylamino, di(Cι.₇-alkyl)amino, carboxy, carboxyamino, Cι_-7-alkylcarbonylamino, cyano, azido, optionally substituted aryl, Cι-7-alkoxy- carbonyl, aminocarbonyl, G^-alkylaminocarbonyl, di(Cι_-7-alkyl)aminocarbonyl, or Cι.7-alkyl- carbonyloxy; or a salt thereof. 5. A compound according to any of the claims 1-4, which is of any of the general formulae II, III, IV or V

π m

IV V

6 A compound according to claim 1 or 5, wherein Ri designates hydrogen, hydroxy, hydroxy- (Cι.7-alkyl), halogen, Cι-7-alkyl, Cι.₇-alkanoyl, Cι.₇-alkanoyloxy or Cι.₇-alkoxy, or Ri may designate two G-7-alkyl groups thereby leading to a quaternarisation of the nitrogen to which Ri (the two alkyl groups) is/are attached, R₂ designates hydrogen, hydroxy, hydroxy-(Cι.₇- alkyl), halogen, Cι.₇-alkyl, Cι_-7-alkanoyloxy, or G_-7-alkoxy, each of R₃, R , R₅, and R independently designates hydrogen, hydroxy, hydroxy-(Cι-7-alkyl), amino, Cι-₇-alkylamino, di(Cι.7-alkyl)amino, Cι.7-alkanoylamino, tri(Cι-7-alkyl)ammonium, halogen, nitro, sulphanyl, Cι.₇-alkylthio, carboxy, cyano, C_2-7-alkenyl, phenyl, Cι_-7-alkylphenyl, Cι_-7-alkyl, Cι_-7-alkanoyl, Cι.₇-alkanoyloxy or Cι.7-alkoxy; with the proviso that at least one of R₃, R₄, R$ and R designate(s) hydroxy, G.₇-alkanoyloxy, or Cι.₇-alkoxy, and at least one of R₃, R₄, R₅ and Re designate(s) hydroxy-(Cι_-7-alkyl), carboxy, cyano, C_2-7-alkenyl, phenyl, G.₇-alkylphenyl, C₁.₇- alkyl or Cι.₇-alkanoyl, or a salt thereof, wherein (a) any alkyl is optionally substituted with one or more, preferably 1-3, substituents selected from hydroxy, Cι_-7-alkoxy, carboxy, G.₇- alkoxycarbonyl, Cι.₇-alkyl carbonyl, formyl, amino, mono- and di(Cι.₇-alkyl)amino, carbamoyl, mono- and di(C_)- -alkyl)aminocarbonyl, guanidino, carbamido, Cι- -alkanoyloxy, sulphono, nitro, Cι.7-alkylthio, trihalogenalkyl and halogen, (b) any alkanoyl group is optionally substituted 1 or more, preferably 1-3, substituents selected from hydroxy, G.₇-alkoxy, carboxy, Cι_₇-alkoxycarbonyl, Cι. -alkylcarbonyl, formyl, amino, mono- and di(Cι.₇- alkyl)amino, carbamoyl, mono- and di(Cι.₇-alkyl)aminocarbonyl, guanidino, carbamido, C₁-₇- alkanoyloxy, sulphono, nitro, Cι-7-alkylthio, trihalogenalkyl and halogen; and (c) any phenyl group is optionally substituted with 1 -3 substituents selected from halogen, methoxy, trifluoromethyl, and methyl.

7. A compound according to any of the claims 1-6, which is one of 4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine

4, 5 -dihydroxy-6-methyl-hexahydropyridazine, 4,5-dihydroxy-6-fluorornethyl-hexahydropyridazine,

4-fluoro-5-hydroxy-6-hydroxymethyl-hexahydropyridazine, l-N-(2-hydroxyethyl)-4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine, 2-N-(2-hydroxyethyl)-4,5-dihydroxy-6-hydroxymethyl-hexahydropyridazine, 4,5-dihydroxy-hexahydropyridazine-6-carboxylic acid; and salt thereof.

8. A compound according to any of the claims 1-7 for use as a medicament.

9. A compound according to any of the claims 1-7 for treatment of or for controlling diabetes.

10. A compound according to any of the claims 1-7 for treatment of or for controlling cancer.

11. A compound according to any of the claims 1-7 for fighting or controlling human immunodeficiency virus.

12. The use of a compound according to any of the claims 1-7 for protecting an agricultura' crop.

13. The use of a compound according to any of the claims 1-7 for inhibiting cellulase activity.