NOVEL GLYCOSIDE COMPOUNDS AND PRODUCTION AND USE THEREOF

Abstract: Steroidal and non-steroidal glycosides and a method of producing them are provided, wherein aglycon compounds are glycosylated with tri-O-acyl glucal using molecular or ionized halogen as reaction catalyst. The aglycon, which may be a non-steroid or a steroid having a reactive functional group (e.g., -OH, -SH, -COOH, -NH2, -NHR1) such as a cholesterol, is glycosylated, wherein the glycosylation is performed in a single step. For example, a steryl pyranoside is oxidized to the corresponding 7-ketosteryl di-O-acyl-pyranoside, which is selectively reduced to provide the corresponding 7-beta-hydroxysteryl 2,3-dideoxy-alpha-D-erythro-hex-2-enopyranoside. These steroidal and non-steroidal glycosides possess valuable pharmacological properties. In particular, the cholesterol glycoside exhibits a selective cell-destructive activity on malignant cell, in vivo, and is substantially free of side effects on normal cells. The glycosides possess useful pharmacological properties which are the same as their respective unglycosylated aglycons, which properties include a drive-enhancing (stimulating) activity and an anti-inflammatory (immunosuppressive or immunoregulatory) activity.

Description: NOVELGLYCOSIDECOMPOUNDSAND PRODUCTIONANDUSETHEREOF Field of the Invention This invention relates to a surprisingly novel method for the production of a broadly novel type of glycoside. The method comprises glycosylation (i.e., glycosidation) of an aglycon compound having a functional group, e.g., a hydroxy compound such as a hydroxy-steroid or hydroxy-non-steroid. The invention importantly relates to the resulting glycosides as novel compounds of diverse application having desired properties including pharmacodynamic properties; and to medicaments containing the compounds. It has been surprisingly found according to the invention that an aglycon compound — in a preferred embodiment, a hydroxy-steroid to be understood as a steroidal alcohol or steroidal phenol — can be reacted in one step with a glycosidic vinyl ether 3,4, 6-tri-O-acyl-D- glucal of formula where Ac is a lower acyl group, preferably a C 1 -4 acyl group, in the presence of molecular halogen as a catalyst, such as iodine I2, chlorine CI2, bromine Br2 and fluorine F2, preferably iodine, to provide the corresponding glycoside in high yield. Thus there is no need for expensive and/or toxic reagents in this reaction step. Further, as a preferred aspect of the invention, a steroidal glycoside — a 3-β-ol cholesterol pyranoside which is cholesteryl 4, 6-di-O-acetyl- 2, 3-dideoxy-α-D-erythro-hex-2-enopyranoside, obtainable by this method — has been found to be applicable as a pharmacologically active agent for use as a medicament, especially as an anti-neoplastic agent, or in geriatric medicine, or as a sedative or activity-enhancing agent. For convenience in describing the invention, the 4, 6-di-O-acyl (or acetyl)-2,3-dideoxy-α-D-erythro-hex-2-enopyranoside will sometimes be referred to herein simply as a DDH pyranoside. In the accompanying drawings with reference to preferred examples of the invention: FIGURE 1 is an infrared spectrum of the glucal used in the reaction of Example 1; FIGURE 2 is an infrared spectrum of the glycosylation product of Example 1; FIGURE 3 is an NMR spectrum of the same glycosylation product of Example 1; FIGURES 4 and 5 are the IR-spectrum and the NMR- spectrum, respectively of the ketone product of Example 2; FIGURES 6 and 7 are the IR-spectrum and the NMR- spectrum, respectively, of the 7β-OHC product of Example 3; FIGURE 8 is a plot showing the tumor cell growth inhibition by selected concentrations of 7β-OH cholesterol in cell culture fluid; According to one preferred embodiment, the invention concerns a method for the production of a pyranoside compound, wherein an aglycon compound selected from aliphatic, alicyclic, aliphatic-aromatic or aromatic compounds having a primary, secondary or tertiary functional group preferably selected from -OH, -SH, -COOH, - H2, and - NHRi, where R^ is C; j __4 lower alkyl or phenyl, is glycosylated, characterized in that the aglycon compound is reacted in a solvent with 3, 4, 6-tri-O-acyl-glucal of formula in the presence of molecular or ionized halogen as a catalyst, such as iodine I2, chlorine CI2, bromine Br2, and fluorine F2, preferably iodine, to produce the corresponding 4, 6-di-0-acyl-2,3-dideoxy-α-D-erythro-hex-2-enopyranoside of said aglycon compound, where acyl is a lower acyl group. In another preferred embodiment a hydroxysteryl compound, preferably a 3β-ol sterol compound, more preferably a delta " -3β-ol steroid compound such as a cholesterol, (e.g., delta5-cholesten-3β-ol) is glycosylated by reaction with 3, 4, 6-tri-O-acyl-D-glucal in a solvent in the presence of molecular or ionized halogen, preferably iodine, as a catalyst. Alternatively, a non-steroidal compound can be thus glycosylated as detailed herein. The reaction is achieved in a single step and in high yield. Thus a double bond which is strongly hindered by the C4, Cg-acyl groups and thus being inert, is introduced between C2=C3 of the glycosidic part of the molecule, whereby as to the hydroxysteryl compound the delta^ double bond of the cyclopentano-perhydro-phenanthrene skeleton is stabilized and remains unchanged. Furthermore, the invention comprises the use of the resulting unsaturated glycoside obtained as an end product or as an intermediate in further reactions to provide functional derivatives which may be steroidal, e.g., cholesterol, derivatives. Thus, in the case of cholesterol, functional groups can be introduced into the perh dro- cyclopentano-phenanthrene skeleton of the unsaturated acylglycoside, wherein the α-bond of the acylglycoside at the same time functions as a protecting group for the original OH-group at C3 of the phenanthrene skeleton. The present method makes use of iodine which is molecularly dissolved in inert solvents. These inert solvents, for example, comprise CH2CI2 dichloromethane, CHCI3 chloroform, CCI4 carbon tetrachloride, CgH4(CH3)2 xylene, C H3(CH3)3 mesitylene, C H5CH(CH3)2 cymene, C Hi2 cyclohexane and methyl derivatives thereof, as well as ligroin, petroleum ether and saturated hydrocarbons, such as for example n- pentane or n-heptane, preferably CgHg benzene or CgH5CH3 toluene. Using the alternative solvent nitromethane CH3NO2, the glycosylation reaction catalyzed by iodine runs quantitatively at room temperature in 2 hours. The method of the invention for the preparation of the DDH pyranoside also makes use of iodine dissolved in ionizing solvents. Using a ketone such as acetone, methyl ethyl ketone (2-butanone) , and cyclohexanone as an alternative solvent, the iodine catalyzed reaction between 3,4, 6-Tri-O-acetyl-D-glucal and cholesterol runs quantitatively at 20°C in only 60 to 90 minutes. As a preferred embodiment, one can also use tetrahydrofuran (THF) ethers and the like. Using a lower alcohol such as methanol, ethanol, propanol ' and the like, the iodine catalyzed reaction between 3, , 6-Tri-O-acetyl-D-glucal and the aglycon runs during 30 minutes time quantitatively at 20°C as well. The glycosylation method according to the mentioned preferred embodiment is directed to the reaction of the vinyl ether of 3, , 6-tri-O-acyl-D-glucal with a cholesterol such as delta5-cholesten-3β-ol, with molecularly dissolved halogen, i.e. iodine, as catalyst in one of the aforementioned solvents. The reaction thereby introduces a double bond between C-atoms 2 and 3 while eliminating the acyl group sited at C3, instead of introducing an iodine atom at C2 in the glycosidic part of the resulting cholesterylglycoside. This reaction is conveniently followed by IR-spectroscopy, and is complete only when the peak of the glucal at 1650 cm -1 has disappeared. The iodine being utilized as catalyst is quantitatively titrated back by a suitable back-titrant reagent such as 0.1 N aqueous sodium thiosulphate ( a2S2θ3) . The method for providing the corresponding di-O-acyl glycoside as exemplified hereinafter for cholesterol compounds is applicable to the glycosylation of not only steroid compounds and cholesterol compounds and precursors but also aglycon compounds in general, i.e., aliphatic, alicyclic, aliphatic-aromatic and aromatic compounds having a primary, secondary or tertiary functional group of the type mentioned. Aglycon compounds that are useful for their pharmacological properties are preferred, since their respective glycosides produced according to the invention have the same utility and useful properties and art-recognized dosage regimens with the possible enhancement of bioavailability, e.g., at cell membranes, due to the presence of the sugar residue. Preferred hydroxy compounds for glycosylation comprise cholesterols, bile salts, steroid hormones, and vitamin D compounds and precursors as described in Stryer's Biochemistry- 3rd Ed. pp. 559-570, Freeman and Company, New York, 1988, incorporated herewith by reference. Examples of such compounds are cholic acid and derivatives, 25-hydroxy-cholesterol, 25-hydroxy-calciferol, pregnenolone, 17α-hydroxy-progesterone, 17α-hydroxy-pregnenolone, 11- desoxy-corticosterone, 11-desoxy-cortisol, corticosterone, cortisol, cortisone, dolichol, androsterone, testosterone, estrone, 17β-estradiol, estratriol-3, 16cc, 17β, 3α, 5β- tetrahydro-corticosterone, serotonin, urocortisol, and allocortolone, and the like, preferably cyclopentano- perhydrophenanthrene compounds having the delta5-3β-OH steryl moiety in which the delta " double bond is stabilized as described by 3β-OH glycosylation resulting in the pyranoside. Other preferred aglycon compounds for glycosylation include a) morphine and morphine derivatives such as codeine, ethyl morphine, dihydrocodeine, hydromorphone, oxycodone, nalorphine, levorphan, and the like; b) α- and β-sympathomimetic (adrenergic) compounds such as -_-hydroxyphenylethanolamine , norf enef r ine , synephrine , etilefrin, phenylephrine, octapamine , isoprenaline , dichloroisoproterenol, metaproterenol, terbutaline, buphenine, and the like; c) antihypertensive compounds such as methyldopa and the like; d) vasoconstrictor compounds such as α- methylnoradrenaline, and the like; e) anticholinergic (parasympatholytic) and antispasmodic compounds such as atropine, homatropine, scopolamine and its methobromide and butyl bromide quaternary compounds, podine methyl sulfate, tropine benzilate, and the like; f) antipsychotic compounds such as acetophenazine, fluphenazine, dixyrazine, perphenazine, hydroxyzine, pericyazine, haloperidol, trifluperidol, moperone, and the like; g) cardiotonic compounds such as digitoxigenin, digoxigenin, diginatigenin, and the like; h) estrogen and contraceptive compounds such as ethinylestradiol, mestranol, quinestrol, and the like; i) antibacterial compounds such as amoxicillin, chloramphenicol , thiamphenicol , tetracycline , chlortetracycline, oxytetracycline, and the like; j) antitussive compounds such as chlophedianol, clobutinol, zipeprol, and the like; k) analgesic compounds such as levorphanol, ciramadol, pentazocine, carbetidine, glafenine, salicylamide, and the like; 1) oxytocic compounds such as ergonovine, prostaglandin F2_., and the like; m) antineoplastic compounds such as fluorouracil, and the like; n) antimalarial compounds such as quinine, and the like; o) fungicidal compounds such as cycloheximide, and the like; p ) an irheuma ic compounds su ch a s oxyphenbutazone, and the like; q) anticholelithogenic compounds such as chenodiol, and the like; r) choleretic compounds such as hymecromone, and the like; s) anti-gout compounds such as allopurinol; t) anti-parkinsonian compounds such as levodopa, carbidopa, droxidopa, and the like; u) anti-spasmodic compounds such as ephedrine, and the like; v) nasal decongestant compounds such as cafaminol, and the like; w) mus cle relaxant compounds such as phenprobamate, guaiacol glycerol ether, and the like; x) ant i-inflammatory compounds such as dexamethasone, beclomethasone, and the like; and y) vitamin and vitamin related compounds such as provitamin D, xanthophyll, vitamin A, vitamin E, thiamin, ascorbic acid, and the like . Other preferred aglycon compounds for glycosylation according to the invention include those listed in the "Therapeutic Category and Biological Activity Index" of The Merck Index XI, pp . THER-5 to THER-29, Merck & Co . , Inc . , Rahway, NJ, incorporated herewith by reference . The glycosylation method and related oxidation and reduction methods described hereinafter may be illustrated by a preferred embodiment employing the starting material cholesterol, as follows: In another method aspect of the invention, the steryl DDH pyranoside product obtained by the glycosylation method can be converted by oxidation of the steroid part into an α-glycosylated 7-keto-sterol such as α-glycosylated 7- keto-cholesterol . The method is applicable to the oxidation of sterol compounds broadly, preferably cyclopentano- perhydro-phenanthrene compounds having the delta-^-3β-OH steryl 4 , 6-di-0-acyl-2 , 3-dideoxy-Ct-D-e rythr o-hex- 2 - enopyranoside moiety to provide the corresponding 7-keto sterols having the corresponding moiety The oxidation is accomplished with an oxidizing agent, which preferably contains chromium, with pyridine-chromium trioxide (C5H5N)2C θ3 or pyridine-chlorochromate (C5H5NHCrθ3)Cl being preferred and t-butyl chromate being especially preferred. The inert glycosidic double bond between C2=C3 thereby remains intact as it is shielded by the C4, Cg acyl (e.g., acetyl) groups. The reduction of this 7-ketone with a suitable reducing agent, preferably a complex metal hydride, such as one or more of Li lH4, NaBH 4 , and KBH4, more preferably LiAlH4, leads to a steroidal glycoside according to the invention. In a preferred embodiment, the method importantly provides 7β-hydroxycholesteryl 2,3-dideoxy-α-D- erythro-hex-2-enopyranoside (7β-OHC) of formula which like cholesterol is systemically biocompatible. The product is obtained after workup of the reaction mixture, e.g. by chromatographic separation of the C7 β-hydroxy isomer from the C7 α-hydroxy isomer, in a suitable solvent mixture, preferably a mixture comprising dichloromethane :acetone preferably in 1:1 mixture. In a preferred aspect, the invention comprises the novel pyranoside compounds having the above formulas . The novel 7β-hydroxycholesterol DDH pyranoside in particular and its 7-keto precursor possess valuable pharmacological properties. For example, a preferred parenteral dosage regimen in treating the proliferative phase growth of the kind described, allowing for ethical considerations and practices exercised in the clinician's judgment, calls for administration of about 10 to about 40 mg. of 7β-OHC DDH pyranoside per 70 Kg. of body weight, once a day or less often while analysis is made of tumor markers such as CEA, TPA, etc. so that the dosage can be adjusted from time to time to normalize the tumor marker level. Whereas the alpha-isomer, delta-^-cholesten-3β, 7α-diol, is formed in the liver as the first degradation product of cholesterol and possesses no physiological activity, the beta-isomer, delta^-cholesten-3β, 7β-diol (as well as its 7-keto analog), is formed in the thymus gland of all mammals as a universal signal substance of the mammalian immune defense. It owes its activity, which is solely directed to malignant cell surfaces, to the fact that it is bound unspecifically by LDL (low density lipoproteins) . The novel steroid and non-steroid compounds of the invention can be used in the form of pharmaceutical preparations comprising each such compound in a pharmacogically effective amount in admixture with a pharmaceutically acceptable carrier which may be conventional per se. These preparations may be formulated by well known procedures. In these respects, see for example Remington's Pharmaceutical Sciences , Chapter 43, 14th Ed., Mack Publishing Co., Easton, PA 18042, USA. These preparations can be administered in any suitable way such as orally, e.g. in the form of tablets, dragees, gelatin capsules, soft capsules, solutions, emulsions or suspensions or parenterally, e.g. in the form of injectable solutions at suitable pH, e.g. ca. 7.5, or topically, e.g. in the form of a cream. The invention and the best mode for practicing the same are illustrated by the following examples . Example 1 Preparation of Cholestervl 4.6-Di-0-acetvl-2.3-dideoxv-α-D- erγthro-hex-2-enopyranoside 5.0 g (=0.02 mole) molecular iodine were dissolved with stirring in 300 ml benzene in a 2-litre three-necked flask fitted with stirrer, reflux condenser and thermometer. To the wine-red solution thus obtained was added the solution of 27.2 g (=0.10 mole) 3, 4, 6-tri-O-acetyl-D-glucal and 38.6 g (=0.10 mole) cholesterol (delta 5 -cholesten-3β-ol) in 700 ml of benzene. In the course of 2 hours the mixture was heated to 70-75°C. The reaction was monitored by IR-spectroscopy; it was terminated only when the peak of the glucal at 1650 cm -1 (Figure 1) had disappeared. The red color of the reaction solution is not significant. After removal of the flask heater the reaction solution is rapidly cooled in a water-bath to about 20-30°C. After transfer into a 2-litre separatory funnel the cooled wine-red reaction solution was thoroughly shaken until complete discoloration with 500 ml + 10% of 0.1 N = 12.5 g + 10% = 13.8 g aqueous solution of Na2S2θ3, washed twice with water, treated with activated carbon, dried over anhydrous Na2Sθ4 and the solvent distilled off, finally in vacuo. Crude yield: 58.3 g (= 97.4% th.) The product, cholesteryl 4, 6-di-0-acetyl-2, 3-dideoxy-Ct-D- erythro-hex-2-enopyranoside, is recrystallized from 2 litres of CH3OH. Yield: 56.95 g (= 95.1% th.) Mp: 118-120-C IR-spectrum: Figure 2 NMR-spectrum: Figure 3 The invention comprises not only the above glycoside product but also other glycoside products made by the above procedure using as starting materials equivalent amounts of the glucal (or other suitable glucal) and the aglycon selected for glycosidation. Suitable aglycon compounds are selected (for their known use as aglycons) from the aforementioned aliphatic, alicyclic, aliphatic-aromatic or aromatic compounds having a primary, secondary or tertiary functional group (i.e., -OH, -SH, -NH2, ~NHRχ) , preferably a compound listed in the foregoing paragraphs a) to y) or in pages THER-5 to THER-29 of the Merck Index XI, supra. Especially preferred glycoside products made by the above procedure are the respective 4, 6-di-O-acyl or acetyl-2,3-dideoxy-α-D- erythro-hex-2-eno-pyranosides of the following aglycon compounds which as DDH pyranoside products are characterized by known IR and NMR spectroscopy data available from the referenced literature. The above glycosylation reaction carried out in benzene at 70°C is selective for the desired alpha isomer rather than the beta isomer. Thus the reaction typically produces a ratio of alpha to beta cholesteryl glycosylate isomers (0t:β) of greater than 20:1. Similarly, in THF at 67°C and at room temperature the reaction in either case typically produces an CC:βratio of about 9:1. Another general procedure was used in which, instead of cholesterol, a starting material (i.e., a substrate) having at least one functional group (e.g., OH, SH, and/or -COOH) was used. The procedure is detailed as follows : GENERAL PROCEDURE The substrate (1 equiv) was added to a stirring solution of glucal (0.5-1.0 g) and iodine (20 mol%) in THF at room temperature. The reaction was monitored by thin-layer chromatography. Reaction times are listed for each compound. Upon completion the solution was diluted with ether and washed with 10% aqueous Na2S2θ3 solution. The layers were separated and the aqueous layer was extracted with additional ether. The combined organic layers were dried over Na2S2θ4, concentrated, and purified by column chromatography on 230- 400 mesh Siθ2. All products were recovered as viscous oils except for those compounds for which a melting point is given. Note: The thiols were added at lower temperatures as indicated for each adduct . ILLUSTRATIVE SUBSTRATES ALCOHOLS (all over 90% yield; α-isomer:β-isomer = 5-8:1) methanol ethanol isopropanol tert-butyl alcohol benzyl alcohol allyl alcohol PHENOLS (α-isomer:β-isomer = 6 - 8:1) phenol (70%) p-methoxyphenol (ca. 65%) p-nitrophenol (ca. 60%) THIOLS (highly reactive) ethyl mercaptan α-isomer:β-isomer = 16:1 (-78°C to -55°C) (35%) isopropyl mercaptan α-isomer:β-isomer = 15:1 (-78°C to 0°C) (77%) tert-butyl mercaptan α-isomer only (-25-C) (68%) thiophenol α-isomer:β-isomer = 20:1 (room temperature) (50%) CARBOXYLIC ACID acetic acid α-isomer:β-isomer = 10:1 (catalytic) (80%) SPECTROSCOPIC DATA FOR ALCOHOL (ROH) PHENOL (ArOH) AND CARBOXYLIC (RC00H) ADDUCTS R = Me (Reaction Time = 30 min. Yield = 87%) X H NMR (CDCl_. 300 MHz, δ 2.087 (s,3H), 2.111 (s,3H), 3.457 (s,3H), 4.05-4.13 (m, 1H) , 4.188 (1H) and 4.266 (1H) (ABq, J AB =12.1 Hz; the 4.188 peaks are further split into d with J=2.5 Hz; the 4.266 peaks are further broken down into d with J=5.3 Hz), 4.934 (s,lH), 5.322 (dd,lH,J=9.7, 1.4 Hz), 5.834 (1H) and 5.894 ppm (1H) (ABq, J AB =10.6 Hz; the 5.834 peaks are further split into dd with J=2.0, 2.0 Hz) . 13- NMR ' (CDCl3_: 75.5 MHz, δ 20.56, 55.70, 63.26, 65.75, 67.35, 95.60, 128.15, 129.38, 170.08, 170.36 ppm. IR (KBr) 1048, 1072, 1107, 1137, 1148, 1188, 1233, 1372, 1438, 1449, 1456, 1744, 2910, 2939, 2956 cm -1 . R = Et (Reaction Time = 50 in. Yield = 97%) m.p. (hexanes = 76-77.5°C. -H NMR (CPC- . 300 MHz) δ 1.256 (d,J=7.1 Hz,3H), 2.084 (S,3H), 2.105 (S,3H), 3.584 (1H) and 3.837 (1H) (ABq, J AB =9.7 Hz; both sets of peaks are further split into q with J=7.1 Hz), 4.09-4.14 (m,lH), 4.174 (1H) and 4.261 (1H) (ABq, J AB =12.2 Hz; the 4.174 peaks are further split into dd with J=2.8, 2.8 Hz; the 4.261 peaks are further split into d with J=5.3 Hz), 5.050 (s,lH), 5.319 (dd,J=9.6,1.4 Hz, 1H) , 5.837 (1H) and 5,891 ppm (1H) (ABq, J AB =10.2 Hz; the 5.837 peaks are further split into dd with J=1.7, 1.4 Hz) . 13 C NMR (CDCl_; 75.5 MHz) δ 15.31, 20.66, 20.83, 63.30, 64.25, 65.75, 67.25, 94.40, 128.37, 129.13, 170.17, 170.53 ppm. IR (KBr) 1019, 1052, 1084, 1108, 1120, 1133, 1137, 1189, 1229, 1239, 1257, 1274, 1372, 1382, 1738, 2902, 2980 cm""!. R = i-Pr (Reaction Time = 65 min. Yield = 96%) 3-H NMR (CDCl_: 300 MHz) δ 1.186 (d,J=6.2 Hz,3H), 1.258 (d,J=6.2 Hz,3H), 2.081 (s,3H), 2.096 (s,3H), 3.988 (dq,J=6.2, 6.2 Hz,lH), 4.12-4.28 (m,3H), 5.133 (s,lH), 5.301 (dd,J=9.5, 1.4 Hz, 1H) , 5.804 (1H) and 5.875 ppm (1H) (ABq, J AB =10.3 Hz; the 5.804 peaks are further split into dd with J=2.6, 2.5 Hz,lH) . ! c NMR (CDCl3_; 75.5 MHz) δ 20.59, 20.83, 22.06, 23.54, 63.42, 65.87, 67.22, 70.83, 93.06, 128.82, 128.91, 170.18, 170.55 ppm. IR (KBr) 1035, 1101, 1125, 1185, 1231, 1372, 1457, 1744, 2933, 2973 cm -1 . R = t-Bu (Reaction Time = 2 hr. Yield = 74%) iH NMR ( CDCI3 300 MHz ) δ 1.292 (s, 9H) , 2.075 9s, 3H), 2.085 (s,3H) , 4.12-4.28 (m,3H), 5.271 (d,lH,J=8.0 Hz) , 5.321 (dd-lH, J=1.3, 1.3 Hz), 5.744 (1H) , and 5.844 ppm (1H) (ABq,J =10.2 Hz; the 5.744 peaks are further split into dd with J=2.8, 2.8 Hz) . 13p. NMR (CDCl3_: 75.5 MHz) δ 20.72, 20.93, 28.79, 63.35, 65.49, 66.72, 75.23, 89.02, 128.13, 129.67, 170.17, 170.57 ppm. TR (KBr) 1025, 1044, 1100, 1183, 1196, 1235, 1370, 1392, 1746, 2935, 2976 cm -1 . R = Ph (Reaction Time = 8 hr. Yield = 70%) H NMR (CDCl_. 300 MH~) δ 1.976 (S,3H), 2.017 (s,3H), 4.10- 4.30 (m,3H), 5.387 (d,J=11.4 Hz,lH), 5.698 (s,2H), 7.00-7.33 ppm (m,5H) . 13c NMR (CDCl3_: 75.5 MHz) δ 20.54, 20.84, 62.66, 65.13, 67.85, 92.12, 117.10, 122.42, 127.14, 129.40, 130.06, 157.08, 170.06, 1740.46 ppm. TR (KBr) 1005, 1029, 1047, 1076, 1096, 1187, 1222, 1371, 1491, 1589, 1598, 1744 cm -1 . R = p-MeO-Ph (Reaction Time = 12 hr. Yield = 75%) .p. (hexanes) = 77-77.5°C. H NMR CDCI3 300 MHz) δ 2.023 (s,3H), 2.109 (s,3H), 3.774 (s,3H), 4.14-4.30 (m,3H) , 5.376 (d,J=9.1 Hz,lH), 5.567 (s,lH), 6.007 (s,lH) , 6.830 (2H) and 7.047 ppm (2H) (ABq,J AB =9.0 Hz) . c NMR (CDCl3_: 75.5 MHz) δ 20.61, 20.85, 55.77, 63.03, 65.60, 67.94, 94.23, 114.85, 115.11, 116.21, 118.90, 127.51, 130.08, 170.32, 170.75 ppm. IR (KBr) 1010, 1037, 1053, 1184, 1227, 1243, 1255, 1270, 1380, 1507, 1741, 2922, 2934, 2958 cm -1 . R = CH2PΪ1 (Reaction Time = 80 min. Yield = 95%) 1H NMR (CDCl3_: 300 MHz) 8 2.081 (s,3H), 2,102 (s,3H), 4.09- 4.29 (m,3H), 4.601 (IH) and 4.809 (IH) (ABq,J AB =11.7 Hz), 5.139 (S,1H), 5.334 (dd,J=9.4, 1.3 Hz,lH) 5.875 (ABq,2H), 7.29-7.40 ppm ( ,5H) . 13c NMR (CDCl3_: 75.5 MHz) δ 20.63, 20.81, 63.19, 65.74, 67.50, 70.35, 93.83, 127.00, 128.56, 128.56, 129.46, 170.17, 170.59 ppm. IR (KBr) 1026, 1038, 1101, 1137, 1151, 1186, 1227, 1370, 1405, 1436, 1454, 1743, 2904, 2937, 2953, 3032 cm -1 . R = CH 2 CH=CH (Reaction Time = 80 min. Yield = 88%) H NMR (CDCl_; 300 MHz) δ 2.086 (s,3H), 2.106 (s,3H), 4.05- 4.30 (m, " 5H), 5.084 (S,1H), 5.212 (d,J=10.3 Hz, IH) , 5.28-5.35 (m,2H), 5.848 (IH) and 5.899 (IH) (ABq, J AB =10.3 Hz), 5.92- 5.99 ppm (m, IH) . 1 c NMR (CDCl3_: 75.5 MHz) δ 20.73, 20.91, 63.00, 65.35, 67.05, 69.22, 93.65, 117.35, 127.86, 129.27, 134.27, 170.18, 170.62 ppm. R = CH2C(CH3>3 (Reaction Time = 30 min. Yield = 97%) H NMR (CDCl3_: 300 MHz) δ 0.931 (s, 9H) , 2.086 (s,3H), 3.133 and 3.481 (ABq, J AB =8.8 Hz, 2H) , 4.07-4.27 (m,3H), 4.985 (S,1H) 5.305 (d,J=9.5 Hz,lH), 5.850 and 5.859 (ABq,J AB =10.5 Hz, 2H; the 5.850 ppm peaks are further split into dd with J=2.7 and 2.6 Hz) . 13c NMR (CDCl_: 75.5 MHz) δ 20.73, 20.91, 63.00, 65.35, 67.05, 69.22, 93.65, 117.35, 127.86, 129.27, 134.27, 170.18, 170.62 ppm. R = OAc (Reaction time 24 h. Yield = 81%) (20 mol% of acetic acid was used) . 1H NMR (CDCI3; 300 MHz) δ 2.082 (s,3H), 2.095 (s,3H), 2.098 (s,3H), 4.06-4.28 (m, 3H) , 5.368 (d,J=10.5 Hz, IH) , 5.850 and 6.011 (ABq, J AB =10.6 Hz, 2H; the 5.850 ppm peaks are further split into dd with J=2.7 and 2.2 Hz), 6.294 ppm (s,lH) . SPECTROSCOPIC DATA FOR THIOL (RSH) ADDUCTS R = Et (-78 to -55°C, Reaction Time = 3.5 hr. Yield = 35%) -H NMR (CDCl_: 300 MHz) δ 1.252 (t,J=7.1 Hz, 3H) , 2.080 (s,3H) , 2.099 (s,3H), 3.581 (IH) and 3.832 (IH) (ABq, J AB =9.7 Hz; both sets of peaks are further split into dq with J=7.1, 7.1 Hz), 4.08-4.29 (m, 3H) , 5.043 (S,1H), 5.315 (dd,J=9.6, 1.3 Hz, IH) , 5.831 (IH) and 5.883 ppm (IH) (ABq,J AB =10.3 Hz) . 13c NMR (CDCl_; 75.5 MHz) δ 15.31, 20.72, 20.92, 63.15, 64.30, 65.52, 67.02, 94.30, 128.09, 129.03, 170.20, 170.66 ppm. IR (KBr) 1049, 1079, 1182, 1232, 1371, 1437, 1451, 1743, 2930, 2964 cm -1 . R = i-Pr (-78 to 0°C, Reaction Time = 6 hr. Yield = 77%) -H NMR ( DCl3_: 300 MHz) δ 1.337 (d,3H,J=6.8 Hz), 1.342 (d,3H,J=6.8 Hz), 2.085 (s,6H), 3.145 (dq, IH,J=6.8, 6.8 Hz), 4.15-4.38 (m,3H), 5.349 (d,lH,J=9.6 Hz), 5.638 (s,lH), 5.761 (IH) and ' 5.935 ppm (IH) (ABq,J AB =10.1 Hz) . 3 NMR . DCl3_: 75.5 MHz) δ 20.66, 20.92, 23.88, 36.3, 63.14, 65.25, 66.93, 79,61, 126.74, 129.39, 170.21, 170.60 ppm. IR (KBr) 1050, 1078, 1122, 1157, 1181, 1229, 1369, 1419, 1452, 1743, 2868, 2927, 2960 cm~l. R = t-Bu (-25 to 0°C, Reaction Time = 2 hr. Yield = 68%) iH NMR DCl3_: 300 MHz) δ 1.404 (s, 9H) , 2.070 (S,3H), 2.080 (s,3H), 4.13-4.37 (m,3H), 5.325 (dd,IH,J=9.3, 1.8 Hz), 5.739 (d, 1H,J=1.7 Hz), 5.753 (IH) and 5.902 ppm (IH) (ABq,J=10.9 Hz) . 13 NMR (CDCl_; 75.5 MHz) δ 20.70, 20.94, 31.51, 44.33, 63.22, 65.16, 66.84, 78.17, 126.61, 129,67, 170.30, 170.72 ppm. IR (KBr) 1054, 1078, 1164, 1236, 1368, 1744, 2962 cm "1 . By the same procedure , but using mevinolin ( lovastatin) as a substrate rather than cholesterol , the resulting adduct in about 60% yield is Lovastatin 4 , 6-di-O- acetyl-2 , 3-dideoxy-α-D-erythro-hex-2 -enopyranoside having the formula : The reaction is carried out under mild conditions, e.g., preferably at room temperature, for one hour, using triacetyl D-glucal (1.0 equiv) , iodine (20 mol %) in THF as the solvent. R = Lovastatin (Reaction Time = 1 h. Yield = 55%) -H NMR (CDCI3; 300 MHz) δ 0.873 (t,J=7.5 Hz,3H), 0.892 (d,J=7.0 Hz, 3H), 1.074 (d,J=7.4 Hz, 3H) , 1.105 (d,J=6.9 Hz, 3H) , 2.091 (s,3H), 2.094 (s,3H), 3.99-4.05 (m, IH) , 4.14-4.28 (m,3H), 4.29-4.34 (m, IH) , 4.52-4.61 (m, IH) , 5.134 (s,lH), 5.323 (dd,J=9.6, 1.3 Hz, IH) , 5.373 (d,J=2.7 Hz, IH) , 5.75- 5.91 (m,2H), 5.909 (part of ABq, J AB =11.1 Hz, IH) , 5.992 ppm (d,J=9.7 Hz, IH) . i3 C NMR (CDCI3; 75.5 MHz) δ 11.67, 13.92, 16.23, 20.70, 20.88, 22.85, 24.23, 26.79, 27.51, 30.77, 32.77, 33.22, 35.01, -35.73, 36.65, 37.46, 41.45, 62.98, 65.26, 67.38, 67.85, 68.65, 76.70, 93.12, 127.44, 128.44, 129.49, 129.72, 131.65, 132.94, 169.32, 170.07, 170.47, 176.39 ppm. By the same procedure, but using either mephenesin or capaicin as a substrate rather than mevinolin, the resulting adduct is mephenesin 4, 6-di-0-acetyl-2,3-dideoxy- α-D-erythro-hex-2-enopyranoside and capsaicin A, 6-di-O- acety1-2,3-dideoxy-α-D-erythro-hex-2-enopyranoside, respectively. R = Mephenesin (Reaction Time = 1 h. Yield = 85%) 1H NMR (CDC1 3 ; 300 MHz) δ 2.068 (s,3H), 2.071 (s,3H), 2.077 (s,3H), 2.080 (s,3H), 2.214 (brs, 3H) , 3.70-3.82 (m,3H) , 3.99-4.40 (m,HH), 5.06-5.12 (m,lH), 5.28-5.39 (m,3H) , 5.78- 5.99 (m,4H), 6.81-6.91 (m,3H), 7.11-7.19 ppm (m,2H) . R = Capsaicin (Reaction Time = 8 h. reflux in THF. Yield = 60%) 1H NMR (CDCI3; 300 MHz) δ 0.834 (d,J=6.6 Hz, 3H) , 0.948 (d,J=6.7 Hz, 3H) , 2.091 (s,3H), 2.093 (s,3H), 3.850 (s) , 5.27-5.35 (m,2H), 5.45-5.90 (m,4H) , 6.71-6.88 (m,3H) . Adducts with cholic acid or a cholic acid analog by this procedure are as follows: Chenodeoxycholic Acid Conditions : triacetyl D-glucal (2.0 equiv) ; iodine (20 mol %) ; THF, room temperature, 4h Results : diadduct (89%) chenodeoxycholic acid 3, 12-di- (4, 6- di-O-acetyl-2,3-deoxy-α-D-erythro-hex-2-enopyranoside) . PgoT-ychoiUc Acid Conditions : triacetyl D-glucal (2.0 equiv); iodine (20 mol %) ; THF, room temperature, 4h Results : diadduct (91%) chenodeoxycholic acid 3, 12-di- (4, 6- di-O-acetyl-2,3-deoxy-α-D-erythro-hex-2-enopyranoside) . Cholic Acid Conditions : triacetyl D-glucal (3.0-5.0 equiv); iodine (20 mol %) ; THF, reflux, 8h Results: mixture of di- and triadducts (85%) : cholic acid 3,7-di-(DDH pyranoside); cholic acid 3, 12-di-(DDH pyranoside); and cholic acid 3,7, 12-tri-(DDH pyranoside) . An adduct with chloramphenicol by this procedure is as follows: Ch_.Qrsmp__er.icol Conditions : triacetyl D-glucal (2.0 equiv); iodine (20 mol %) ; THF, room temperature, 6h Results : diadduct (85%) chloramphenicol l,3-di-(DDH pyranoside) . Having described the invention, the embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows :

Claims: Claims 1. Method for the production of a pyranoside compound, wherein a aglycon compound selected from aliphatic, alicyclic, aliphatic-aromatic and aromatic compounds having a primary, secondary or tertiary functional group is glycosylated, characterized in that the aglycon compound is reacted in a solvent with 3, 4, 6-tri-O-acyl-glucal of formula in the presence of halogen as a catalyst to produce the corresponding 4, 6-di-0-acyl-2,3-dideoxy-α-D-erythro-hex-2- enopyranoside of said aglycon compound, where acyl is a lower acyl group. 2. Method according to claim 1, characterized in that the aglycon compound is a delta-^-3β-ol steryl compound. 3. Method according to claim 2, characterized in that a cholesterol compound is reacted with 3,4,6-tri-0- acetyl-D-glucal in a solvent to yield the corresponding 4,6- 0-di-0-acetyl-2,3-dideoxy-α-D-erythro-hex-2-enopyranoside. 4. Method according to claim 3, characterized in that benzene or toluene is used as a solvent. 5. Method according to claim 3, characterized in that the 4, 6-di-0-acetyl-2, 3-dideoxy-α-D-erythro-hex-2- enopyranoside is oxidized with an oxidizing agent to produce 7-ketocholesteryl 4, 6-di-0-acetyl-2, 3-dideoxy-α-D-erythro- hex-2-enopyranoside which in turn is reduced with a metal hydride as a reducing agent to produce 7-β-hydroxycholesteryl 2,3-dideoxy-α-D-erythro-hex-2-enopyranoside. 6. Method according to claim 5, characterized in that the oxidizing agent is selected from the group consisting essentially of t-butyl chromate, pyridine-chromium trioxide, and pyridine chlorochromate. 7. Method according to claim 5, characterized in that one or more of LiAl^, NaBH4, and KBH4 is used as a reducing agent. 8. Method according to claim 5, characterized in that the 7β-hydroxycholesteryl pyranoside is isolated by chromatography. 9. Method according to claim 8, characterized in that the isolation is carried out by chromatography using a solvent mixture which comprises dichloromethane and acetone. 10. A hydroxysteryl 2, 3-dideoxy-α-D-erythro-hex- 2-enopyranoside compound of formula 11. A hydroxysteryl 2,3-dideoxy-α-D-erythro-hex- 2-enopyranoside of formula where R is a steryl moiety of formula 12. A ketosteryl 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside of formula 13. A ketosteryl 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside of formula where R is a ketosteryl moiety of formula 14 . A medicament for inhibiting the growth of neoplastic cells which comprises in pharmaceut ically acceptable dosage form an effective neoplastic cell growth inhibiting amount of a pyranoside of formula 15. A medicament according to claim 14 that is effective for inhibiting the growth of leukemic cells. 16. A medicament according to claim 14 comprising a liquid carrier selected from a) water, b) ethanol, c) propylene glycol or a mixture of two or more of a) , b) and c) . 17 Cholesteryl 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside. 18. Cholesteryl 4, 6-di-0-acetyl-2,3-dideoxy-α-D- erythro-hex-2-enopyranoside. 19. A 2, 3-di-0-acyl-α-D-erythro-hex-2- enopyranoside of a hydroxy compound selected from cholesterols, bile salts, steroid hormones, and vitamin D compounds. 20. A pyranoside according to claim 19 wherein the aglycon compound is selected from cholic acid and derivatives, 25-hydroxy-cholesterol, 25-hydroxy-calciferol, pregnenolone, 17α-hydroxy-progesterone, 17α-hydroxy- pregnenolone, 11-desoxy-corticosterone, 11-desoxy-cortisol, corticosterone, cortisol, cortisone, dolichol, androsterone, testosterone, estrone, 17β-estradiol, estratriol-3, 16α, 17β, 3α,5β-tetrahydro-corticosterone, serotonin, urocortisol, and allocortolone. 21. Cholic acid 3, 7, 12-tri- (4, 6-di-0-acyl-2, 3- dideoxy-α-D-erythro-hex-2-enopyranoside) . 22. Clavulanic acid 4, 6-di-0-acyl-2,3-dideoxy-α-D- erythro-hex-2-enopyranoside. 23. Amoxicillin 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside. 24. Daunorubicin 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside. 25. Lovastatin 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside. 26. Mevastatin 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside. 27. Simvastatin 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside. 28. Mephenesin 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside. 29. Capsaicin 4, 6-di-0-acyl-2, 3-dideoxy-α-D- erythro-hex-2-enopyranoside. 30. The 4, 6-di-0-acyl-2, 3-dideoxy-α-D-erythro-hex- 2-enopyranoside of an aglycon selected from the group of aglycon compounds listed in paragraphs a) to y) supra. 31. Cholic acid 3, 7-di- (4, 6-di-0-acyl-2, 3-dideoxy- α-D-erythro-hex-2-enopyranoside) . 32. Cholic acid 3, 12-di- (4, 6-di-0-acyl-2, 3- dideoxy-α-D-erythro-hex-2-enopyranoside) . 33. Chenodeoxycholic acid 3, 7-di- (4, 6-di-0-acyl- 2,3-dideoxy-α-D-erythro-hex-2-enopyranoside) . 34. Deoxycholic acid 3, 12-di- (4, 6-di-0-acyl-2,3- dideoxy-α-D-erythro-hex-2-enopyranoside) . 35. Chloramphenicol 1,3-di- (4, 6-di-0-acyl-2,3- dideoxy-α-D-erythro-hex-2-enopyranoside) . AMENDED CLAIMS [received by the International Bureau on 8 December 1992 (08.12.92) ; original claim 1 amended ; other claims unchanged ( 1 page)] 1 . Method for the production " of a pyranoside compound, wherein an aglycon compound selected from aliphatic, alicyclic, aliphatic-aromatic and aromatic compounds having a primary, secondary or tertiary functional group selected from -OH, -SH and -COOH is glycosylated, characterized in that the aglycon compound is reacted in a solvent with 3, , 6-tri-O-acyl-glucal of formula in the presence of molecular halogen or molecular iodine as a catalyst to produce the corresponding 4 , 6-di-0-acyl-2 , 3- dideoxy-α-D-erythro-hex-2-enopyranoside of said aglycon compound, where acyl is a lower acyl group . 2 . Method according to claim 1 , characterized in that the aglycon compound is a delta-5-3β-ol steryl compound . 3. Method according to claim 2 , characterized in that a cholesterol compound is reacted with 3, 4 , 6-tri-O- acetyl-D-glucal in a solvent to yield the corresponding 4 , 6- O-di-O-acetyl-2 , 3-dideoxy-α-D-erythro-hex-2-enopyranoside . 4 . Method according to claim 3 , characterized in that benzene or toluene is used as a solvent . Method according to claim 3 , characterized m

Inventors: KLEMKE R ERICH, KOREEDA MASATA, KLEMKE, R., ERICH, KOREEDA, MASATA
Applicants: HARRIER INC, HARRIER, INC.
Application year: 1992
Main classification: C07H15
IPC codes: A61K31, A61P35, C07H15, C07H17, C07J9, C07J17, C07J75