CN1566109A - Aldose reductase inhibitor, preparation method and use thereof - Google Patents

Abstract

The invention provides compounds having a general formula (I), their preparation method and pharmaceutical compositions containing them, wherein said compounds and pharmaceutical compositions containing them can be used for treating diabetes and syndromes. The invention also provides two novel microorganisms.

Description

Aldose reductase inhibitors, process for their preparation and their use

Technical Field

The present invention relates to a novel aldose reductase inhibitor, a process for preparing the same and use thereof, and more particularly, to a compound of the general formula (I) as an aldose reductase inhibitor, a pharmaceutical composition containing the same, a process for preparing the compound of the general formula (I) by microbial fermentation and a novel microorganism used therein, and use of the compound of the general formula (I) or the pharmaceutical composition containing the same in the preparation of a medicament for preventing diabetic complications.

Background

Diabetes is a syndrome caused by absolute or relative attenuation of insulin secretion and/or insulin action, and is mainly manifested as hyperglycemia. Diabetes is classified into insulin-dependent diabetes mellitus (type I diabetes) and insulin-independent diabetes mellitus (type II diabetes), and complications may occur in the late stage of type II diabetes, in which microvascular complications include ocular disease, nephropathy, and peripheral and autonomic neuropathy, and macrovascular complications include atherosclerotic coronary heart disease and peripheral arterial disease. Diabetes, especially its complications, causes great pain to the patient and their relatives.

According to the data published by the world health organization diabetes expert committee 2002, the number of diabetes mellitus is approaching 2 billion worldwide, with 96% of patients dying from chronic complications. Chinese diabetic patients are reported to have more than 5000 million. With the acceleration of the industrialization process, the number of patients is on the rising trend.

Since insulin was used to treat diabetes, ketoacidosis, infection no longer threatened the life safety of diabetic patients. Complications from long-term diabetes remain a serious problem for diabetics as headache. Through researches on the abnormal polyol metabolic pathway of diabetic patients, the accumulation of sorbitol in cell tissues is found to be an important cause of the change of tissue structure and function, and the key enzyme in the polyol metabolic pathway is Aldose Reductase (AR). Aldose reductase uses reduced coenzyme II (NADPH) as coenzyme, and catalyzes the reduction reaction of hexose, and can convert glucose and galactose into corresponding reduction products, namely sorbitol and galactitol. Then, sorbitol is oxidized into fructose by sorbitol dehydrogenase (sorbitol dehydrogenase). Sorbitol dehydrogenase is the second enzyme in the polyol metabolic pathway, which can oxidize a variety of sugar alcohols, but not galactitol. Therefore, accumulation of galactitol is likely to occur in vivo, leading to the development of galactosemia (Zhang Lemna et al, study of aldose reductase inhibitors, division of antibiotics for foreign medicine, 1997, 18 (1): 5-10). It is noteworthy that neither glucose nor galactose are optimal substrates for AR, which is not activated when blood glucose concentrations are maintained at normal physiological levels. In physiological conditions of hyperglycemia (e.g., diabetes) hexokinase, which catalyzes the conversion of glucose to glucose-6-phosphate, is saturated, and AR is activated, which promotes the conversion of glucose to sorbitol in the body. However, the activity of sorbitol dehydrogenase is not proportionally increased, and sorbitol is not easy to pass through cell membranes due to its own polarity factor, so that sorbitol is accumulated in cells. Numerous animal and clinical trials have demonstrated that Aldose Reductase Inhibitors (ARIs) are effective in ameliorating polyol metabolic disorders in diabetic patients, thereby preventing and delaying the onset and progression of diabetic complications such as cataract, neuropathy, renal disease, etc. (Yang Hui, aldose reductase inhibitors and diabetic complications, division of foreign medicine and pharmacy, 1999, 26 (4): 217-222).

Some studies have been made on aldose reductase inhibitors, and torestat (Tolrestat), a carboxylic acid aldose reductase inhibitor developed by Ayerst, was marketed in Ireland in 1989. But subsequently failed to pass FDA approval for marketing in a large-scale randomized double-blind clinical trial for the treatment of diabetic complication neuropathy by failing to exhibit sufficient efficacy.

The first hydantoin-type ARI sorbinil (sorbinei) with higher activity both in vitro and in vivo was developed by the company fevered, which is effective against rat crystalline AR and human placental AR and has been marketed in the us, europe and japan, but was later found to cause severe allergic reactions and was terminated in clinical use (Sarges r. german Patent 2746244, 1977).

Varma et al investigated aldose reductase inhibitory activity of various flavonoids. The results show that they are active to varying degrees. However, to date, no compound has been marketed (Var SD, et al, Flavonoids as inhibitors of lens aldehyde reaction, Science NY, 1975, 188: 1215-.

Genistein with an isoflavone structure was found to have the effect of treating diabetic cataracts by Eugene de Juan et al (US5919813, US5980929, US6208099 and US 6399655).

(Genestein)

However, they did not find the activity of Genestan-forming glycosides as aglycones in the treatment of cataracts.

Although there are drugs for preventing diabetic complications, the market demand is still not satisfied. There is still a need in the market for alternative drugs, in particular of natural origin, for the prevention or treatment of diabetic complications.

Accordingly, it is an object of the present invention to provide a novel compound for treating or preventing diabetes or its complications, a pharmaceutical composition containing the same, and use thereof for treating or preventing diabetic complications.

It is another object of the present invention to provide a process for preparing such compounds and novel microorganisms for use in the process.

The invention also aims to provide a pharmaceutical composition for treating diabetes and preventing diabetic complications.

Summary of The Invention

The present inventors have conducted extensive studies on aldose reductase inhibitors, and as a result, have surprisingly found that metabolites of certain microorganisms have aldose reductase inhibitor activity, thus completing the present invention.

In one aspect, the present invention provides a compound of the following general formula (I), and pharmaceutically acceptable salts, solvates, stereoisomers or prodrugs thereof:

wherein,

R₁is OH, or

α -L-fucopyranosyl as shown or a group in which the hydroxyl group is substituted with other glycosyl or protecting group;

R₂is H or-OH

R₃is-OH or a hydroxy protecting group, or is of the formula

α -L-rhamnosyl, or a group in which the hydroxyl group is substituted with another sugar group or a protecting group, with the proviso that R₁And R₃At least one of which is a sugar radical orGlycosyl derived group.

In another aspect, the present invention provides a process for the preparation of a compound of formula (I) as defined above, which process comprises fermenting a selected actinomycete ina culture medium and isolating and purifying the resulting fermentation broth.

In another aspect, the present invention provides two novel microorganisms which can be used in the above fermentation process and which have the accession numbers CGMCC 0834 and CGMCC 0885.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) as described above or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may further comprise a commonly used known drug for treating diabetes.

In another aspect, the present invention relates to the use of a compound or composition of formula (I) as described above for the preparation of a medicament for the prevention of diabetic complications, as well as the use of a pharmaceutical composition containing known drugs for the preparation of a medicament for the treatment and prevention of diabetic complications.

Description of the drawings:

FIG. 1 is an optical micrograph of strain N99-253.

FIG. 2 is an optical micrograph of strain N99-596.

FIG. 3 phylogenetic tree of strain N99-596 and related species constructed from the 16S rDNA sequence

FIG. 4 is a phylogenetic tree of strain N99-253 and related species constructed based on the 16S rDNA sequence.

FIG. 5 is a dose-response curve for inhibition of aldose reductase by N99-596A, B.

FIG. 6 is a dose-response curve of N99-253A for aldose reductase inhibition.

Detailed description of the invention

As noted above, the present invention relates to compounds of formula (I), pharmaceutically acceptable salts, solvates, stereoisomers or prodrugs thereof, which are aldose reductase inhibitors:

wherein R is₁Is OH, or

α -L-fucopyranosyl as shown or a group in which the hydroxyl group is substituted with other glycosyl or protecting group;

R₂is H, -OH

R₃is-OH or a hydroxy protecting group, or is of the formula

α -rhamnosyl, or a group wherein the hydroxyl group is substituted with another sugar group or a protecting group, with the proviso that R₁And R₃At least 1 is a glycosyl group or a glycosyl-derived group.

According to a preferred embodiment of the invention, R in the compound of formula (I)₃Is OH, R₂Is H or-OH, R₁Is α -L-fucopyranosyl.

The above compounds can be used as aldose reductase inhibitor for preventing diabetic complication.

The aldose reductase inhibitor of the present invention may be the above-mentioned compound itself, or a pharmaceutically acceptable derivative thereof, such as a hydroxyl protecting group or a derivative in which a hydroxyl group on a sugar group is further substituted with another sugar group or protecting group, as appropriate.

Examples of the hydroxyl-protecting groupinclude monohydroxy-protected methyl, t-butyl, allyl, benzyl, trityl substituted with a methoxy group or the like, trimethylsilyl, t-butyldimethylsilyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, thienyl, formaldehyde acetal, cyclohexanone acetal, methylthiomethyl, acetoxy, benzoyloxy, o, m, p-nitrobenzoyloxy, formyloxy, trifluoroacetyloxy, chloroacetyloxy, methoxyacetoxy, phenoxyacetoxy, methoxycarbonyl, ethoxycarbonyl, isobutoxycarbonyl, benzyloxycarbonyl, 2, 2, 2-trichloroethoxycarbonyl, 2, 2, 2-tribromoethoxycarbonyl, p-nitrobenzyloxycarbonyl, phenylaminocarbonyl, benzylthiocarbonyl, pivaloyloxy, 3-benzoylacetoxy, benzoyl, succinyloxy, each acyloxy, o-benzyloxycarbonylbenzoyl, 3-phenylpropyloxy, nitro, p-toluenesulfonyloxy, 2, 4-dinitrosulfonyloxy, alkoxyacetylphenoxy, methoxymethyl, 1-ethoxyethyl, benzoyl methyl, trimethylsilyl acetal, triethylsilyl acetal, β -cyclohexylidene, isopropylidene-substituted with a carboxy-protecting group or the like, and other conventional hydroxy-protected cyclohexylidene benzoate, isopropylidene, benzylidene, benzoic acid, and the like.

In the case where the protecting group contains an acidic or basic group, the above compounds may also form a pharmaceutically acceptable salt form with a non-toxic base or acid. Examples of the acid include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid; organic acids such as acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, glycolic acid, maleic acid, malonic acid, oxalic acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, trifluoroacetic acid, fumaric acid, succinic acid, malic acid, tartaric acid, citric acid, salicylic acid, p-aminosalicylic acid, pamoic acid, benzoic acid, ascorbic acid and the like. Examples of base addition salt forms are sodium, potassium, calcium salts, and salts with pharmaceutically acceptable amines such as ammonia, alkylamines, anilines, and amino acids such as arginine and lysine, and the like.

The above compounds, derivatives and salts may form solvates, such as hydrates, alcoholates and the like. The aldose reductase inhibitors described above may also be prodrugs or forms which release the active ingredient after metabolic changes in the body. The selection and preparation of suitable prodrug derivatives is well known to those skilled in the art.

Hereinafter, for convenience, any of the compounds defined in the present invention, pharmaceutically acceptable salts thereof, solvates thereof, and stereoisomers thereof will be simply referred to as the compounds of the present invention.

In another aspect, the present invention provides a process for preparing the above compound, which comprises:

fermenting a culture of a microorganism capable of producing a compound of formula (I) by fermentation, and optionally isolating and purifying the resulting culture.

In the case where a specific compound having a specific substituent is desired, it can be obtained by adding a specific inducer to the culture broth or inducing a microorganism to produce a specific fermented product according to a method familiar to those skilled in the art.

In a specific embodiment of the present invention, the method comprises optionally culturing the microorganism on a seed culture medium to obtain a preculture, culturing (fermenting) the preculture in a fermentation medium, and isolating and purifying the resulting fermentation broth to obtain the compound of the present invention.

The obtained compound of the present invention can be further derivatized to obtain various derivatives.

The separation comprises centrifuging the fermentation broth, collecting the biomass, extracting the biomass with a solvent and removing the solvent, and the purification comprises silica gel column chromatography and selective HPLC single-component preparation, which are well known to those skilled in the art.

The method of the invention is not limited by the order described above and the medium components may employ variations thereof within the purview of one skilled in the art.

The microorganism producing the compound of formula (I) of the present invention can be isolated from Streptomyces Yunnanensis (Streptomyces diannanensis) CGMCC No.0834(N99-596) or Streptomyces murinus (Streptomyces huayunensis) CGMCC No.0885(N99-253), for example, by the present inventors. However, the present invention is not limited to these two Streptomyces species, and the compounds of the general formula (I) of the present invention can be used in fermentation production as long as they are obtained from the metabolites thereof.

The strain N99-596 was isolated from a soil sample from Yunnan province, China (FIG. 1).

^*And (3) feature description: the bacterial colony of the strain on a yeast extract-malt extract culture medium is gray, and the surface of the strain is flat and villous. Observing the developed hyphae of the strain under a microscope, wherein the hyphae are multi-branched and have no septum; spore chains or sinuous or irregular helicesAnd a hook shape. Good growth on all the media tested, air silks andthe basal filaments were all predominantly gray-brown or brown-gray, and the culture characteristics are shown in tables 1 and 2.

TABLE 1 cultural characteristics of Strain N99-596

Culture medium	Aerial hypha	Intrabasal hypha	Soluble pigment
				Sword agar	Light brown gray	Light grey brown	Is free of
Glycerol-asparagine agar (ISP5)	Light grey brown	Grey colour	Is free of
				Starch agar inorganic salt (ISP4)	Light brown gray	Light grey brown	Is free of
Oatmeal agar (ISP3)	Light grey yellowish brown	Light brown gray	Is free of
				Yeast extract-malt extract agar (ISP2)	Grayish yellow-brown	Dark yellow-brown	Is free of
Potato extract agar	Light brown gray	Grayish brown	Is free of

TABLE 2 physiological and biochemical characteristics and carbon Source utilization status of Strain N99-596

Physiological and biochemical processes	N99-596	Carbon source utilization	N99-596
				Liquefaction of gelatin	+	Glucose	+
Milk coagulation	+	Arabinose	+
				Milk peptone	-	Sweet mellow wine	+
Starch hydrolysis	-	Sucrose	+
				Nitrate reduction	+	Glycerol	+
Hydrogen sulfide generation	-	Fructose	+
				Melanogenesis	-	Galactose	+
Cellulose growth	-	Maltose	+
				Utilization of urea	+	Mannose	+
		Cotton seed candy	+
				Antimicrobial spectrum		Xylose	+
E.coli	-	Sorbitol	+
				B.subtilis	-	Sodium oxalate	+
Candida albicans	-	Sodium acetate	+
				Aspergillus niger	-	Melibiose	+

^*16S rDNA sequence and phylogenetic analysis:

^*the 16S rDNA partial sequence of the strain N99-596 is shown as sequence 1. The 16S rDNA of N99-596 is compared with the known 16S rDNA of a plurality of strains, and the obtained strains N99-596 constructed according to the 16S rDNA sequence and phylogenetic trees of related strains are shown in figure 3.

The strains aligned were as follows:

TABLE 3

Name of scholars	Plant strain	Numbering
			Streptomyces sp	N99-596
Streptomyces argenteolus	JCM 4623	AB045872
			Streptomyces ornatus	DSM 40307	X79326
Streptomyces caviscabies	ATCC 51928	AF112160
			Streptomyces setonii	ATCC 25497	D63872
Streptomyces avermitilis	NCIBM 12804T	AF145223
			Streptomyces virginiae	IFO 3729	D85119
Streptomyces lavendulae	IFO 12341	D85110
			Streptomyces cyaneus	NRRL B-2296	AF346475
Streptomyces indigocolor	NRRL B-12336	AF346474
			Streptomyces mirabilis	ATCC 27447	AF112180
Streptosporangium roseum	DSM 43021	X89947

In conclusion, the strain No. N99-596 liquefies gelatin, coagulates milk, reduces nitrate, is positive, does not grow on cellulose, does not produce hydrogen sulfide and melanin, does not produce urease, and can utilize almost all tested carbon sources. The cell wall contains L-DAP, cell wall type I. According to the morphological characteristics and the phylogenetic analysis based on the 16S rDNA sequence, the strain N99-596 is determined as a new species of streptomyces yunnanensis (Streptomyces diannanensis). N99-596 has been preserved in China general microbiological culture Collection center (CGMCC) at 11.15.2002 with the preservation number of CGMCCN 0.0834.

Strain N99-253 was isolated from a soil sample from Yunnan province, China (FIG. 2).

^*And (3) feature description: the bacterial colony of the strain is gray on a yeast extract-malt extract culture medium, and a small hydrolysis ring is arranged around the bacterial colony. Observing the developed hyphae of the strain under a microscope, wherein the hyphae are multi-branched and have no septum; spore chains or sinuous or irregular helices. Good growth was achieved on all media tested, both air and basal filaments were predominantly gray-brown or brown-gray, and the culture characteristics are shown in Table 4.

TABLE 4 culture characteristics of Strain N99-253

Culture medium	Aerial hypha	Intrabasal hypha	Soluble pigment
				Sword agar	Light brown gray	Light grey brown	Is free of
Glycerol-asparagine agar (ISP5)	Light grey brown	Grey colour	Is free of
				Starch agar inorganic salt (ISP4)	Light brown gray	Light grey brown	Is free of
Oatmeal agar (ISP3)	Brown grey	Light brown gray	Is free of
				Yeast extract-malt extract agar (ISP2)	Brown grey	Dark yellow-brown	Is free of
Potato extract agar	Light brown gray	Grayish brown	Is free of

^*16S rDNA sequence and phylogenetic analysis:

^*the 16S rDNA partial sequence of the strain N99-253 is shown as a sequence 2. The 16S rDNA of N99-253 was compared with the known 16S rDNA of several strains, and the phylogenetic tree of the strain N99-253 and related strains constructed based on the 16SrDNA sequence was shown in FIG. 4.

The strains aligned were as follows:

TABLE 5

Name of scholars	Plant strain	Numbering
			Streptomyces sp.	N99-253
Streptomyces argenteolus	JCM 4623T	AB045872
			Streptomyces flavogriseus	CBS 101.34＝DSM 40323T	AJ494864
Streptomyces caviscabies	ATCC 51928T	AF112160
			Streptomyces setonii	ATCC 25497T	D63872
Streptomyces ornatus	DSM 40307T	X79326
			Streptomyces cyaneus	ISP 5108T	A0899460
Streptomyces venezuelae	JCM 4526T	AB045890
			Streptomyces galilaeus	JCM 4757T	AB045878
Streptomyces bobili	JCM 4624T	AB045846

Phylogenetic trees of strain N99-253 and related species constructed based on the 16S rDNA sequence are shown in FIG. 4.

N99-253 was able to utilize almost all of the carbon sources tested. The cell wall contains L-DAP, cell wall type I. According to the morphological characteristics and the phylogenetic analysis based on the 16S rDNA sequence, the strain N99-253 is a new species of streptomyces and is named as streptomyces huayunensis (Streptomyces huayunensis). N99-253 is preserved in China general microbiological culture Collection center (CGMCC) at 21.1.2003 with the preservation number of CGMCC No. 0885.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising as an active ingredient a compound of formula (I) as defined above and a pharmaceutically acceptable carrier. The preparation of pharmaceutical compositions is a method commonly used in the art.

The compounds described herein, or pharmaceutically acceptable addition salts or hydrates thereof, can be delivered to a patient using a variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, enteral and parenteral administration, including intramuscular, subcutaneous and intravenous injection.

The term "administering" as used herein includes all means of directly and indirectly releasing a compound to its intended site of action.

The compounds described herein or pharmaceutically acceptable derivatives thereof may be administered alone or in combination with other compounds of the invention, and/or in combination with other known diabetes or diabetic complication therapeutic agents.

The active compounds of the present invention may be administered as such, or in the form of a pharmaceutical composition in which the active compound is in admixture with one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutical compositions for use in accordance with the present invention are generally formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen and may be manufactured in accordance with common general knowledge in the art.

For example, for the treatment of diabetes, it is often advantageous to employ an oral formulation. The oral pharmaceutical preparation comprises capsules, tablets and the like. When the patient has difficulty swallowing, the medicine can also be administered by sublingual tablet or other non-swallowing mode.

The compounds of the invention may also be formulated for parenteral or transdermal or transmucosal administration. Or by means of suppositories or implants.

It will be appreciated by those skilled in the art that, based on the compounds of the present invention, suitable Drug Delivery Systems (DDS) may be employed to achieve a more advantageous effect.

Since diabetes is a chronic disease, an oral dosage form that can be taken for a long period of time is economically advantageous to patients.

Preferably, the composition is in unit dosage form, such as a tablet or capsule.

The mode of administration and the choice of effective dose will vary depending, inter alia, on the condition to be treated. The choice of mode of administration and dosage is within the ability of those skilled in the art.

Unit dosage forms of the compounds of the invention will typically contain from 0.1 to 99% by weight active, more typically from 5 to 75% by weight active. For example, a unit dosage form may contain 1mg to 1g of the compound, more typically 10mg to 500mg, for example between 50mg and 400mg, typically at a dose of 100mg to 200 mg.

Each dosage unit or each oral administration preferably contains from 1 to 250mg (in relation to parenteral administration, preferably from 0.1 to 25mg) of a compound of formula (I) or a pharmaceutically acceptable derivative thereof, administered 3 times daily or as determined by the number of meals.

The compounds of the present invention will be administered in an amount effective to provide the desired therapeutic effect. The concentration necessary to provide the desired therapeutic effect will vary depending on, inter alia, the precise nature of the disease, the age, weight and severity of the disease in the patient.

In general, the compounds of the invention will be administered in an amount in the range of from 0.01mg/kg to 100mg/kg body weight, more preferably from 0.1mg/kg to 10mg/kg body weight, especially from 1mg/kg to 5mg/kg body weight.

Pharmaceutically acceptable compounds of the invention will normally be administered to a subject according to a daily dosage regimen. In the case of adult patients, this may be, for example, an oral dose of between 1mg and 500mg, preferably between 1mg and 250mg, or an intravenous, subcutaneous or intramuscular dose of between 0.1mg and 100mg, preferably between 0.1mg and 25mg, of a compound of formula (I) or a pharmaceutically acceptable salt thereof, administered in 1 to 4 divided doses per day, calculated as the free compound. Thus, for an average human weighing 70kg, a typical daily dose of a compound of the invention will be in the range of 70mg to 700 mg. Such a dose may be administered, for example, 3 times or 2 to 4 times per day, depending on the number of meals eaten.

Ultimately, however, the size of the dose administered and the frequency of administration will be determined and judged by thephysician treating the patient.

The compounds of the invention may also optionally be administered in combination with known agents for the treatment of diabetes. An effective amount of a medicament for treating diabetes can be added into the composition. Drugs for the treatment of diabetes are known to those skilled in the art, for example: tolbutamide, acetohexamide, glyburide, glipizide, gliclazide, gliquidone, glimepiride, phenformin, metformin, repaglinide, nateglinide, rosiglitazone, pioglitazone, acarbose, voglibose, eprapastat, chlorpropamide, glibornuride, gliquidone, glimepiride, xylitol, chitosan, or the like.

When the compounds of the present invention are administered in combination with known agents for the treatment of diabetes, they may be administered simultaneously, separately or sequentially. Or making into compound preparation.

The compounds of the present invention are useful for the prevention or treatment of diabetic complications, wherein the diabetic complications are selected from the group consisting of: diabetic nephropathy, diabetic eye disease, diabetic nervous system disease, diabetic heart disease, diabetic arteriosclerosis and diabetic microangiopathy, preferably diabetic nephropathy, diabetic eye disease, diabetic nervous system disease or diabetic microangiopathy.

The principle of the compounds of the present invention for preventing diabetic complications is described in the background section, and there is an inositol loss hypothesis as to the relationship of the polyol pathway to diabetic complications. This hypothesis explains the present invention in principle, but the present invention is not limited by this theory in any way, because the compounds of the present invention have actually played a role in preventing diabetic complications.

Inositol loss hypothesis

Greene D found that accumulation of sorbitol in nerve cells leads to loss of myo-inositol, and that the use of ARI can effectively prevent myo-inositolLoss of alcohol, and thus proposed the inositol loss hypothesis that inositol is a major component in the synthesis of a cell membrane lipid called inositol polyphosphate phospholipid, which is closely related to the structure and function of the cell membrane. Glucose has a similar spatial three-dimensional structure to that of inositol, so an increase in glucose concentration in tissues can competitively inhibit the inositol-transporting system. Phosphorylated derivative of inositol phosphoacyl inositol diphosphate (PIP)₂) After hydrolysis, two compounds with specific properties can be produced-diglyceride and inositol 1, 4, 5-triphosphate. Among them, diglycerides activate protein kinase C in the lipid bilayer of cell membranes. Protein kinase C can activate Na⁺/K⁺ATP-ase, and entry of myo-inositol into the cell depends on Na⁺/K⁺-an ATPase. Therefore, hyperglycemia due to diabetes causes a decrease in the intracellular entry of inositol, an increase in the activity of AR, and accumulation of sorbitol, which in turn form a circulation, eventually leading to loss of inositol. Loss of inositol from peripheral nerves Na in the interstitium of neurites⁺Accumulate, causing block of conduction of nerve electrical impulses. Raskin studies in diabetic mice and galactose-fed rat models also demonstrated inositol loss and Na in the glomeruli and retina⁺/K⁺Decrease in ATPase Activity and sorbitol accumulationThe product is related.

The above hypothesis is only used for understanding the present invention.

In another aspect of the present invention, the present invention provides the use of a compound of the present invention and a composition comprising a compound of the present invention for the manufacture of a medicament for the prevention or treatment of diabetic complications, wherein the complications are as described above.

In another aspect of the present invention, the present invention provides the use of the composition of the present invention containing a drug for treating diabetes for the preparation of a drug for treating diabetes and preventing diabetic complications, wherein the diabetic complications are as described above.

The invention also provides a screening method of the aldose reductase inhibitor.

The activity of aldose reductase inhibitors has been generally measured by catalyzing an aldehyde group-containing saccharide with aldose reductase in the presence of NADPH as a coenzymeSimultaneous conversion of NADPH to NADP⁺(nicotinamide adenine dinucleotide phosphate), and NADP⁺The characteristic absorption of the reduced NADPH of the contained pyridine ring at 340nm can be indirectly measured by measuring the decrease rate of optical density at 340 nm. The source of aldose reductase may be bovine, porcine, monkey, murine, human, etc., or may be from different organs such as seminal vesicle, brain, placenta, etc. However, according to the operation reported in the literature, the screening model has the following difficulties:

(1) aldose reductase is extracted from pig eyes dug from slaughterhouses by itself, and has different activity each time.

(2) The coenzyme NADPH is extremely unstable.

(3) The measurement index is the change of OD value per minute, the accuracy is required to be 0.001, and the precision of a general spectrophotometer is not enough.

(4) Some samples are turbid when added to the test living system, which causes interference.

A biochemical analyzer with higher automation degree is selected, and a screening and activity measuring method is improved aiming at the difficulties. The total volume of the assay system was reduced to 200 microliters, which reduced the amount of enzyme and reagent used. Automated programming reduces accidental errors in manual operations and improves the precision of the measurements. Since a large number of samples are assayed simultaneously, which requires a high system stability for each assay, the activity of the enzyme is assayed before each assay, and the enzyme is used for screening after confirming that the change in OD value is within a suitable range as a control. NADPH was newly added each time, and a blank was simultaneously measured. And a solvent with good solubility on the sample and small influence on the enzyme activity is selected, so that the turbidity is reduced. In order to identify whether or not a sample that was initially positive had an effect on NADPH and had an increased OD value, the change in OD value was recorded without addition of aldose reductase, and if the OD value had increased, it was false positive and rejected. The method for efficiently screening the aldose reductase inhibitor is used for screening microbial metabolites, and the screening accuracy and stability are improved.

Specifically, the activity measuring method of the invention uses DL-glyceraldehyde as a substrate and NADPH as a coenzyme to measure the enzyme activity:

aldose reductase catalyzes the reduction of DL-glyceraldehyde to glycerol in the presence of NADPH, while NADPH is converted to NADP⁺And the characteristic absorption of the reduced NADPH at 340nm can be measured, and the reduction rate of optical density at 340nm can be measured to indirectly measure the activity of aldose reductase.

The specific operation is that buffer solution, enzyme solution and samples are added into each sample well in a 96-well plate; the control wells were solvent substituted for the samples, the rest being the same; blank wells were replaced with solvent for sample and buffer for enzyme solution, and the rest was the same. Mixing, keeping the temperature for 15 minutes, and adding Li₂SO₄DL-glyceraldehyde and NADPH were reacted, and the decrease in optical density (. DELTA.OD/min) per minute at a wavelength of 340nm was measured at the same temperature as the incubation.

The present invention will be described in further detail with reference to the following examples, which are not to be construed as limiting the invention

Any limitation of the scope of the invention.

Instruments and reagents are commonly used by those skilled in the art.

Materials:

NADPH (Beijing Xin Jingke biotech Co., Ltd.)

Fresh porcine eyeball (Shijiazhuang meat Co-factory).

Mercaptoethanol, PMSF (phenylmethylsulfonyl fluoride, Sigma Co.)

1. Slant culture medium: 4% of glucose, 4% of yeast powder, 5% of malt extract, 2.0% of agar powder, 0.035% of compound VB, 0.001% of trace salt and pH 7.2;

2. seed culture medium: contains starch 2.4%, glucose 0.1%, peptone 0.3%, beef extract 0.3%, yeast extract 0.5%, CaCO₃0.4％，pH7.0；

3. Fermentation medium:

medium No. 46: solubility in waterStarch 4.0%, defatted Soybean flour 2.0%, 0.1mol/LNaS₂O₄3.2 microliter, FeSO₄·7H₂O 0.05％，K₂HPO₄0.05％，KCl 0.03％，pH6.5；

Medium No. 48: contains glucose 4.7%, peptone 0.4%, yeast powder 0.1%, beef extract 0.4%, NaCl 0.2%, CaCO₃0.5％，pH7.0

Example 1

Culture method

Inoculating actinomycete N99-596 slant to seed culture medium, culturing at 27 deg.C for 72hr, inoculating to 750ml triangular flask containing 140ml culture medium, and culturing in fermentation medium at 27 deg.C under shaking for 6 days.

Separation method

Centrifuging 5000mL of N99-596 fermentation broth at 3000rpm for 15min, collecting thallus and supernatant, extracting thallus with 2000mL acetone, evaporating acetone, extracting with 2500mL ethyl acetate twice, passing ethyl acetate layer through anhydrous Na₂SO₄After dehydration, concentration and suction drying, 2.2g of brown substance was obtained.

A2.2 g sample was taken, dissolved in a small amount of methanol, and further separated and purified by column chromatography on silica gel (. phi.2.5X 25cm) under 100% CHCl₃Elute to 100% MeOH, collect the combined active fractions, concentrate and pump to dryness to give 85.2mg of a brown solid.

Taking the above active substance, using ODS reverse phase column (Phenomenexods. phi. 20X 250mm), single component preparation was performed on preparative HPLC (mobile phase 30% CH)₃CN at a flow rate of 6ml/min and a detection wavelength of 254nm) to give yellow substances N99-596A (12.1mg) and B (9.6 mg).

The analysis method comprises the following steps:

1mg of each N99-596A, B is taken, 1ml of methanol is added, the volume is regulated to 1mg/ml, and the HPLC analysis conditions are as follows:

sample preparation: N99-596A, B (1mg/ml)

HPLC: high-pressure liquid phase pump: waters 515 (double pump)

A detector: 2487 ultraviolet detector

A chromatographic column: kromasil 100_ C₁₈4.6mm×250mm

Mobile phase: 25% acetonitrile-water

Flow rate: 1ml/min

Detection wavelength: 254nm

Column temperature was room temperature (22-25 ℃ C.)

Sample introduction amount: 10 μ l

Retention time: a: 9-10 min, B: 19-20 min

The purities of N99-596A, B are all more than 98 percent

Physical and chemical properties of N99-596A, B:

molecular weight: a is as follows: 416, B is: 432

The molecular formula is as follows: a is C₂₁H₂₀O₉B is C₂₁H₂₀O₁₀

Ultraviolet absorption: λ of A_max249nm, lambda of B_max260nm

Structure of N99-596A, B

According to the physicochemical property and the nuclear magnetic resonance spectrum, the chemical structures of the component A and the component B are determined as follows:

N99-596A N99-596B

of component N99-596A, B¹³C-NMR、¹H-NMR was as follows:

N99-596 A：

¹H-NMR(δ，ppm)8.29(1H，s，2-CH)7.95(1H，d，J＝9Hz，5-CH)7.28(1H，d，J＝2Hz，2’-CH)7.05(1H，dd，J＝8.5，2Hz，5’-CH)6.93(1H，dd，J＝9，2.5Hz，6-CH)6.87(1H，d，J＝8.5Hz，6’-CH)6.86(1H，d，J＝2.5Hz，8-CH)5.27(1H，s，1”-CH)3.90(1H，m，3”-CH)3.65-3.79(2H，m，2”，5”-CH)3.16(1H，m，4”-CH)1.13(3H，d，J＝6.5Hz，6”-CH₃)

¹³C-NMR(δ，ppm)174.59(4-C)162.51(7-C)157.39(9-C)154.584(2-C)147.85(4’-C)143.69(3’-C)127.31(5-C)123.45(6’-C)123.20(1’-C)122.97(3-C)119.45(2’-C)116.63(5’-C)116.31(10-C)115.15(6-C)102.09(8-C)99.94(1”-C)71.94(4”-C)70.33(3”-C)70.17(2”-C)69.48(5”-C)17.85(6”-C)

N99-596 B

¹H-NMR(δ，ppm)8.34(1H，s，2-CH)7.28(1H，d，J＝2Hz，2’-CH)7.07(1H，dd，J＝8.5，2Hz，5’-CH)6.88(1H，d，J＝8.5Hz，6’-CH)6.38(1H，d，J＝2.5Hz，8-CH)6.22(1H，d，J＝2.5Hz，6-CH)5.27(1H，s，1”-CH)3.90(1H，m，3”-CH)3.65-3.79(2H，m，2”，5”-CH)3.16(1H，m，4”-CH)1.05(3H，d，J＝6.5Hz，6”-CH₃)

¹³C-NMR(δ，ppm)179.90(4-C)164.09(5-C)161.82(7-C)157.36(9-C)153.76(2-C)147.90(4’-C)143.69(3’-C)123.29(2’-C)121.91(3-C)121.51(1’-C)120.99(6’-C)115.96(5’-C)106.73(10-C)99.92(6-C)98.83(1”-C)93.48(8-C)71.86(4”-C)70.28(3”-C)69.96(2”-C)69.25(5”-C)17.49(6”-C)

example 2

Preparation of aldose reductase

The following operations are all carried out at 0 to 4 ℃. Approximately 90g of porcine lenses were removed from each batch, homogenized in three volumes of cold NaPi buffer, and centrifuged at 10000 Xg for 50 minutes to remove insoluble material. (NH) is added to the supernatant₄)₂SO₄To 40% saturation, gently stirring for 15 minutes, centrifuging to remove the precipitate, and adding (NH)₄)₂SO₄To 50% saturation, gently stirred for 15 minutes, centrifuged to remove the precipitate, and (NH) added₄)₂SO₄To 75% saturation, the precipitate was collected by centrifugation with gentle stirring for 15 minutes, and washed with cold 0.05mol/L NaPi (containing 0.5mmol/L PMSF and 0.5 mmol/L)EtSH) and dialyzed overnight against a 0.05mol/L NaPi solution (containing 0.5mmol/L PMSF and 0.5mmol/L EtSH). After dialysis, the enzyme solution was added to glycerol to a concentration of 40% and stored at-20 ℃ for further use.

Method for determining activity of compound:

taking appropriate amount of N99-596A, B, preparing 6mg/ml solution with methanol, and gradually diluting to 3mg/ml, 1.5ml/ml, 0.75mg/ml and 0.375 mg/ml. 50 microliters of each of the samples were added to a total of 1000 microliters of the activity-measuring system at final concentrations of 0.3mg/ml, 0.15mg/ml, 0.075mg/ml, 0.0375mg/ml and 0.01875mg/ml, respectively, and the inhibitory activity against aldose protozyme was determined.

Sample tube addition for viability assay reactions: 60mmol/L NaPi buffer solution (pH6.2) and a proper amount of enzyme solution, 50 μ L of methanol solution of the sample; control wells were added: 60mmol/L NaPi buffer solution (pH6.2) and appropriate amount of enzyme solution, 50 μ L methanol; blank well addition: 60mmol/L NaPi buffer (pH6.2), 50. mu.l methanol. The sample, control and blank wells were all moisturized at 37 ℃ for 15 minutes, and 334. mu.l of 1.2mol/L Li was added to each tube₂SO₄The reaction was started with 83. mu.l of 36mmol/L DL-glyceraldehyde and 83. mu.l of 1.5mmol/LNADPH, and the optical density decrease (. DELTA.OD/min) at 37 ℃ per minute was measured at a wavelength of 340nm using a biochemical analyzer.

As a result:

inhibitory Rate (%) of aldose reductase activity
				Drug concentration (mol/L)	N99-596A	Drug concentration (mol/L)	N99-596B
720	89.1	696	90.3
				360	82.3	348	81.6
180	56.8	174	51.5
				90	37.5	87	21.2
45	22.5	43.5	18.5
				22.5	15.8	21.75	11.8

FIG. 5 shows the IC of N99-596A, B for aldose reductase₅₀170. mu. mol/L and 165. mu. mol/L, respectively.

Derivatives of component N99-596A, B have similaractivity.

Example 3

The culture method of N99-253 was the same as that of N99-596 in example 1.

The separation method comprises the following steps:

n99-253(46) fermentation broth 5000mL was centrifuged at 3000rpm for 15 minutes, the cells and supernatant were collected separately, after the cells were extracted with 2000mL acetone, acetone was evaporated, extracted twice with 2500mL ethyl acetate, the ethyl acetate layer was purified over anhydrous Na₂SO₄After dehydration, concentration and suction drying, 1.2g of brown substance was obtained.

Taking 1.0g sample, dissolving with small amount of methanol, and further separating and purifying by silica gel column (phi 2.5 × 25cm) chromatography under CHCl elution condition₃Performing gradient elution with MeOH of 100: 0-100, collecting and combining active components, and concentrating and draining to obtain brown solid of 118 mg.

Taking the above active substances, and using ODS reversed phaseColumn (Phenomenex ODS. phi. 20X 250mm) Single component preparation on preparative HPLC (mobile phase 30% CH)₃CN flow rate of 6ml/min, detection wavelength of 210nm) to give yellow substances N99-253A (11.1mg) and N99-253B (9.6 mg).

2.2.3 analytical methods:

1mg of each of N99-253A, B was taken, 1ml of methanol was added to the solution to be dissolved in 1mg/ml, and the HPLC analysis conditions were as follows:

sample preparation: N99-253A, B (each 1mg/ml)

HPLC: high-pressure liquid phase pump: waters 515 (double pump)

A detector: 2487 ultraviolet detector

A chromatographic column: kromasil 100_ C₁₈4.6mm×250mm

Mobile phase: 25% acetonitrile-water

Flow rate: 1ml/min

Detection wavelength: 254nm

Column temperature was room temperature (22-25 ℃ C.)

Sample introduction amount: 10 μ l

Retention time: a: 10-12min, B: 13-15min

The purities of N99-253A, B are all more than 98 percent.

2.2.4 physicochemical Properties of N99-253A, B:

molecular weight: A. b is as follows: 416

The molecular formula is as follows: A. b is all C₂₁H₂₀O₉

Ultraviolet absorption: λ of A_max261nm, lambda of B_max261nm

2.2.5 Structure of N99-253

According to the physical and chemical properties and the nuclear magnetic resonance spectrum, the chemical structure of the A, B component is determined as follows:

253A

253B

of N99-253A, B¹H-NMR and¹³C-NMR was as follows:

N99-253 A

¹H-NMR(δ，ppm)8.42(1H，s，2-CH)7.40(2H，d，J＝8.5Hz，2’，6’-CH)6.84(1H，d，J＝2Hz，8-CH)6.67(2H，d，J＝8.5Hz，3’，5’-CH)6.43(1H，d，J＝2Hz，6-CH)5.26(1H，d，J＝8.0Hz，1”-CH)4.16(1H，m，3”-CH)3.84(1H，m，5”-CH)3.71(1H，m，2”-CH)3.37(1H，m，4”-CH)1.12(3H，d，J＝6.5Hz，6”-CH₃)

¹³C-NMR(δ，ppm)180.49(4-C)163.22(7-C)161.66(5-C)157.51(9-C)157.25(4’-C)154.56(2-C)130.18(2’，6’-C)122.55(3-C)121.00(1’-C)115.09(3’，5’-C)105.98(10-C)99.37(6-C)98.36(1”-C)94.28(8-C)71.88(4”-C)71.47(2”-C)69.92(3”-C)66.93(5”-C)16.08(6”-C)

N99-253B

¹H-NMR(δ，ppm)8.42(1H，s，2-CH)7.39(2H，d，J＝8.5Hz，2’，6’-CH)6.86(1H，d，J＝2Hz，8-CH)6.83(2H，d，J＝8.5Hz，3’，5’-CH)6.48(1H，d，J＝2Hz，6-CH)5.57(1H，s，1”-CH)3.85(1H，m，3”-CH)3.65-3.79(1H，m，5”-CH)3.29-3.654(1H，m，2”-CH)3.17(1H，m，4”-CH)1.12(3H，d，J＝6.5Hz，6”-CH₃)

¹³C-NMR(δ，ppm)180.52(4-C)161.75(7-C)161.68(5-C)157.51(9-C)157.21(4’-C)154.58(2-C)130.18(2’，6’-C)122.58(3-C)121.02(1’-C)115.10(3’，5’-C)106.09(10-C)99.65(6-C)98.38(1”-C)94.59(8-C)71.57(4”-C)70.24(3”-C)70.07(2”-C)69.83(5”-C)17.85(6”-C)

2.2.7 biological Activity of N99-253A

The activity was measured according to the method of example 2.

The measurement result shows that the inhibitory activity IC of N99-253A on AR₅₀It was 200. mu. mol/L. Derivatives of component N99-253A have similar activities.

Sequence listing

<110>limited liability company of North China pharmaceutical group

<120>aldose reductase inhibitors, preparation method and use thereof

<130>IDC020023

<140>CN

<141>2003-07-09

<160>2

<170>PatentIn version 3.1

<210>1

<211>1487

<212>DNA

<213>Streptomyces diannanensis

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cgcctcgcgg catcgcagct cattgtaccg gccattgtag cacgtgtgca gcccaagaca 300

taaggggcat gatgacttga cgtcgtcccc accttcctcc gagttgaccc cggcagtctc 360

ctgtgagtcc ccatcacccc gaagggcatg ctggcaacac agaacaaggg ttgcgctcgt 420

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accgaccaca aggggggcac catctctgat gctttccggt gtatgtcaag ccttggtaag 540

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gcggcaccga cgacgtggaa tgtcgccaac acctagttcc caacgtttac ggcgtggact 720

accagggtat ctaatcctgt tcgctcccca cgctttcgct cctcagcgtc agtaatggcc 780

canagatccg ccttcgccac cggtgttcct cctgatatct gcgcatttca ccgntacacc 840

aggaattccg atctccccta ccacactcta gcctgcccgt atcgactgca gacccggggt 900

taagccccgg gctttcacaa ccgacgcaac aagccgccta cgagctcttt acgcccaata 960

attccggaca acgcttgcgc cctacgtatt accgcggctg ctggcacgta gttagccggc 1020

gcttcttctg caggtaccgt cactctcgct tcttccctgc tgaaagaggt ttacaacccg 1080

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cccactgctg cctcccgtag gagtctgggc cgtgtctcag tcccagtgtg gccggtcgcc 1200

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ccagactcct acgggaggca gcagtggggaatattgcaca atgggcgaaa gcctgatgca 360

gcgacgccgc gtgagggatg acggccttcg ggttgtaaac ctctttcagc agggaagaag 420

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attggggtct gcaactcgac cccatgaagt cggagttgct agtaatcgca gatcagcatt 1320

gctgcggtga atacgttccc gggccttgta cacaccgccc gtcacgtcac gaaagtcggt 1380

aacacccgaa gccggtggcc caaccccttg tgggagggag ctgtcgaagg tgggactggc 1440

gattgggacg aagtcgtaac aaggtagccg tacgga1476

Claims (12)

1. A compound of the following general formula (I) and derivatives, pharmaceutically acceptable salts, solvates, stereoisomers or prodrugs thereof:

wherein,

R₁is hydroxy, or

α -L-fucopyranosyl as shown or a group in which the hydroxyl group is substituted with other glycosyl or protecting group;

R₂is H or hydroxy;

R₃is a hydroxyl group or a hydroxyl protecting group, or is represented by the formula

α -L-rhamnosyl, or a group in which the hydroxyl group is substituted with another sugar group or a protecting group, with the proviso that R₁And R₃At least one of which is a glycosyl.

2. A compound according to claim 1, wherein R₁is-OH, R₃Is composed of

3. A compound according to claim 1, wherein R₃Is H, R₂is-OH, R₁Is α -L-fucopyranosyl.

4. A process for preparing a compound of claim 1 comprising

Culturing a microorganism producing a compound of the general formula (I) by fermentation, and then isolating and purifying the resulting fermentation broth.

5. The process according to claim 4, wherein the microorganism is Streptomyces WAYUNNIANSIS (Streptomyces huayunnensis) CGMCC No.0885 or Streptomyces Diannanensis (Streptomyces diannannensis) CGMCC No. 0834.

6. The method of claim 4, wherein the isolation method comprises centrifugation of the fermentation broth, collection of the biomass, extraction of the biomass with a solvent, and removal of the solvent, and the purification method comprises silica gel column chromatography and selective HPLC single-component preparation.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 as an active ingredient and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, further comprising a known agent for treating diabetes.

9. Use of a compound according to any one of claims 1 to 3 or a composition according to claim 7 or 8 for the manufacture of a medicament for the prevention or treatment of diabetes and/or diabetic complications.

10. The use of claim 9, wherein the diabetic complication is selected from the group consisting of: diabetic nephropathy, diabetic eye disease, diabetic nervous system disease, diabetic heart disease, diabetic arteriosclerosis and diabetic microangiopathy.

11. The use of claim 10, wherein the complication is diabetic nephropathy, diabetic eye disease, diabetic neuropathy or diabetic microangiopathy.

12. Use of a compound of the following general formula (I) and derivatives, pharmaceutically acceptable salts, solvates, stereoisomers or prodrugs thereof in the manufacture of a medicament for the prophylaxis or treatment of diseases for which an aldose reductase inhibitor is indicated:

wherein,

R₁is hydroxy, or

α -L-fucopyranosyl as shown or a group in which the hydroxyl group is substituted with another glycosyl group or a protecting group, or

α -rhamnosyl shown or a group wherein its hydroxyl group is substituted with other glycosyl or protecting group;

R₂is H or hydroxy

R₃Is a hydroxyl group or a hydroxyl protecting group, or is represented by the formula

α -rhamnosyl, or a group wherein the hydroxyl group is substituted with another sugar group or a protecting group, with the proviso that R₁And R₃At least one of which is a glycosyl group.

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