IE913228A1 - Monosaccharides having anti-proliferation and¹anti-inflammatory activity, compositions and uses thereof - Google Patents

Abstract

Derivatives of simple monosaccharides which exhibit anti-proliferative and/or anti-inflammatory activity and are useful for treating mammals having inflammatory disorders and/or autoimmune disorders. This invention also encompasses pharmaceutical compositions containing these compounds and methods of treating inflammatory and/or autoimmune disorders.

Description

The compound· of this Invention axe derivatives of simple monosaccharides which exhibit anti-proliferation and antiinflammatory activity and are useful for treating mammals having inflammatory disorders and/or autoimmune disorders. This invention also encompasses pharmaceutical compositions containing these compounds and methods of treating inflammatory and/or autoimmune disorders.

II.

Certain monosaccharides and their derivatives are known to have therapeutic value in the treatment of inflammatory and autoimmune disorders· Derivatisation of monosaccharides at specific hydroxyl groups may be accomplished by synthetic techniques which axe known in the art. For example, it is common to block or protect one or more of the hydroxyl groups leaving oneor more hydroxyl groups free to undergo derivatisation, such as formation of an ether group. Various blocking groups and methods are described, for example, in U.S. Patent Bos. 2,715,121 and 4,056,922, the disclosures of which are incorporated herein by reference.

Various derivatives of five and six carbon monosaccharides, as well as synthetic methods, are described, for example, in 0.8. Patent Nos. Re. 30,354, Re. 30,379, Re. 32,258, 4,055,322, 4,735,934, 4,738,953, 4,995,195 and 5,010,054. The therapeutic activity of the various substituted monosaccharides is also disclosed in the above documents. The disclosures of these patents are also incorporated herein by reference. λ known derivative of a -D-glucose having beneficial therapeutic properties is amiprilose, 1,2-O-lsopropylidene 3-03*-(H,N*-dimethylamino-n-pxopyl)-e-0-glucofuranose, and its hydrochloric acid salt, aaprllose BC1 (THERAieCTIN*). These two compounds are known to have anti-inflammatory activity and demonstrated utility in managing the signs and symptoms of rheumatoid arthritis. More generally, these compounds have immunomodulatory activity, and therefore have a therapeutic effect on other autoimmune disorders such as psoriasis, ecsema or systemic lupus erythematosus.

Unfortunately, though some derivatives of the monosaccharides have shown beneficial therapeutic activity, high doses of these derivatives, such as TSJSRAlTSCTm·, are often needed to produce effective results. Because therapy for inflammatory or autoimmune disorders is often mid-term or longterm, there is a need to develop more potent, non-toxic compounds which can be orally administered, and thereby promote patient compliance. This invention describes additional monosaccharide derivatives with increased, potency.

It ie therefor· an object of the present invention to provide new compound· and compositions which exhibit antiproliferation and anti-inflammatory activity.

It ie also an object of the invention to provide compound· and compositions which are useful in the treatment of mammals having inflammatory and/or autoimmune disorder·· It is a further object to provide new compounds that exhibit significantly increased potency over available compounds, such as THERAFBCTIN·, in order to provide ease of oral administration. ii. swmftgy tiXft.SffmUaa To achieve the foregoing objects and in accordance with the purposes of the invention as embodied and broadly described herein, there is provided mionOsaaccharides, having the following formulae! λ compound of formula Z IXMlSjJbf w* v5^*10 R ie H, Cj—alkyl, Cj—C^q alkenyl, Cj—C^q nyi/ benzyl, or Cj-C^q ester/ (XX) λ compound of formula II wherein: ,1 X* i· 0 and R5 ia C12-C2Q alkyl, or C^^T, wherein a- 1,2,3 or 4 and T la selected from cyano, pyrrolyl, pyrrolidinyl, joethylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxatolyl, ozazolidinyl, or and and R$ ieooxaiolyl, iiootazolidinyl, imidazol idinyl, piper idinyl, piperazinyl, morpholinyl, O(CB2)3N(CH3)2, (C5C10 aUcoxy)' CH2CH(C»3)CH2H(Ca3)2, CH3CH2N(Cj-C10 alkyl)2, or (C3-C7 alkenyl), ia NH ia C2-C10 alkyl, or cnH2nT wherein n- 1,2,3 or 4 and Y la selected from hydroxy, cyano, pyrrolyl, pyrrolidinyl joethylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxa tolyl, ozazolidinyl, iaoxasolyl, .. ieoozatolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CB2)3N(CH3)2, (Cj- C1Q alkoxy), Or phenyl? and R7 are hydrogen or form an iaopropylidene ring? - 5 IE 913228 λ compound of formula III» \ 'Λ ^-¾ Λ* (III) wherein r ie 0, R® la Cg-CjQ alkyl, or wher-a »· or 4 and T la «elected from phenyl/ cyano, pyrrolyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolldinyl, oxazolyl, oxazolidinyl, iaooxaaolyl, iaooxazolldinyl, iaidaaolldinyl, piperidinyl, piperazinyl, zerpholinyl, OCCHp^MfCHpg, (C5-C10 alkoxy), NH or N(CH3)2? ot x2 ii ra and R® ia H, or ^Η^ϊ, wherein n- 1,2,3 or 4 and T ie selected from OB, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, iaidaaolyl, pyrazolyl, pyrazolinyl, pyrazolldinyl, oxazolyl, oxazolidinyl, iaooxaaolyl, izoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, aorpholinyl, O(CH2)3»{CH3)3 or (Cj-C^ alkoxy) or phenyl; or X2 la $ and R® ia Cg-C^ alkyl, or CnH3nT wherein n« 1,2,3 or 4 and T ia «elected from OH, phenyl, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, iaidaaolyl, pyrazolyl, pyrazolinyl, pyrazolldinyl, oxazolyl, oxazolidinyl, iaooxazolyl, - 6 IE 913228 isoosasolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3W(CH3)2 or (C5C10 alkoxy); and R9 and R10 are hydrogen or form an isopropylidene group; and A confound selected from (38) 1,2-0-isopropylidene-a-D-xlborhexos-3-uloee-l, 4 »3,6dlfuranose; and Methyl 3-0-3' - (Ν'-dimethylamino-n-propyl, -6-deoxy-Dglucopyranoside.

Other embodiment· in accordance with the present invention are pharmaceutical compositions containing an effective amount of one or more of the above compounds, and a method of treating an inflammatory disorder and/or an autoimmune disorder comprising administering an effective amount of a compound described above.

XV. Detailed Description of the Preferred Embodiments Monosaccharides are known to exist in ah equilibrium between hemiacetal cyclic structures and an open chain sugar. The preferred cyclic structures are furanose· (3-membered ring structures) and pyranoses (6-membered ring structures). Other ring structures may be formed but are not as thermodynamically stable and generally rearrange to form the pyranose or furanose . structures. When a cyclic hemiacetal is reacted with an alcohol, . ··. ah acetal Is formed.

A glycoside can be defined as a cyclised derivative of a atonoeaccharide having two ether (0-R groups) substituents on the acetal carbon of the sugar. One of these ether substituents is the carbocylic ring. The second ether substituent is formed by the reaction with the alcohol and is termed the aglycon. Because of the second ether substituent, the resultant glycoside is stable and does not exist in an equilibrium with its open chain structure. Glycosides having a 5-membered ring are known as furanosides, those with 6-membered ring as pyranosides.

One embodiment oi the present invention relates to derivatives of the simple monosaccharide fructose, in particular to f ructof uranosides. Fructofuranosides can be in an »-D or #D configuration. The f ructof uranosides of the present invention, shown below in formula I, encompass both « or ft configurations and are substituted at one or more of the free hydroxyl groups, but, preferably, have at least one free hydroxyl group. As discussed above, the techniques to form these derivatives of the present invention are generally known in carbohydrate chemistry.

The fructofuranosides of the present invention are represented by formula (i)t R1 and r2 9χλ g, methyl, ethyl, alkenyl or together form an isopropylidene ring; R3 is H, C5-CXq alkyl, Cj-C10 alkenyl, C5-C10 alkynyl, 2 octyne, bensyl, or Cj -=10 ester; and μ ^3^io 2 octyne, benzyl, or Cj-Cjq ester.

Preferred fructofuranosides of formula Z where λ Is methyl are shown in Table z. - 9 IE 913228 «UN Z Compound &x S? I3 &* (I·) Rl 1 R2 leopropylidene B C7H15 (lb) C2B5 C2H5 B . C7H15 (Ic) rI 4 R2 - leopropylidene C7H13 H (Xd) R1 6 R2 - leopropylidene 2-octynyl 2-octynyl CI·) R1 4 R2 « leopropylidene trane-2-octenyl trrane-2octenyl («) R1 4 R2 leopropylidene . cls-2-octenyl cie-2- octenyl (I*) Oil ι-2-octene B H cie-2- octenyl 0 (lb) R1 1 R2 leopropylidene CC7B15 H 0 (ID Rl 4 R2 leopropylidene H CC7H15 0 0 (ID R1 4 R2 · leopropylidene CC7B13 cc7b15 The compound· included in Table x ares ·. ·* * · methyl 1,3-O-ieopropylidene-S-O-heptyl-a-D- 'fructofuranoeid· (I·), methyl l,3-di-0-ethyl-6-0-heptyl-e-D-fructofurano»ide («>)> methyl 1,3-0-ieopropylidene-4-0-heptyl~a-Dfructofuranoeide (Ic); - 10 IE 913228 methyl l,3-0-isoprogylldene-4,6-dl~0-(2-octynyl)-a-Dfructofuranoside (Id); methyl 1,3-0-ieopropylldene-4,6-dl-O- (trans, 2-octenyl) α-D-fruotofuranoside (Ie); methyl 1,3-0-isopropylidene-4/ 6-dl-O- (cis, 2-octenyl) a-D-fructofuranoside (12); methyl 1,5-di-0- (cis, 2-oatenyl) -e-0-f ruotof uranoside methyl l,3-0-iaopropylidene-4-0-octanoyl-e-D- . fructofuranoeide (lh); methyl l,3-0-isopropylidene-6-0-©ctanoyl-«-Dfzuctofuranoeide (ii); and methyl 1,3-0-isopropylidene-4, 6-di-O-octanoyl-a-Dfructofuranoside (Ij,.

The present invention also relates to f ructof uranoses.

These compounds have the same substituents as defined in formula I where λ ia hydrogen, λ particularly preferred compound 1st 2,3-0-isopropylidene-4-0-heptyl-/-D-fructofuranose (Ik).

The following Examples 1-4 illustrate the preparation of representative compounds of formula I according to thia invention. The activity of theae compounds le illustrated in Example 5.

Example 1 Preparation of methyl 1,3-0-ieopropylidene-6-0-n-heptyl-a-Dfructofuranoside, (la). step 1» The preparation of Methyl a-D-fructofuranoside.

Thia compound was prepared by the method taught by CortezGarcia, R., L. Hough, and A.C« Richardson (Journal of the - 11 IE 913228 Chemical Society, Perkin 1, pp. 3176-3181, 1881) except that a ailica gel column was used, eluted with chloroform-methanol (9:1 to 8»2) to gives methyl e-D-fructofuranoside 23.8%; («Jq25 +81.6· (o 1.02, ethanol). {Cortes-Garcia, 22.3%; C«)D25 *80· (c 2, water)} methyl l-D-fructofuranoside 28.2%; [a]p25 -47.8· (c 1.23, ethanol). {Cortex-Garcia, 35%; (e)025 -48· (e 2, methanol)}. step 2: The preparation of Methyl 1,3-0-isopropylldene-a-Dfructofuranoside.

Method on· ι The reaction procedure was described by CortesGarcia, except the longer reaction time was used (three days).

The best yield was 50%. («)D25 +40.1· (c 1.78, chloroform).

{Cortez-Garcia 33%, («]& +42.3· (methanol)}. The compound had physical properties in agreement with literature values.

Method twos A mixture of methyl a-D-f ructof uranoside (0.8g, 4.1mmole), dimethoxyprcpane (5g, 48nsole, 6ml) and ptoluenesulfonic acid (O.lg, 0.5mmole) in 20ml of DMF was stirred at 25°C for 24 hours. Sodium bicarbonate was added to neutralize the solution. The solvent was filtered and the filtrate was evaporated to dryness. Column chromatography {petroleum etherethyl acetate (8:2 to7:3) then chloroform-methanol (9:1)} gave methyl 1,3-O-ieopropylidene-a-D-fructofuranosids (0.3$g, 39%). step 3: The preparation cf Methyl l,3-0~lsoprbpylldene-6-0pivalcyl-o-D-fructofuranoside. .

Methyl 1,3-O-isopropylidene-a-D-fructofuranoside (1.3g, 5.55mmol·) was dissolved in methylene chloride (10ml) and pyridine (7ml). To this solution, pivaloyl chloride (0.68g, 5.68mmole, 0.7ml) was added at 0eC. The solution was stirred and the temperature roeo to 25 *C. After 18 hour·, another O.lal of pivaloyl chloride (0.8mmole) va· added and stirring waa continued for 24 hour·. Water va· added to the mixture and advent was evaporated. The residue waa extracted with chloroform, washed with water, brine, dried over aodium sulfate, and concentrated. Column chromatography {petroleum ether-ethyl acetate (9:1 to 713) > gave methyl 1,3-0-ieopropylidene-6-0-pivaloyl-e -Dfructofuranoaide (1.3g, 83%), (D2S +30.9* (c 2.34, methylene Chloride). J'H-N.m.r. (CDClj): I 4.4-3.9 (m, 8H), 3.3 (a, 3B, OCgj), 1.45, 1.38 (2 «, each 3B, C(CS3)2), 1,23 (a, 9B, CO(CHj)j) PP®· Step 4: The preparation of Methyl l,3-0~isopropylidene-4-0benzyl-6-O-pivaloyl-a-D-fructofuranoside.

Methyl l,3-0-iaopr0pyl±dene-6-0-pivaioyl-a-Dfructofuranoaide (l,3g, 4,08mmole) was dissolved in DMF (15ml) and 5.2ml of benaylbromide (7.4g, 43.7anole) was added. To thia solution, a total 5.2g of silver (X) oxide (2?.4nmole) was added in three.portions during one hour with stirring. Stirring was continued at 25 *C for two days. The silver salt waa filtered and washed with DMF and methylene chloride, and solvent waa removed under reduced pressure. The residue waa extracted with methylene chloride and the extract waa washed with water, brine, dried and concentrated. Column chromatography {petroleum ether-ethyl • acetate (50:1)} gave methyl l,3-0-isopropylidene-4-0-bensyl-6-0* **. •25 ' pivaloyl-α-D-fructofuranoaide [D *50.7* (c 1,35, chloroform). (CDClj): f 7.33 (m, SB, C^), 4.80 (dd, 2H, OCS2Ph), 4.3-4.1 (m, 4H), 3.9 (d, IB, £12.1 Bs, B-l), 3.8 - 13 IE 913228 (β,ΙΒ), 3.7 (d, IB, i«.l Hi, H-l), 3.3 3), 1.4-1.3 (2 s, each 3B, C(Q|3)2), 1.2 (s, 9B, CO(C&3)3) ppm.

Step 5» Th· preparation of Methyl l,3-0-izopropylidzaie-6-0-tbutyldimethylsilyl-e-b-f ructof nranoeide.

To a solution of methyl l,3~O-isppropylidene-a-Dfructofuranoside (0.25g, 1.07nnnole) in DMF (5ml) wae added imidazole (0.16g, 2.3mmole) and t-butyldimethylsilyl chloride (0.2g 1,32mmole). The mixture wae stirred at 25°C for <8 hours.

Water was added and solvent, was evaporated under reduced pressure. Column chromatography {petroleum ether-ethyl acetate (9il to 8s2)} gave methyl l,3-0-isopropylidene-6-0-tbutyldimethylsilyl-e-O-fructofuranoeide (0.26g, 97%), then using chloroform-methanol (lOOtl) gave starting material methyl 1,3-0isoprppylidene-e-D-fructofuranoslde (70mg). Methyl 1,3-0ieopxopyiidene-6-O-t-butyldimethylsilyl-a-D-fructofttranoside» (e]02S +21.3· (c 1.01, chloroform). ^H-B.m.r. (CDC13)s 9 4.13.7 (m. 7H), 3.3 (s, 3H, 0C®3), 2.6 (d, IB, OB), 1.45-1.35 (2 s, each 3B, C(Cg3)2), 0.9 (s, 9B, t-Bu), 0.1 (β, 6B, 8i(Cfl3)2) ppm.

Step 6» The preparation of Methyl l,3-0-isopropylidene-4-0benzyl-6-O-t-butyldmethylsilyl-e-D-f ructofuranoside i To a solution of methyl i,3-0-i»opropylidene-6-0-tbutyldimethylsilyl-s-D-fructofuranoside (0.26g, 0.75aaaols) and bensyl bromide (0.15g, 0.9mmole, 0.1ml) in DMF (2ml) was added 80% sodium hydride (25mg, 0.83mmole) slowly. The mixture was stirred at 25*C under nitrogen for 40 minutes. Methanol was added to destroy the excess sodium hydride and solvent was removed under reduced pressure at 25®C. The residue was extracted with chloroform, washed with water, brine, dried and - 14 IE 913228 concentrated. Column chromatography {(petroleum ether-ethyl acetate (20 si to 8i2)} gave methyl l,3-0-lsopropylideae-4-0benayl-8-O-t-butyldimethylailyl-e-D-iruotofuranoside (0.28g, 85.6%) and methyl l,3-0-isopr©pylideae-4-0-bensyl-a-Dfructo furano aide (lOmg, 4%). Methyl l,3-0-ieopropylIdene-4-0bensyl-6-O-t-butyldimefchylailyl-«-D-f ructof uranoside t [«)D2S +48.7· (c 1.58, chloroform). ^-M.m.r. (CDClj)i < 7.3 (a, 5B, Cfo}. 4.6 (·, 2H, Qfijyh), 4.1-3.7 (m,7H), 3.3 (a, 3H, 0CH3), 1.4-1.3 (2 a, each 3H, C(Ca3)j), 0.9 (·, 9H, t-Bu), 0.06(a, 6H, Sl(Cfi3)2) pp·· Step 7 s The preparation of Methyl l,3-0-isopropylidene-4~0bensyl-e-D-f ructof uranoside.

Method ones Methyl l,3-0-iaopropylidene-4-0-benxyl-6-0pivaloyl-«-D-f ructof uranoside was dissolved in methanol (20ml) and sodium methylate (0.2g) was added and stirred at 25®C for 20 hour·. The reaction solution was neutralised with Amberlite IR120 (H+) ion exchange resin, filtered and concentrated.; Column chromatography {petroleum ether-ethyl acetate (9tl to 8s2)} gave methyl 1,3-0-ieopropylidene-4-0-benayl-e-D-f ructof uranoside (1.15g, 86.8% yield).

Method twos A solution of methyl l,3-0-ieopropylidene-4-0benzyl-6-O-t-butyldimethylsilyi-e-D-fructofuranoside (0.2g, 0.45moole) and IX t-Bu^NF-THF (0.5ml, 0»5mmole) in THF (5ml) was 8.stirred at 25’C for 18 hours. Solvent was removed and the residue was chromatographed {petroleum ether-ethyl acetate (8:2)} to give methyl 1,3-0-isopropylidene-4-0-bensyl-«-Dfructofuranoside (0.14g, 93%).

Method three ι To a eolation of methyl 1,3-O-isopropylidene 6-0-pivaloyl-a-D-fructofuranoside (0.2g, 0.63mnole) and beneyl bromide (0.14g, 0.81maole, O.lal) in η» (2ml) wee added 801 eodium hydride (27mg. 0.9mmole) slowly. The mixture was stirred at 25*C under nitrogen for 15 minutes. Methanol was added to destroy the excess sodium hydride and solvent was evaporated under reduced pressure at 25*C. The residue was dissolved in methanol (5ml) and sodium methylate (O.lg) was added. The mixture was stirred at 25°C for 10 hours and neutralised with Amberlite /R-l20 (B+) resin, filtered and concentrated. Column chromatography {petroleum ether-ethyl acetate (9il to 8i2)> gave methyl 1,3-0-ieopropylidene-4-0-benxyl-a-D-f ructofuranoeide (0.13g, 64%). (e)D^5 +83.7· (c 0.98, methylenechloride). 1HB.m.r. (CDClj)ι 9 7.3 (a, 5B, Cgflj), 4.6 (dd, 2Β, OCfljRh), 4.1 (m, 2B), 3.9-3.8 (a, 43),3.6 (dd, XB), 3.3 (s, 3B, OCHj), 2.1 (s, IB, OB), 1.4—1.3 (2s, each 3B, C(CMj)2) ppm.

Steo 8» The preparation of Methyl 1,3-0-isopropyl idene-4-Obenxyl-6-O-n-hsptyl-e-D-f ructofuranoeide.

To a solution of methyl l,3-0-ieopropylidene-4-0-benxyl-aD-fructofuranoeide (1.63g, S.03mmole) and bromoheptane (4.lg, 23mmole, 3.6ml) in DMT (35ml) was added 80% sodium hydride (0.6g, 20mmole) slowly. The mixture was stirred at 25*C for 1.5 hours. Methanol was added to destroy the excess sodium hydride and solvent was evaporated. The residue was extracted with chloroform, washed with water, brine, dried, over anhydrous sodium sulfate and concentrated. Column chromatography {petroleum ether-ethyl acetate (9»1)} gave methyl l,3-0-isopropylidene-4-0bensyl-6-O-n-heptyl-a-D-fructofuranoeide (2g, 94%). [«Id25 +SX.»· (β X.J», MthyXea. ehXorid·,. ^B-M.a.s. (COClj)t ( 7.3 (a 5B, CjHj), <·« {«, 2Sr OCgjPh), <·* (*/ 3»), 3.9 (d, IB), 3.8 (d, IB), 3.6-3.4 (a, 48), 3.3 (·, 3B, OCHj), 1.6 9m, 2B, OCB2Ca2C5H11), 1.4-1.3 (2 s, each 3H, C(CB3)2), 1.2 (br, 8B), 0.9 (t, 3B, ch3) ppm.

Stap St The preparation of Methyl l,3-0-isopropylidene-6-0-nheptyl-a-D-fructofuranoside· Method one» λ mixture of methyl l,3-O-ieopropylidene-4-Obensyl-6-O-n-heptyl-a-D-fructofuranoeide (0.13g, 0.3mmole), 10% palladium on activated carbon (26mg) in methanol (5ml) was shaken under hydrogen (25 lba. per sg. inch) for 8 hour·. Catalyst was filtered and solvent was removed. Column chromatography {petroleum ether-ethyl acetate (9tl)> gave methyl 1,3-0i»opropylidene-6~0-n-heptyl-a-D-f ructof uranoside (la) (70mg, 70%). ’ ’ Method two» A mixture of methyl 1,3-0-isopropylidene-4-Obensyl-6-O-n-heptyl-a-D-f ructof uranoside (0.22g, 0.52mmole), 10% palladium on activated carbon (0.2g, 400mg/naaole per Bn) and ammonium formate (0.3g) in methanol (10ml) was refluxed for 2 hours. Another 0.25g of ammonium formate was added and refluxed for 3 hours. A final part of 0.2g of ammonium formate was added and refluxing was continued for another 2.5 hours. After cooling, catalyst was filtered and washed with methanol, filtrate ' ...was evaporated to dryness. Column chromatography {petroleum ether-ethyl acetate (9tl)> gave methyl l,3-0-isopropylidene-6-0n-heptyl-a-D fructofuranoeide (la) (0.14g, 81%). (oi]D28 +27.4· (c 1.01, methylene chloride). ^B-M.m.r. (CDClj)» t 4.2-3.9 (a, 5H), 3.4-3.7 (a, 4H), 3.3 (e, 3H, OCB3), 2.7 (d, IB), 1.6 (m, - 17 IE 913228 38), 1.5-1.4 (2 s, each 38, C(Cg3)2), 1.3 (br 8, 88), 0.9 (t, 38, CBj) ppm. 13C-MMR (CDClj)I < 102.0 (£-2), 98.6 (£(¢83)3), 85.9, 79.7, 78.7 (£-3, £-4, £-5), 71.7, 71.5 (£-6, , 61.7 (£-1), 48.7 (O£H3), 31.8, 29.5, 29.1, 27.6, 25.9, 22.6, 19.7 14.1 ppm. ».·.» m/s 333 (8+8), 301 (M+H-C83Q-), 260 (Mch2oc(ch3,2). 3 Preparation of methyl l,3-0-isopropylidene-4,6-di-0-(2nntynyi ructof uranoside, (Id).

Methyl 1,3-O-isopropylidene-e-D-fructofuranoside was prepared by the procedure of Cortex-Garcia et al. a* described in Sxample 1. To a solution of methyl 1,3-O-isopropylidene-w-Df ructof uranoside (0.3 g, 1.28 mmole) and 1-bromo,2-octyne (0.73g, 3.85'nmole) in DMF (4 mb) was added 80% HaB (0.12g, 4.0 mmole) slowly and the mixture was stirred at 25* under nitrogen for 40 min. Methanol was added to destroy the excess sodium hydride and solvent wax removed. The residue was extracted with methylene chloride and this solution washed with water, brine and sodium bicarbonate. This solution was then dried with anhydrous sodium sulfate, filtered and evaporated to a residue. This residue was dissolved in eluant and chromatagraphed on a silicagel column using petroleum ether-acetone (24:1) which gave methyl 1,3-0isopropylidene-4,6-di-0-( 2-cctynyl) -β-D-fructofuranoside (Id) (0.41 g, 71%) as a colorless oil. The compound was treated with sodium bicarbonate and kept in freeser. The compound was identified by its specific rotation, HMR and mass spectral analysis.

Kramole 3 Preparation of methyl l,3-0-lsopropylidene-4,6-di-0-(trans,2 octenyl)-a~D-fruetofuranoside, (I·)· The procedure of Kxample 2 was followed except that 1-bromotrans-2-octene was substituted for l-bromo-2-octyne. Methyl 1,3O-isopropylidene-4,6 -di -0- (trans, 2-octenyl} -a-D-fructof ur anoside was obtained in 70.0% yield as a colorless oil and identified by its specific rotation, nhk and mass apwLfal analysis, gXW*» 1 Preparation of 2,3-0-isopropylidene-4-0-heptyl-4,Dfructofuranose (Ik): Step 1: Preparation of 1,8-Di-d-trityl-D-fructose.

To a solution of D-fructose (10 g, 55,5 mmole) in pyridine (40 mL) and chloroform (25 mb) was added trityl chloride (35 g, 128.3 mmole) and the mixture was stirred at 25* for 2 days, another 5 g of trityl chloride was added and stirring was continued for 2 days. Solvent was removed under reduced pressure and the residue was extracted with chloroform, washed with aqueous saturated cupric sulfate solution, brine, dried over anhydrous sodium sulfate and concentrated. Column chromatography (chlorofmmethanol (100:1)] gave 1,8-Di-O-trityl-D-fructose (11 g, 30%), This compound was identified by its specific rotation and by NMR and Mass spectrometry.

Step 2: Preparation of l,8-Di-0-trityl-2,3-0-isopropylidenel-D-fructosfuranose. . λ mixture of 1,6-Di-O- trityl -D-fructose (2 g, 3 mmole), . dimethoxypropane (S mL) and p-toluenesulfonic acid (0.1 g) in DMF (20 mL) was stirred at 25* for 3 h.. sodium bicarbonate was added - 19 IE 913228 to neutralise the solution. After removal of the salt and solvent, the residue was extracted with chloroform, washed with water and brine, dried and concentrated. Column chromotagreaphy [petroleum ether-ethyl acetate] gave 1,6-Di-O-trityl-2,3-0ieopropylidene-#-D-fructosfuranoee (0.9 g, 42%). This compound wae identified by its specific rotation and by NMR and Mass spectrometry.

Step 3» preparation of 2,3-0-Xsopropylidene-4-0-heptyl-l,6-di-0trityl-/-D-f ructof uranose. 1,6-Di-O-trityl-2,3-O-isopropylidene-i-D-f ructosf uranose (0.5 g, 0.71 mmole) was dissolved in DMF (10 mL) and sodium hydride (60%, 0.12 g) was added. The mixture was stirred at 25®C for 20 min. then 1-bromoheptane (0.62 g, 3.5 mmole) was added and stirred for 1 h. Methanol was added to destroy the excess sodium hydride and solvent was evaporated. The residue was extracted with chloroform, washed with water, brine, dried over anhydrous sodium sulfate and concentrated. Column chromatography [petroleum ether-ethyl acetate (100il)] gave 2,3-0-lsopropylidens-4-0-heptyl1,6-di-O-trityl-#-D-fructofuranose, (0.47 g, 82.5%). This compound was identified by its specific rotation and by NMR and Mass spectrometry.

Step 4t Preparation of 2,3-0-Iaopropylidene-4-0-heptyl~j-Dfructofuranose.

' Ammonia (about 100 mL) was passed through a potassium hydroxide tower and cooled into liquid with dry ice to a solution of 2,3-0-l»opropylidene-4-0-heptyl~l,6-di~0-trityl-A-bf ructof uranose (1.35 g, 1.68 mmols) in dry THF (30 mb) cooled* with dry ice. The solution was stirred and pieces of lithium was added - 20 IE 913228 until th· blue color persisted. The solution was stirred until the reaction was finished (checked with TLC). Bthanol was added to discharge the reaction and ammonia was allowed to evaporate overnight. The the residue, chloroform was added and salt was removed. Column chromatography (petroleum ether-acetone (8*2)} gave 2,3-0-lsopropylidene-4-O-heptyl-l-D-fructofuranose (Ik) (0.45 g, 84.1%). This compound was identified by its specific rotation and by NMR and Mass spectrometry.

Pharmacological Activity of compounds of Formula I.

The pharmacologic assays performed to determine the immunomodulatory effects of the experimental compound· in vitro include the Mixed Lymphocyte Reaction. (MLR) and the ConA blastogenesis assay. These assays were used to determine the inhibitory effects of the compounds of the invention On T lymphocyte activation and proliferation. Since inflammation at the cellular level is characterised by T lymphocyte recruitment, activation and proliferation, these assays are appropriate to use as screens for novel compounds having therapeutic potential in the treatment of disorders ia which inflammatory mechanisms are involved.

The MLR is a classical assay used to .measure T cell function by studying the proliferative response of T cells which are activated in vitro by genetically disparate stimulator cells.

This is accomplished by co-culturing spleen cells from two different strains of mice. Splenic T cell proliferation occurs as a result of cellular activation signals generated by the ongoing cellular interactions.

Specific Method» MLR ASSAY Balb/c mice were euthanised by cervical dislocation and their spleens removed· Single cell suspensions of the spleens were prepared in culture medium (hopes buffered RPMI-1640 supplemented with 10% fetal calf serum, 2mM glutamine, 500 units penicillin/ streptomycin, and 4 x ΙΟ*5 X 2-mercaptoethanol) using a Teflon pestle. The cells were centrifuged at 1500 rpm and the pellets resuspended in ACT (0.15 M tris, 0.14 X ammonium chloride, pH 7.2) in order to lyse the red blood cells. After a 5 minute incubation in a 37 C waterbath, the cells were resuspended in culture medium and counted. C57B1/6 spleen cells which were used as stimulator cells, were also prepared by this method. The stimulator cells . were treated with 100 ug/ml of mitomycin c for 1 hour at 37 C (to inhibit stimulatory cell proliferation) and were then washed 5 times in culture medium. The proliferative responses were measured by culturing 2.5 x 105 responder cells with 5 x 195 stimulatory cells in 96 well microtiter plates ih the presence or absence of various doses of test compounds or vehicle (DMSO). Syngeneic control cultures using mitomycin c treated Balb/c spleen cells as stimulator cells were also run. All cultures were run in triplicate.

Solutions of compounds of the present invention in DMSO were prepared at a stock concentration of 120 fflM. Dilutions were made in culture medium to the following concentration·! 3, 10, 30, 100, and 300 uM. Th· vehicle (DMSO) was used as a negative control.

After incubation of for 5 days at 37 C with 5* C02, the amount of cell proliferation wae measured by adding 20 ul of MTT (3-(4,5-dimethylthiasol-2-yl}~2,5diphenyl-tetrasolium bromide) (10 mg/ml in phosphate buffered saline) to each well. Plates were incubated for 4 hours at 37 C, after which 180 ul of 10* sodium dodecyl sulphate in phosphate buffered saline was added. After an overnight incubation, the optical density (OD) of each well was read on a Molecular Devices microplate reader et 570 - 650 na.

The results were determined by calculating the difference between the means of the allogeneic cultures', and the means of the «yngenic cultures for each test article concentration. Differences of the test* article groups were compared to the control group and the percent change from the control was determined. Test articles which were found to inhibit proliferation are presented as -()% of control with inhibitions greater than -20% of control being considered active.

Mouse Spleen cell ConA Blastogenesis Assay it is well known that several plant lectins, when cultured in vitro with lymphocytes, stimulate cellular activation and proliferation. Concanavolin, (ConA) selectively stimulates the blastcgenic response of τ lymphocytes. Therefore, the ConA blastogenesis assay is useful for screening the immunomodulatory and anti-proliferative activities of experimental compounds. - 23 IE 913228 gMQlflc Itothodi Con* A...V Six to 8 week old male C57B1/6 mice were purchased from Sarian Sprague Dawley (Indianapolis, IS), Spleens were removed and were homogenised to obtain a single cell suspension. Srythrocytes were lysed by hypotonic shook.

Upon determination of the viability and concentration of the lymphoid cells, they were adjusted to 4 x 108 cells/ml in culture medium (ftFXX-1640) supplemented with 10% fetal bovine serum; 100 ug/ml streptomycin; 100 U/ml penicillin, 0.2 x hepes buffer; S x ΙΟ’5 X 2-mercaptoethanol and 2 aX glutamine). Spleen cells were seeded into microtiter plate wells at 2 x 105 cells/0.050 ml/well. To these cultures were added various doses of experimental compounds and ConA at a final concentration of 4 or 1 ug/ml. Control cultures consisted of cells, ConA and culture medium containing the vehicle, DMSO only. Zn some assays the positive controls cyclosporin A (CSS) and AST were also run. For the testing of the methyl l,3-0-isopropylidene-$-0-n-heptyl-a-Dfructofuranoside compound indomethacin and MOQA were used as controls. All cultures were run in triplicate.

Solutions of compounds of the invention in DMSO were prepared and dilutions were made in culture medium. . Assay concentrations were either» 1, 2.5, 10, 25, 100, 300 and 750 ug/ml or 0.001, 0.0Ϊ, 0.1, 1, 2.5, 10, 25 and 100 ug/ml. .

Cultures were incubated for 3 days at 37*C in a humidified atmosphere of 5% of COj in air. For the last 18 hours of culture, 1 uCi of 3H-thymidine was also incubated in each well. The cells were precipitated by a multi-channel harvester. The amount of InoozpoxaMd by the oaltuxM, a· a aaaaoxa ot call proliferation, was measured in a liquid scintillation counter.

The amount of radioactive incorporation is proportional to the amount of cellular proliferation in individual wells. The students T test was used to determine the significance of the difference between experimental and control values.

IL-1 Assay Interleukin 1 (IL-1) is a potent immunomodulatory cytokine that has a broad range of pro-inflammatory activities. IL-1 ie known to be produced by activated accessory cells such as macrophages. Accessory cell production of IL-1 results in the activation and proliferation of T and B lymphocytes. Therefore, by inhibiting macrophage activation and production of IL-1, the activation and proliferation of the cellular effectors of inflammation can be modulated. Compounds of the invention were screened for inhibitory activity in a classical IL-1 assay. Specific Method» IL-1 Assay Six to 8 week old male C57B1/6 mice were purchased from Harlan Sprague Dawley. Peritoneal macrophages were elicited by the administration to mice of a single intraperitoneal injection of 0.2 ml of complete Freund's adjuvant. After 48 hours, elicited macrophages were removed from the mice by lavage with the use of Hank's balanced salt solution.

Macrophages were washed and seeded into microtiter wells at a density of 2 x 105 cells/well in culture medium (as described in the ConA blastogenesis assay). To the macrophage cultures were added various doses of the compounds of the invention and 10 ug/ml of the macrophage activator lipopolysaccharide (to stimulate IL-1 production). Control culture· consisted of macrophages, lipopolyaaccharid· and culture medium containing various doses of DM8O only. Cultures were incubated for 24 hours at 37 *C in a humidified atmosphere of 5% COjin air. Compounds of the invention were prepared in DMSO and diluted to the following concentrations in culture mediums 0.001, 0.01, 1, 2.5, 10, 25 and 100 ug/ml.

The amount of IL-1 produced in the individual wells was determined in a bioassay for IL-1. This involved the removal cf thymuses from mice less than 8 weeks of age. Thymocytes were isolated by passing each thymus through a stainless steel mesh screen. Thymocytes were placed in culture at 1.5 x 10$ cells/well in RPMI-1640 medium containing 54 fetal bovine serum and 1 ug/ml PHA in microtiter plates. Triplicate cultures were cultured with dilutions of macrophage supernatants in the same medium at 37fC in a humidified atmosphere of 5% CO^ in air* After 48 hours, wells were pulsed with 0.5 uCi of 3H-thymidine. After 18 hours cells were harvested and radioactive incorporation (as a measure of call proliferation) was quantitated in a liquid scintillation, counter. The students t test was used to determine the significance of the difference between experimental and control values.

Results of Screening Assays Performed on Representative of Ponnula I ._ A summary of the inhibitory effects of compounds (lb) - (XJ) on T lymphocyte proliferation as measured in the ConA blastogenesis assay is given in Table XX. As demonstrated by - 26 IE 913228 these data, the fructofuranosides inhibit dose dependent inhibition of ConA stimulated T lyaqphocyte proliferation with strong inhibition (great than -50% of control) by these compounds of the present invention at 100 ugZnl with the exception of (Zi) which was found to be a less potent inhibitor of the mitogen induced τ lymphocyte proliferative response.

Tables III and iv are specific examples of the inhibitory effects of compounds of the invention on T lymphocyte proliferative activity. As seen in Table XXX, compound (Ik) is inhibitory in a dose dependent manner with significant inhibition of T lymphocyte proliferation observed at concentration· ranging from 1 ug/ml to 100 ug/ml. Table XV demonstrate· that compound (Xi) exert· significant, non dote dependent modulation of T lymphocyte proliferation with significant inhibition observed at all dose levels on the 1 ug/ml ConA cultures. The results shown in Table V indicate that methyl l,3-0-isopropylidene-8-O-n-heptyla*D-iructofuranoside produced a doe «-dependent, significant, inhibitory effect upon the ability of normal, splenlcally-derived, mouse τ-cells to proliferate in response to mitogenic stimulation. There were less T-cells in the treated cultures at the end of the assay in comparison to the untreated control cultures.

IE 91322» I * · . . . ’ 1 e— * • · w· 1 8» li# Ik XX I 1 28 i§ xi HM 88 ι L 88 MW 88 ii • · · • t ι ι 88 1 1 COX x Jk *-· 1 S3 £x 82 ii ii 1 I 88 88 88 ii | I 1 1 1 »28 1 f w 88 ii §h W 1 w** IB ii 1 1 XX 4» <0 88 252 1 1 82 1 1 §8 $8 X, 85 SB 8® S8 ii 1 1 B2 ii 1 « BS 88 ii ϋ 1 w* 8 S3 ¢5 1 « 4k® si 1 1 23 1 1 3 31 1 1 2$ as ii w«> ••«-..I 83 X • k* 1 83 B8 82 Si i\»C3 Si &4| ϋ ii I 1 SB 1 w 8S w XX SB HW ON ®o Bi ii Φ* ®2 38 1 1 r»ut 38 Ml MCB ( WX ii ϋ » iS 3i 88 EFFECT OF THE INVENTION ON A MIXED LYMPHOCYTE RESPONSE1 Concentration of Con-A -- % Change fro· Controlb CfiNCBltnitlon of Invention fuo/en 100 25 10 2.5 1 o.l 0.01 0.01 IE 91322« I o IE 91322b IH O *8 ι ι ί < ι ι ϊ I 5 · ϊ ι <4 I , 3L 3 K 8w β ►· is» · «4 5* *© ££ ’ 8b s ♦ 'Si » I £ ) Η» ' • ’ τ£ K 1« Ii 3* «Κ §* 3? I i$ ♦ ι»» *<λ *w Ί»θ *»- · 2L b· fig V & e © »♦ I Ul 14* ι · 5P 5P S2 »1— »* »·„ <*K 5*8 t*S g Q?“ 8& *£ I Ooj w· . — ϊ°« ?b *b L -L ^.o *—· · <~»u> ι S ι rs» ι * 8& · Sm^ · p·· * w *> ·© *·*?* ι w · 8* *S §i Ύ$ -Sh» IS» W— N <4 Sk • M ©· ISJ —.; >-tn -**· *^· S-S ?*K Ii |S |g || §| 3g I 1 Sg ·** *W1 b · '8. bo to ~ » ij Wl %0 *0 is Is s sS Tf if a* 8f «Μ»** t* ~8 5& ,?Ύβ ΓΟΜΟΙ* Sk • Μ» wen u> £ · *-*· *M 8& »«-» ο» r- © IS» 8 Κ» ~s • ' • »>· T?* + w A? ©h> it • *·—i»►— s& 8b ii IW • tat * in ♦b *♦ *4 © © *4 · M Oik re s S II 4k W· U% (J) e 8 />♦ I «a o e 8 off SS. £g w ' C • ♦ Ch 9 £ is» .(4 * 2« H TABLE IV EFFECT OF COMPOUNDS ON THE CON-A RESPONSE OF NORMAL SPLEEN CELLS wound Con-A Blastogonlc response of norrnl spleen cells to Con-A x i0“? qm; % effect Dose of DMSO In solution 0.375% 0.094% 0.0375% 9x3x10?% 3.75x10?% 3.75x10“*% 3.75x10-5% 3.75X1O76* I w N ι I · ♦ · I ·.

“S *s • · · rs, 4 « ·· · 0» +>. a $ tt r a «? w B Effect ef Methyl l,3-0-I«ogropy1idene-6-0-h-heptyl-ft,t>-fnictof«rano$fl (la) on the Con-A Response ef normal Spleen Cells » Mb w Compounds of formula 1 were found to also bs Inhibitory to T lymphocyte activation, and proliferation when assessed for activity in mixed lymphocyte reactions. As is seen in Table VI, when compared to controls, fructofuranosides of formula Z exhibit non dose dependent reductions in MLR generated proliferation with strong inhibition defined as greater than -50% of control cultures and moderate inhibition defined as -20% of controls.

Further immunomodulatory effects of compounds (la), (Id), and (le) were demonstrated by testing for inhibitory effects of these compounds on macrophage IL-1 production, in results given in Table VIZ, the compounds were observed to inhibit macrophage IL-1 production in a dose dependent manner with significant inventory effects observed in all compounds at concentrations ranging between 2.5 to 100 ug/ml.

In summary, compounds of formula I have been found to have, immunomodulatory and anti-proliferative effects which predict that these compounds of the present invention have utility, from a therapeutic standpoint, in the treatment of a variety of inflammatory and/or autoimmune diseases. - 35 IE 913228 §885- §§8So. §885u. §885 ooooo » » « · · H->WWW ooooo • . * · · * * M . mw «δι>ί ι. 4*0 β w A, u> ooooo J* *5?*?5ϊ?^»ΰ> NWWWW w s ssssS sSasss £ ooooo < * « · · ooooo • · » « »—» !_.>_· *M »— 38338 ooooo • · ♦ » · QWMMH 32388 ooooo ··'··· ggM UJ silba I til 'MfOr-WUI »-» (a> W ι 0» I I I J.

S88S£ SS5SMe o »? IE 91322» ooooo ooooo • I · ♦ » · · · · · SSSSS XS88S Group B Vehicle Control B 0.182 ooooo ooooo o » · 4 · · ···»· * §'O fr* ►* η* Ο η* *5 9 <3 asss B3sss s I I I I I I t 8-8KJS g*«ES EFFECT OF COMPOUNDS ON THE PROOWTION OF INTERLEUKIN-i BY PERITONEAL A second embodiment of this invention are derivatives of glucose and allose. Glucose and sllose are six carbon monosaccharides which differ from each other by the configuration of the 3-carbon in the sugar. More particularly, these compounds include mono-substituted ether derivatives of α-D or β-Ό glucofuranoses and e-D or F-D allofuranoses as well as analogs thereof. The generic formulae ΙΪ and HI, below, encompasses both λ or p configurations. The compounds are substituted at the 3or 6-position of the monosaccharide. The. analogs of these compounds are those where the oxygen at the 3- or 6-positon has been replaced by an amino group or by sulfur.

As will be apparent from the following discussion, the compounds of this embodiment can be broadly classified into two groups: fully blocked mono-substituted compounds, I.e.. those having two isopropylidene protecting groups, and partially blocked mono-substituted compounds, i.e.. those having only one isopropylidene protecting group. Applicants have found that fully blocked and partially blocked derivatives of glucofuranose and allofuranose are effective in the treatment of autoimmune and/or inflammatory disorders. - 40 IE 913228 The 3-subatituted derivatives of giucofuranoa· and allofuranose are shown by the following general formula II» and R5 is ^12*^20 alJc7^' 07 CnH2nY' *^®rein n“ 1,2,3 or 4 and 7 is selected from cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidasolyl, pyrasolyl, pyrasolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isooxasolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, OfCH^)2, (¢5-^0 alkoxy), ch2ch(ch3)ch2H(CH3)2, ch2ch2h(c5~c10 alkyl)2, or (Cj-Gy alkenyl; or X1 is NH and R5 is C2-C10 alkyl, or CnH2nY n® 1,2,3 or 4 and 7 is selected from hydroxy, cyano, pyrrolyl, pyrrolidinyl methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrasolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (C$- C1Q alkoxy), and phenyl; and R$ and R7 are hydrogen or fora an.isopropylidene ring.

Selective removal of the protecting group at the 5,6-positlon by the following general procedure yields partially blocked fi 7 compounds of formula ZZ where R and R are hydrogen. Preferred compounds of formula ZZ are» 1.2- 0-Ieopropylidene-3-0-n-dodecyl-e-D-glucof uranoee (ZZa); , 2-O-Ieopropylidene-3-O-n-pentadecyl-e-D-glucof uranoee (lib) j 1.2- q-leopropy lidene-3-O-n-ootodecyl-a-D-glucof uranoee (Ho); 1.2- o-Ieopropylidene-3-0-3'-(phenylpropyl)-α-D-glucofuranoee (lid); 1,2»3,6-Di-0-ieopropylidene-3-0-3 ’ - (morpholinylpropyl) -«~D— glucofuranoee (lie); 1,2»5,6-Di-0-iaopropylidene-3-0-3' -n-propoxy-n-heptyl-a-Dglucofuranose (Ilf); 1.2- 0-I»opropylidene-3-0-3 *-n-propoxy-n-hoptyl-e-Dglucofuranose (Hg); 1.215.6- Di-0-isopropylidene-3-0-2 * - (ethylpyrrolidyl) -α-Dglucofuranose (Xlh); 1.2- 0- Ieopropylidene-3-0-2 ' - ( ethylpyrrolidyl, -e-Dglucofuranoee (III); 1.2- O-ieopropylidehe-3-0-3'-(propan-1 *-ol)-a-D-glucofuranose (iii); 1.215.6- Di-0-ieopropylidene-3-deoxy-3-amino-l '-(3' -hydroxy-npropyl)-«-D-glucofuranose (Ilk); 1.2- 0-isopropylidene-3-0-3' - (Ν' ,N' -dimethylamine-n-propyl, a-D-allofuranose (III); 1,2(5,6-Di-0-isopropylidene-3-0-3' - (phenylpropyl) -e-Dallofuranoee (Ilm); - 42 IE 913228 1.2- 0-Isopropylidene-3-deoxy-3-R-3' (H' ,R'-dimethylamino-npropyl-e-Drallofuranose (Un); 1.2- 0-lsopropylidene-3~deoxy-3-amino-3-h-heptyl-a-D-allofuranose (Πο), 1.2- 0-isopropylidene-3-deoxy-3-aaino-3'-(propaa-l-ol,α-D-glucofuranose (Up); 1.2- 0-Isopropylidene-3-deoxy-3-N-3'-(phenylpropyl) -o-Dallofuranose (iig); 112-0-isopropylidene-3-deoxy-3-amino-n-heptyl-e-I>-glucof uranose (Hr); and 1.2- 0-isopropylidene-3-deoxy-3-amino-n-3'-(phenylpropyl)-e-Dglucofuranose (Ila).

Representative examples Cf the above-mentioned compounds of formula II were prepared by one of the following procedures in Examples 6-11. The pharmacological activity of the compounds of formula II is illustrated in Example 12.

WAU General Procedure for the Selective Hydrolysis of 5,6poeitions.

The fully blocked monosaccharide (1;Og) is dissolved in tetrahydrofuran (1 ml) and to thia was added dropwise a perchloric acid solution (30%, 1 ml), with stirring, at 0-5°C. The reaction is monitored by TLC and GC. The normal reaction time varies from 20 minutes to 1 hour. After the completion of the reaction, it is neutralised with a saturated solution of potassium carbonate to a pH of 9.0. The solid salt formed is filtered through Celite and washed well with tetrahydrofuran (50 ml) in several portions·. The combined filtrate is subjected to rotary evaporation and the - 43 IE 913228 residue obtained is purified by flash chromatography using silica gel and appropriate solvents. The yield of the final products varies from 75-98%.

Preparation of l,2»5,6-di-0-isopropylidene-3-0-n-dodecyl-oD-giucofuranose and l,2-0-isopropylideno-3-0-n-dodecyl-e-Dgluoofuranose, (Ila).

Step It 1,2ι5,6-Di-0-isooropylidene-3-0-n-dodecyl-e-Dglucofuranose (R®Ci2H25)· A mixture of 1,2* 5,6-Di-O-isopropylidene-a-D-glucofuranose (5.2 g; 0.02 mol) and dry powdered sodium hydroxide (3 equivalents) was heated together at 120-130° in an oil bath. This reaction was conducted under diminished pressure so as to get rid of water formed during the reaction. This reaction takes about 30-45 minutes depending upon the batch size. The vacuum line was then disconnected and 1-bromododecane (1,2 eq.) was added in one portion. The reaction mixture was stirred at the same temperature for 30 minutes to 2 hours. The reaction flask was then cooled, dichloromethane (100 ml) was added and the mixture was filtered through Celite and washed with 100 ml more of the solvent.

Solvent was removed using rotary evaporator and the residue purified by flash chromatography using ether* hexane (10*9.0).

Step 2* Hydrolysis according to the general procedure described in-Sxample 6 gave the partially blocked compound 1,2-0isopropylidene-3-O-n-dodecyl-o-D-glucofuranose, (Ila).

Preparation of i,2*5,6-Di-0-isopropylidene-3-0-3'(npropoxyheptyl)-e-D-glucofuranose, (Xif) and 1,2-0-Isopropylidene3-0-3' - (n-propoxy-heptyl) -α-D-glucofuranose, (iig).

Step li Preparation of l,2»5,6-Di-0-isopropvlidene-3-0-3*(npropoxyheptyl)-a-D-giucofuranose (R5 -(CH2)3OC7Hi5) 1,2*5,6-Di-o-isopropylidene~3-0-3 '-propanol-a-Dglucofuranoae was prepared by reacting l,2*5,6-di-Oisopropylidene-a-D-glucofuranose with sodium hydroxide (equivalents) at 120 - 130eC under vacuum. After 30 minutes, the vacuum line was disconnected and 1-bromopropanol (3 eq) was added.

The reaction mixture was heated at the same temperature for 45 minutes. The flask was then cooled and ether added (100 »L). The * solution was filtered through Celite, washed with 100 &L more of ether and the solvent removed. The residue was purified by flash chromatrography using 10% ether in hexane to afford the title compound in 84% yield.

A mixture of l,2*5,6-Di-O-isopropylidene-3-0-3'-propanol-aD-glucofuranose(0.02 mole) and dry powdered sodium hydroxide (3 equivalents) was heated at. 120-130® under vacuum. When the evolution of water had ceased, the vacuum line was disconnected and heptyl bromide (1.2 eq.) was added in one portion and the mixture heated at the same temperature for 1 hour. The reaction flask was cooled and ether (100 ml) was added. The solution was filtered -through Celite and washed with 100 ml of ether. The combined solvents were subjected to rotary evaporation to remove the ether and then purified by flash chromatography using 5% ether in hexane. 1,2*5,6-Di-0-isopropylidene-3-0-3' (n-propoxyheptyl) -e-D- 45 IE 913228 glucofuranose was obtained and characterized by HMR and mass spectral analysis.

Step 21 Hydrolysis according to the general procedure described in Example 6 gave the partially blocked compound 1,2-0Isopropylidene-3-0-3 · - (n-propoxyheptyl )-«-D-glucof uranose.

Preparation of l,2-0-Isopropylidene-3-deoxy-3-amino or substituted amino-e-D-glucofuranoses.

Step 1» Preparation of l,2»5,6-di-0-isopropylidene-3-deoxy-3asido-a-D-glucofuranose: A mixture of 1,2:5,6-di-O-isopropylidene-3-tosyl-a-Dallofuranose (prepared as described in Methods in Carbohydrate Chemistry, vol. 6, p. 197 (20g, and sodium aside (2.5-eguivalents) in anhydrous DMP (100 ml) was heated at 80*C for 3 hours. The progress of the reaction was monitored by TLC and GC. When the reaction was complete, DMP was removed under reduced pressure and the residue was extracted with ether (150 ml), and washed with water (2x25 ml), saturated solution of sodium bicarbonate (2x20 ml) and then with brine (1x25 ml). The organic layer was dried over MgSO*, filtered and solvent was removed using a rotary evaporator. This compound (95% yield) was found to be sufficiently pure by TLC and GC and hence was used in the next step.

Steo 2« Preparation of l,2»5,6-di-0-isopropylidene-3-deoxy-3amino-a-D-glucof uranose.

The asido compound (5g) obtained in step 1 was reduced catalytically using Hj, Palladium-charcoal (10%, 50 mg) and methanol (100 ml) in a Parr-hydrogenator at a pressure of 35 psi for 6 hours. The reaction mixture was then filtered using Celite, washed with methanol (100 ml) and the solvent removed using a rotary evaporator. The residue obtained showed a single homogenous spot on TLC and complete disappearance of aside group peak by IR. The yield of the pure compound was 95*.

Steo 3» Hydrolysis according to the general procedure described in Example 6 gave the partially blocked compound 1,2-di-oisopropylidene-3-deoxy-3-amino-e-D-glucofuranose.

Step 4t Preparation of l,2t5,6-Di-0-isopropylidene-3-deoxy-3amino-n-heptyl-«-D-glucofuranose. 3-Deoxy-3-amino compound obtained in step 3 was heated with l-bromoheptane at 70-80°C in the ratio of 1:2.2 for 3-4 hours.

The progress of the reaction was followed by TLC and GC. After the completion of the reaction, the product was extracted with ethyl acetate, washed with a saturated solution of sodium bicarbonate, brine and then the organic layer dried over anhydrous MgSO^. The removal of the solvent gave the crude compound which was purified by flash chromatography. Other amino substituents can be synthesised by the same procedure using an appropriate alkyl halide compound. The alkyl halide itself can be further functionalised..

Step 4t Hydrolysis according to the general procedure described in Example 6 gave the partially blocked compound 1,2-Di0*ls.opropylidene-3-deoxy-3-amino-n-heptyl-0-D-glucofuranose, - 47 IE 913228 Example IQ Preparation of 1,2 »5,6-DI-0-iaopropylidene-3-0-suba tituted ο-D-allofuranoses and l,2-0-Ieopropylidene-3-0-substituted e-Dallofuranose.

Step li 1,2:5,6-di-0-ieopropylidene-«,D-allofuranose wae treated with dry powdered sodium hydroxide and a suitable alkyl halide or substituted alkyl halide in the same manner and ratio as described for the glucofuranose derivative in Example 7, step 1.

Step 2« Hydrolyeis according to the general procedure described in Example 6 gave the partially blocked compounds.

Sample. H Preparation of l,2i5,6-Di-0-isopropyI±dene-3-deoxy-3-amino-nheptyl-a-D-allofuranose and l,2-0-lsopropropylidene-3~deoxy-3amino-n-heptyl-a-D-allofuranose, (IIo).

Step It Preparation of l,2»5,6-Dl-0-isopropylidene-3-deoxy-3amino-n-heptyl a-D-allofuranose. 1,2:5,6-Di-0-isopropylidene~a-D-ribo-hexof uranose-3-nlose (prepared according to the literature procedure Methods in Carbohydrate Chemistry, Vol. VI pp. 125) and heptylaaine, in the ratio of 1:2, were mixed and heated at 50-80°C for 30 minutes to 2 hours under diminished pressure. When the evolution of water had ceased (the progress of the reaction was monitored by TLC and GC), the vacuum line was disconnected. The product was dissolved in anhydrous THF and added dropwise to a stirred suspension of lithium aluminum hydride (LAH, 2 equivalents) in anhydrous THF.

The reaction was carried out at 5-10’C with rigorous stirring and was complete in 2 to 3 hours. The excess LAH was then decomposed by careful addition of water and 15% sodium hydroxide solution (1 ml of each per gram of LAH used). The reaction mixture was then filtered through Celite, washed with THF and solvent removed. The residue was dissolved in ethylacetate, washed with water, dried and solvent removed. Zt was purified by flash chromatography using appropriate solvents. Other amino substituted compounds can be prepared by substituting other appropriate amine compounds for heptylamine in this procedure.

Step 2i Hydrolysis according to the general procedure described in Example 6 gave the partially blocked compound 1,2-0iaopropyl±dene-3-deoxy-3-amino-n-heptyl-a-D-allofuranose, (Ilq). gxampAft.12 Pharmacological Activity of Compounds of Formula XI.

Compounds were tested for immunomodulatory, antiproliferative and anti-inflammatory activities in screening assays which measure T-cell proliferation, activation and macrophage IL-1 production as described in Example 5. Compounds of the present invention were also tested for effects on fibroblast proliferation and production of pro-inflammatory mediators.

Since the early 1970'a it has been known that important mediators of the inflammatory process are the leukotrienes and prostaglandins which are synthesised by tissue cells and macrophages at the site of inflammation (Flower et al., Analgesics-antipyretics and Anti-inflammatory Agents; Drugs Employed in the Treatment of Gout,* The Pharmacological Basis of Therapeutics. Hew York, 1985). in inflammatory disorders damage to mammalian cells occurs by physical trauma or the combination of an antigen with antibody and thia ia thought to initiate the the biosynthesis of these mediators of inflammation, which are, in - 49 IE 913228 turn, responsible for the physiological and visible signs of inflammation. This correlate* with the recruitment, activation and proliferation of T-lymphocytes to the localised area of inflammation. In psoriasis, there is an Ιλοι&&β· in the formation of arachidonic acid in the psoriatic skin that results in mildly increased production of prostaglandins, and a several-fold increase in the concentration of leukotrienes, principally LTB*. LTS4 is the principal biological mediator which is responsible for the promotion of .the Inflammatory process that exacerbates the disease (Anderson, T.F., New Reasons for Being Time-Honored Empiric Therapy, Consultant. 1985. In the autoimmune diseases with arthritic components, proliferating synovial fibroblasts are responsible for the production of inflammation mediators. The data below demonstrates that the compounds of the present invention have pharmacological activity in reducing fibroblast production of LTBj and PG82, which have an effect in regulating the activity of the infiltrating T-lymphocytes, and are antiproliferative agents in skin fibroblast cultures. Moreover, this activity indicates that physiologically acceptable doses of these claimed compounds can be used, either topically or systemically, to inhibit T-oell and human fibroblast proliferation.

Assays were conducted to demonstrate the ability of the compounds of the present invention to modulate the proliferation of BUD—8 human skin fibroblasts and to modulate the production of and I»TB^. - 50 IE 913228 Specific Method: ribrblast Assay The human ekin cell fibroblast line, BUD-8 was obtained prior to each assay from the American Type Culture Collection. This is a fibroblast-like cell line which was originally derived from the normal skin of a 58 year old white female.

BUD-8 cell cultures were expanded for use in 25 cm flasks at 37*C in an atmosphere of 5% C02 in air. At approximately <-5 five day intervals, or when confluence was reached, the cells were passaged. This was accomplished by detaching the cells With a Teflon scraper, washing and reseeding the cells at a lower density into fresh tissue culture flasks.

The effect of the compound of the present invention on the proliferative capacity of human BUD-8 skin fibroblasts was measured with the use of a 3a-thymidine incorporation assay using culture conditions which were similar to those used for a Con-A blastogenesis assay, described previously. Cultured skin cells were detached from the surface of tissue culture flasks mechanically with a Teflon scraper. The cells were washed, resuspended in incubation medium and the viabilities were determined. These cells were then plated in triplicate at a density of 2X103 cells/0.1 ml/microtiter well for the proliferation assay and a density of 1X10* cells/0. lml/microtiter . well for the assays to quantitate PGB2 and LTB*. To these cells was added incubation medium containing indomethacin to inhibit prostaglandin production, or nordihydroguaiaretic acid (HDGA) to inhibit leukotriene production (positive controls).

SFP-91 WED 19:51 202 408 4400 P. 55 - 51 IE 913228 After 18 hours of culture, samples of the BUD-8 skin cell supernatants were collected from one set of microtiter plates and frozen until assayed for PGB? or for LTB* content using the radioimmunoassays described below.

After 3 days of culture, 1 uCi 3H-thymidine was added in a 50 ul volume to each culture well of the microtiter plates. Eighteen hours later, each of the BUD-8 cultures was examined morphologically for evidence of compound-induced toxicity such as cell rounding or granularity. The thymidine-pulsed cells were then precipitated and the amount of 3H-thymidine incorporated was counted in a liguid scintillation counter.

The concentrations of the compounds of the invention which were used in these assays were» Group 5: 100 ug/ml Group 6» 300 ug/ml Group 7t 750 ug/al Group 1. 0 ug/ml Group 2» l ug/ml Group 3i 10 ug/ml Group 4: 25 ug/ml The incubation medium used for culturing the BUD-8 cells was RPMI-1640 medium containing 10* fetal bovine serum, 100 ug/ml streptomycin, 100 U/ml penicillin, 0.2 X Hepes buffer solution, 5x10’$ K 2-mercaptoethanol and 2 mM glutamine.

.. A radioimmunoassay (Rew England Nuclear) was used to quantitate PGS2 levels In BUD-8 skin cell culture supernatants. Briefly, into each polypropylene tube were mixed 0.1 ml anti-PGE2, 0.1 ml 1251-PGB2 and 0.1 ml PGKj standard or P6S2 containing sample. The BUD-8 skin culture supernatants were diluted 1:2 prior to addition to the radioimmunoassay. Tubes were refrigerated overnight. A polyethylene glycol solution (16%, 8,ύυΰ m.w.) wa* then added to precipitate immune oomploxoc and the radioactivity in the precipitate was counted in a gamma counter.

Levels of LTB^ in aliquots of BUD-8 skin cell cultures supernatants were quantitated by radioimmunoassay (New England Nuclear). Briefly, into each polyproplyene tube were mixed 0.1 ml anti-LTB*, 0.1. ml ^H-LTB^ and 0.1 ml LTB^ standard or LTB^ containing sample. The BUD-8 skin cell culture supernatants were used directly in the radioimmunoassay. Tubes were refrigerated overnight. A charcoal solution (0.5 ml of 0.5% charcoal Norit λ) was added and each tube was centrifuged. The radioactivity in the supernatant was then counted in a liquid scintillation counter.

The Student's t test was used to determine the significance of the difference between values for T-lymphocytes or skin cells cultured in the presence of experimental compounds versus in control medium alone.

The inhibitory effects of compounds (Ila), (Hg), and (Ilk) on the T lymphocyte blastogenic response to ConA are illustrated in Table VIII. These compounds were found to exhibit non-dose dependent inhibition of T lymphocyte proliferation with all compounds tested being strongly inhibitory (greater than -50% of control response) at the 100 ug/ml concentration of compound. A specific example of a compound of the present invention mediated modulation of the T lymphocyte proliferative response to ConA is given in Table IZ. Statistically significant inhibition of τ cell proliferation was exerted by compound (Ilg) at concentrations ranging between 100 and 750 ug/ml. -53-.

Further immunomodulatory effects of these compounds were demonstrated in the MLR assay (Table X). Strong non-dose dependent inhibition (greater than -50% of control) was observed at several concentrations of compounds (Iig) and (Ila). This indicates that the compounds inhibit T lymphocyte function in activated cultures. tr ι ςπ lp ϊ a £ s £ A 1Λ s A I a o ίΛ «-Η ° 5? c a. •5 >· ,© -n ?? S £ £ B· B §eS *<« Λ M I 33 33 fea 8 i3 33 33 33 » ·ί» »i »» ·£·£ A.S Si £8 ££ £3 38 38$ 3 ££ 38 S« £$ -5 W ££ S3 £* 53 SS S ££ H io SS r\j ♦ Ο» tz» m co tz> as SSi Si 28 38 IE 913228 or I cn O' I ft r* —« r+ “*» I V» A —-1 is» £ ta s c a * (VI *4 »- * * » w (VI s ♦ M U>L*> UJ s ί a (Λ •W Ol * £ S3 ΑΪ 3. -* r+ -H p ι/» a ooooo • · · · ft Ο Ο »— 5ί I ixsror tn 882 μ>» ooooo a · · · .

OO w ►— ►* MO'MOW k> oooo • ft · ft 8&3έ ooooo • · · * * ooooo F3 MOtMOO • I I I I O W W wo mQ o tn ►— o o •M tn EFFECT OF THE INVENT I »1 ON A MIXED LYMPHOCYTE RESPONSE4 Mouse peritoneal macrophage IL-1 production was also found to be inhibited by compounds of the present invention as described in Table XI. Compound (Hg) exerted dose dependent inhibition of macrophage IL-1 production with significant decreases in IL-1 activity observed at 10, 25 and 100 ug/ml of compound. Since IL-1 is a potent stimulator of B and T lymphocytes, both of which are active in inflammatory and autoimmune diseases, these data are indicative of the extensive immunomodulatory effects of the compounds of the present invention. Because uncontrolled fibroblast proliferation and biosynthetic activity are hallmarks of inflammatory diseases in which Joint damage is observed (such as rheumatoid arthritis), compounds of the present invention were tested for activity in fibroblast cultures. Studies were performed to determine the anti-proliferative effects of the compounds, and these effects were correlated with levels of the pro-inflammatory mediators TGBj and LTB* in the fibroblast cultures.

Compounds of the present invention were also tested for potential effects on fibroblast proliferative and biosynthetic activity. Compound (Ilg) was observed to exert non dose dependent anti-proliferative effects on Bud-8 skin cell fibroblasts with significant anti-proliferative activity seen at 100, 25 and 10 ug/ml of compound (Table XII). Evidence of cell toxicity was observed at the two highest concentrations of compound tested.

This anti-proliferative effect correlated well with significant non dose dependent inhibitory effects on fibroblast production of pro-inflammatory mediators. LTB^ levels were significantly decreased in Bud 8 ekin call cultures at concentrations of 1-100 ug/ml of compound (Ilg) (Table XIII).

Cell toxicity was again observed in cultures which included 300 and 750 ug/ml of the compound. Non dose dependent Inhibitory effects on fibroblast Production are exhibited in Table XIV where concentrations of 1, 10 and 25 ug/ml of compound are seen to correlate with significant decreases in PGBg levels in Bud 8 skin cell fibroblasts cultures. Compound induced cell toxicity was observed only at the highest dose level tested.

Since uncontrolled fibroblast proliferation and activation are dominant features in inflammatory diseases such as rheumatoid arthritis and psoriasis, these data strongly suggest the potential for the use of the compounds of the present invention as therapeutic agents for the treatment of these diseases. ι σN ι EFFECT OF COMPOUNDS ON THE PRODUCTION OF LTB4 BY BUD-8 SKIN CELL FIBROBLASTS3 Compound pg LTB4/IOOO ul supernatant (LTB4 produced/10^ BUD-8 cells); % Effect control 38.8*1.6 (749) Dose of compound added to BUD-8 skin cells; -η κ* Ο 4Λ <* The fully blocked 6-substituted derivatives of glucofuranose and allofuranose are shown by the following general formula III» rx‘f* (III> wherein If is o, R® is Cg-C20 alkyl, or CnH2nY' wherein n- 1,2,3, or 4 and 7 Is selected from phenyl, cyano, pyrrolyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozasolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)jN(CHj)2, (C5-C10 alk0*y)» 1,11 or n(CH3)2? or X2 is NH and R® is H, or CnH2nY, wherein η» 1,2,3 or 4 and T is selected from OH, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, I imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CHj)2 or (C5-C1Q alkoxy) or phenyl; or X2 1. s 0 and Re is C5-C10 alkyl, or CnH2nY wherein n- 1,2,3 or 4 and T is selected from OB, phenyl, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolldinyl, oxazolyl, oxazolidinyl, isooxazolyl, izooxazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2 or (C5-C1Q alkoxy); g in and R and R are hydrogen or form an isopropylidene group.

Selective removal of ths protecting group at the 3,S-position iz achieved , by the general procedure described in Example 6 to yield 9 10 partially blocked compounds of formula III where R and R are hydrogen. Preferred compounds of formula III are: 1.2- 0-lsopropylidene-6-0-heptyl-a-D-glucofuranose (Ilia); 1.2- 0-Isopropylidene-6-0-nonyl-e-D-glucof uranose (Illb) ; 1.2- 0-Isopropylidene-6-0-dodecyl-a-D-glucofuranose (IIIc); 1.2- o-leopropylidene-$-0-pentadecyl-a*D-giucofuranose (Illd); 1.2- 0- Isopropylldene-6-0-3’ - (phenylpropyl) -e-Dglucofuranose (IHe); 1.2- 0-Isopropylidene-6-0-3'-(N',N' -dimethylamino-n-propyl) -o-Dglucofuranose (Illf); 1,2:3,5-Di-0-isopropylidene-6-0-methoxyoctyl-a-Dglucofuranose (Illg); 1.2- 0-Isopropylidene-6-0-propionitrile-e-Dglucofuranose (Illh); - 65 IE 913228 1,2:3,5-Di-O- Isopropyl idene-6 -deoxy-δ-amino- [ 2' -aminoethyl- 2 - (N me thyl pyrrol idyl )-«-D-glucof uranose {Illi); 1,2:3,5-Di-O-Isopropyl idene-6 -deoxy-6-amino-3 *.- (phenylpropyl) -a D-glucofuranose (Hlj)? 1,2:3,5-Di-0-Isopropylidene-6-deoxy-6-N-(N# -propylpipecolinyl) -a D-glucofuranoee (IHk); 1,2:3,5-Di-0-Isopropylidene-6-deoxy-6 -amino-ethoxyethanola-D-glucofuranose (1111)? 1.2- 0-IsopropylIdene-6-deoxy-6~amlno-3·-(propan-1'-ol,-«-Dglucofuranose (Illm)? 1,2:3,5-Di-0-Ieopropylidene-5-deoxy-6-thio-n-heptyl-A-Dglucofuranose (Illn); 1.2- 0-Isopropylidene-6-deoxy-6-thio-n-heptyl-a-Dglucofuranose (IIIo); 1,2:3,5-Di-0-I»opropylidene-6-deoxy-6-thio-2'-(ethyl-Npyrrolidyl)-«-D-glucofuranose (Illp); 1.2- 0-Isopropylidene-6-deoxy-6-thio-2'-(ethylpyrrolidyl)«-D-glucofuranose (Illg)f 1,2:3,5-Di-0-Isopropylidene-6-deoxy-6-thio-3'-(Ν',N'-dimethyl amino-isobutyl) -«-D-glucofuranose (Hlr) ? 1,2:3,5-Di-0-Ieopropylidene-6-deoxy-6-thio-3' - (propan-1 * -ol) «-D-glucofuranose (Ills); and 1.2- 0-Ieopropylidene-6-deoxy-6-thio-3'-(phenylpropyl)-e-Dglucofuranose (lilt).

Synthetic procedures for representative compounds according to this embodiment are shown in the Examples which follow. The activity of selected compounds is shown in Example 17. - 66 IE 913228 fiEWfcg 13 Preparation of l,2:3,5-di-0-isopropylIdene-6-deoxy-6-thio-nhepty1-a-D-glucofuranose and l,2-0-Isopropylidene-6-deoxy-6-thion-heptyl-a-D-glucofuranose, (Illo).

St9P .1.» The preparation of l,2:3,5-di-0-isopropylidene-6-deoxy-6thio-α-D-glucofuranose has been described in U.S. Patent No. 4,996,195, the disclosure of which is incorporated by reference.

Step 21 The preparation of l,2:3,5-di-0-isopropylidene-6-deoxy-6thio-n-heptyl-α, D-glucof uranose : A mixture of 1,2:3,5-di-0-isopropylidene-6-deoxy-6-thio-a-Dglucofuranose (2.76g; 0.01 mol) and dry, powdered sodium hydroxide (1.20g) were mixed together and heated at 90°- 95°C, in an oil bath, under diminished pressure (0.1am Hg). When the formation of water bubbles in the flask ceased (40 minutes), the vacuum line was disconnected and l-bromoheptane (2.15g, 0.012 mol) was added. The mixture was heated at the same temperature for 45 minutes (the progress of the reaction as followed by TLC) and then the flask was cooled to ambient temperature. Dichloromethane (75mL) was added and stirred well. The resultant mixture was filtered and the residue was washed with 75mL more dichloromethane in small portions. The solvent was removed and the residue was purified by flash chromatography using etherihexane » 30:70. The yield of the pure compound was 3.33g (89.24). NMR (CDCl^): S 6.01 (d, 1H, Hx), 4.59 (d, 1H, Hj), 1.34 (m, 12H) 0.89 (t, 3H, CH2CS3)· CIMS: 375 (M + 1). 2£S&_3.‘ On· gram of l,2,3,5-di-0-isopropylidene-6-deoxy-6-thio-nheptyl-e-D-glucofurano·· was dissolved in tetrahydrofuran (lmf) and hydrochloric acid (311, ImL) was dropwise-added, over a period of 10 minutes, at 0-10’C. The reaction mixture was stirred at the same temperature for 1.5 hours, and then neutralized with saturated potassium carbonate solution to pH 9.0. The mixture was extracted with ethylacetate (lOOmL) and the solvent removed using a rotary evaporator. Evaporation of the solvent gave the crude product which was purified by flash chromatography using EtjOihexane - 60:40. The yield of the pure compound waa 0.77g (86.2%). NMR (CDClj), S 5.96 (d, IH, Ηχ), 4.54 (d, IH, Hj). 0.87 (t, 3H, CHjCKj). CIMSj 335 (M + 1), 277 (M - C^).

EXAMPLE 14 Preparation of 1,2,3,5«Di-0-Isopropylidene-6-0-n-nonyl-a-Dglucofuranose and 1,2-0-Isopropylidene-6-0-n-nonyl-aD-glucofuranose, (Illb). step 1, 1,2,3,S-Di-O-isopropylldene-6-O-n-nonyl.-a-Dglucofuranose (Rg CgHxg).

The synthesis of 1,2«3,5-Di-0-isopropylidene-a-Dglucofuranose (as described in U.S. Patent Ho. 4,996,195) was achieved by reacting pivaloyl chloride with 1,2-0-isopropylideneα-D-glucofuranose at the 6-position, followed by cyclisation of 3 and 5-positions with dimethoxypropane and finally hydrolysis of 6pivaloyl ester. This compound was treated with dry powdered sodium hydroxide and nonylbromide by exactly the same procedure as described in Example 7, step 1. - 68 IE 913228 Step 2t Hydrolysis according to the general procedure described in Example 6 gave the partially blocked compound 1,2-0Isopropylidene-6-0-n-nonyl-a-D-glucofuranoee, (IHb).

Preparation of l,2»3,5-Di-0-ieopropylideno-6-deoxy-6-aminoα-D-glucofuranoee and 1,2-0-isopropylidene-6-deoxy-6-amino-a-Dglucofuranoee. step 1» Preparation of l,2i3,5-Di-0-isopropylidene-6-deoxy-6amino-a-D-glucofuranoee.

A mixture of 1,2j3,5-Di-O-ieopropylidene-6-tosyl-a-Dglucofuranose (prepared ae described in U.S. Patent No. 4,996,195) (lOg) and eodium aside (2.5 equivalents) in dry dimethylformamide (100 ml) was heated at 80-90°C for 2 hours. The progress of the reaction was monitored by TLC and GC, After the completion of the reaction, the DMP was removed under diminished pressure. The residue was dissolved in ether (150 mi), washed with water (1 x 50 ml), sodium bicarbonate solution (1 x 50 ml), the organic layer dried (KgSO*) and the solvent removed. The product 1,2»3,5-di-0isopropylidene-6-deoxy-6-azido-a-D-glucofuranoee so formed was found to be > 98% pure by GC, TLC and NMR. l,2:3,5-di-Oisopropylidene-6-deoxy-6-asido-«-D-gluco£uranose was then reduced catalytically with hydrogen using Pd/C by a procedure analgous to that described in Example 9, Step 2. The yield of the pure product, 1,2:3,5-di-0-ieopropylidene-6-deoxy-6-amino-a-Dglucofuranoee was 96%.

Step 2» Hydrolysis according to the general procedure described in Example 6 gave the partially blocked compound 1,2-0isopropylldene-6-deoxy-6-amino-a-D-glucof uranoee. - 69 IE 913228 SXA1PLB lg Th· general procedure for the preparation of 6-deoxy-$-amino or 6-amino substituted glucofuranose was to react l,2:3,5-di-0isopropylidens-6-tosyl-a-D-glucofuranose with an appropriately substituted amine (2.2 equivalents), for example 3-pheayl-lpropylamine (Illj), at 80-90”C for 2-3 hours. The progress of the reaction was followed by TLC and GC. After, the completion of the reaction, the product was dissolved in ethylacetate (100 ml), washed with a saturated solution of sodium bicarbonate (2x20 ml), brine (1x20 ml), dried (MgSO*) and solvent removed. The 1,2:3,5Di-0-isopropylidene-6-deoxy-6-am±no-3' - (phenylpropyl) -a -Dglucofuranose was then purified by flash chromatography using the appropriate solvent system.

Step 2: Hydrolysis according to the general procedure described in Example 6 gave the partially blocked compound 1,2-0l»opropylidene-6-deoxy-6-amino-3 ' - (phenylpropyl) -*-Dglucofuranose.

Example. 17 Pharmacological Activity of Compounds of Formula ill. Compounds of formula III were tested for immunomodulatory and anti-proliferative effects. Results of these studies are described in the data tables below.

Compounds (Hie), (ZHi), (IHn) and (Hip) were found to inhibit the mouse spleen cell blastogenic response in a dose dependent manner, with strong antiproliferative effect (greater than -SOI of control) with all compounds tested (Table XV).

A specific example of the inhibitory effects on the ConA mediated blastogenic response of mouse T lymphocytes is given in Table XVI. Compound (IUp) significantly decreased the proliferative response of splenic T lymphocytes at concentrations ranging from 10 to 750 ug/ml. Additional immunomodulatory effects of the compounds of the present invention were observed in MLR cultures (Table XVII) where compounds (Illd), (Ille) and (Illi) were demonstrated to exert strong non dose dependent inhibition of T lymphocyte proliferation. Compound (IXXd) was found to exhibit strong inhibitory effects on the MLR at concentrations ranging from 10 to 300 uM whereas compounds (III·) and (Hid) were active at the highest dose levels only.

Results of the testing of compounds of the present invention for modulatory effects on mouse peritoneal, macrophage production are presented in Table XVIII. IL-1 activity was demonstrated to be significantly lowered in cultures containing 2.5, 10, and 100 ug/ml of compound (Illi). Compound (Ule) was significantly inhibitory to IL-1 activity at 2.5, 10, 25 and 100 ug/ml. Both compounds appeared to exert Inhibitory effects on IL-1 activity in non dose dependent manner. -j % IA B M £ Q) (A a r* £ < -* f w a v> « •A Ό V> s « w> rr a· a -•ο n 0 m 09« ΦΌΗ 3* ai h ί “*· 15 i 3 n s • Ο.Λ b a <|p IiM a rr ia *ίΣξ *T *4· S η o s S*r-O ’'’«z'S'· * · -*· a i ΪΦ A 5, AS is?3 S’* » e o <* w» Q> 7 P· i B r“* «.S 0 0 3(A -fc « o ids «•a e » r>ft rt *8tf* -fcS S 3s;s ^3 £3 8 2. *lb $ a •S.P-, ?8y» 3 r> Je. 33 88 33 88 < II-* It II 88 88 22 II II II ia«a xo> nh »O«O 090» xo» II II II «ate tn cn nh »0U9 ox *CN ii 33 33 ii 33 SS I I H» l I It «00 ee *— tn eo hoi ‘-•tn II II + I 88 s£ Si BS ii si 33 —< £ I” ΓΠ ι I O 10 0» X I I SB I I Si Si Ki S'. — I ti sM -a? 1: Or* M • tt s § ooooo © • · · · · « OOMfc-Γΰ 5— ►“· R Ο © ►— OB tn »-» A ~<4 © nJ ooooo O • « * · · · II··· Q®WMW OOOOO • · · · · © tntn ?8cngh» ooooo * · · · · £ tn ooooo ooooo I I ►-© ©S3 I · vj r· (4© tn r> EFFECT OF THE INVENTION ON A NIXED LYNPHOCYTE RESPONSE® Ln I f-Ο « vi o v; nt 1/3 ΐ!<Γ£ · £ 3* 3 3 tt SerrtSS S i***_<» — J* Φ «a·_wM i -5^s ’ 33·3»8ί ¢8-8 »M SS =3-3 5 $Π» V· £ «ϊίί’δ -2.S «2-S ι/» vi *, re —w ™ » 0 9 0 9 X 9 o e QΓ-Π 5TV· VI iri ’ v c-fl S' 3gJ ~ Φ VI — V» —«-*· «.* ~ra 9 Silk PJ^S g£ss£ a—s s. w* □£ 3*£ IS®!15· ji?£* ^SRS-r = ^«s“g S 3T®25« fr-L s ««d £ -«» tt f* «Λ §?£ 1 X» rt O -*< νι vi O 9 vi a Ο Λ O tt ft 3iO.O<< A <5 o » er η o A -*»o o 2 — Ο «1 9 tt —•A aro r+ e 4 ® ±· —· —· tt 9 O ftt Ui —>r* 9 ”3 ^5*2 v* oi <8 ?* »♦ y *$? s* * O -4 0 O s t/> «e* O f*O et -I Pl m m-j s re Q-x -2-2 5-.81 £|^rtSoS ι σ· 3» r+ 3* A -H -* J 3, 3£ ·· * « *3 Δ l-rf ·· * A © ’ * n iS & —· ?g=:g£§l S' · V» r* A * 5 -«.-nA o *ι/ι->.(ί τ □ o.g’srV’* nx« 2 1 u>£ A |.'o * bA-S'ESp'S sffi =1 ft VI Ί «*3 3 -* arA ftrt —3 ^2.Srn ν» 0.-1 —’M 3- O r> .. ..rfsl r* A A j* M, A r- M A Λ -h0 OB A ft ii -t> __ o A C A —-r>3« Ο V» A--> «+ Q 3 »— -4» »“· O 09 A _ ^«O s tn ai A w. «Λ - A Q A>2 Hl The compounds of the present invention also include the following monosaccharidesι - (38) l,2-0-isopropylidene-a-D-ribo-hexos-3-ulose1,4j3,6-difuranose; and (IV) - Methyl 3-0-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-Dglucopyranoside.

EXAMPLE 18 Preparation of 1,2-0-Isopropylidene-a-D-ribo-hexos-3-ulose1,4:3,6-difuranose, (IV).

Two grams of l,2:5,6-di-0-isopropylidene-a-D-ribohexofuranos-3**uloee were dissolved in tetrahydrofuran (ZmL) and cooled to 0 - 5°C. Agueous perchloric acid (2mL, 30%) was added dropwise with stirring over a period of 10 minutes. The progress of the reaction was monitored by TLC. When the hydrolysis was complete (40 minutes), the solution was neutralised with a saturated solution of potassium carbonate and extracted with ethyl acetate. Removal of the solvent gave a crude product which was purified by medium pressure column chromatography using silica gel G (10-40^) and eluting with ether,hexane 50,50. The yield of the white crystalline material was 1.48g (87.6%), m.p. 81-82· C. IR (KBr): 3390cm“l (broad OH), Ho > C 0 (stretching). NMR (CDC13): S 5.97 (d, 1H), 4.51 - 4.41 (a, 4H), 4.26 (m, 1H), 3.78 (m, 1H), 3.07 (d, 1H), 1.58 (d, 3H), 1.40 (s, 3H).13C NMR (CDC13, APT): S 113.99, 110.93 (C-3 and C-9), 106.98, 84.04, 82.78, 71.07 (C-l, C-2, C-4, C-5), 73.56 (C-6), 27.22, 27.18 (C-8, C-9). CIMS: 219 (M + 1).

SIWffbg. U Pharmacological Activity of 1,2-O-Xsopropylidene-a-D-ribo-hexos3-ulose-l,4:3,6-difuranose, (IV).

Compund IV was teated in the Bud-8 skin cell fibroblasts assays as described in Example 12. As can been seen from Tables XIX, XX and XXI below, the compounds of the invention produced significant, non dose dependent decreases in the Bud-8 skin cell fibroblast proliferation in those cultures that contained 1, 10, and 100 ug/ml, respectively, when compared to the control cultures that did not contain the compounds of the invention. Cytotoxicity was not observed at these concentrations of the test compound. Cytotoxic effects were observed in the highest concentrations tested (300 ug/ml and 750 ug/ml). These antiproliferative effects correlates with decreases in Bud-8 skin cell fibroblast production of PGBj and LTB* (Tables XX and XXI) where significantly decrease of levels of both pro-inflammatory mediators are seen at several non-toxic doses of compound IV. expressed as cpm of tri pi tcates ±S0.

^The effect of experimental compounds on the proliferation of BUO-8 cells is expressed as percent change from the amount of ^H-thymidine incorporated in the absence of experimental compounds. Significance of the effect of experimental compounds: *t P<0.05; #, P<0.01, **, P<0.00.

Effect of Conpounds IV on the Proliferation of BOO-8 Skin Cells rt « rt > either cell rounding or granularity. y S' «1 vt o TABLE XX Effect of Compound IV PGEjj Production by BUM Skin Cells Compound no P6E?/50 ul supernatant (P6E? production/10^ BUD-β cells): % effect __ i5.g,3.5(i >033) Dose of experimental compound added to BUM cells: amount of PGE2 of cells cultured In the absence of experimental coepounds. Significance of the effect of experimental compounds:*, P<0.05; f, PO.Ol, *♦ PO.OOl.

I Φ Γ» t either cell rounding or granularity. y V» 9, I nC I— «3 y Λ -* £ 4? ι CP I VI e ? ί VI o Λ «*· s sr ·· ♦ ’W Δ « 3» * . Ό a g fc— Λ X δ EXAMELB 20 Preparation of Methyl 3-0-3(Ν' ,N'-dimethylamino-n-propyl) -6deoxy-D-glucopyranoside (V).

Step ii The preparation of l,2-0-isopr©pylidene-3-0-3'-(N',N*dimethylaminopropyl) -6-0-p-toluenesulfonyl-a-D-glucof uranose, (l)· Ten grams of l,2-0-ieopropylidene-3-0-3*-(H*,M*dime thy laminopropyl)-a-D-glucof uranose were dissolved in 30mL of anhydrous pyridine and cooled to 5°C in an ice-water bath, pToluenesulf onyl chloride (6.25 g, 1 eq), dissolved in 20mL of pyridine, was added to the stirred solution over a period of 30 minutes. The reaction was then allowed to attain ambient texoperature over a period of 1 hour. After a total reaction time of 3 hours, the solvents were removed under vacuum, and the residue was dissolved in dichloromethane (200mL), and the organic phase washed with 25mL of saturated sodium hydrogen carbonate solution. The combined organic phase was washed with water and brine, dried over anhydrous magnesium sulfate and evaporated to give 14.3 g (95%) of the tpeylate as a glassy material, that solidified on trituration with ether, cimbi 460 (M + 1, Step 21 The preparation of l,2-0-isopropylidens-6-deoxy-3-0-3'(N' ,N'-dime thy laminopropyl)-a-D-glucofuranose, (2).

Ten grams of l,2-0-Isopropylidene-3-0-3'-(N',N'dimethylaminopropyl) -6-0-p-toluenesulf onyl-a-D-glucof uranose (1) were dissolved in 60ml of freshly distilled tetrahydrofuran and the mixture was gradually added to a stirred suspension of 1.7 g (2 eq) of lithium aluminum hydride in 50mL of THP. After the addition was complete, the mixture was refluxed for 2 hours. It was then cooled in an ice bath and the excess lithium aluminum hydride - 85 IE 913228 was quenched by th· addition of 2ttl of water, followed by 5mJ of 15% sodium hydroxide solution. The mixture was filtered through Celite and the filtrate was evaporated to give 5.91 g (94%) of th· 1,2-0-isopropyl idene-6-deoxy-3-0-3'-{Ν' ,N' dimethylaminopropyl)-a-D-glucofuranose (2) product which was used without purification for th· next step. CIHS, 290 (M + I) NMR (CDClj), 4 5.92 (d, 1H, Ηχ), 4.55 (d, IN, Hj), 3.95 (m, 2H, Hj and H4), 3.83 (a, IH, H5), 3.48 (m, 2H, OCfij), 2.28 (s, 3H, Cfij), 1.48 and 1.32 (s, 3H each, C(OS3)2).

Steo 3, The preparation of 6-Deoxy-3-0-3'-(Ν',N'-dimethylaminopropyl )-D-glucopyranose, (3).

Three grams of l,2-0-isopropylidene-3-0-3'-(N',H'dimethylaminopropyl)-6-deoxy-α-D-glucof uranose (2) was dissolved in 10nL of tetrahydrofuran and 5mt of 3 N hydrochloric acid was added. The mixture was stirred for 2 hours at 50°C, cooled and neutralised with 20% sodium hydroxide solution. The solvents were removed under vacuum and the residue was extracted with hot ethyl acetate. The organic phase was dried over magnesium sulfate and evaporated to give 2.2 g (85.4%) of the free sugar, the 6-Deoxy3-0-3*-(N',N'-dimethylaminopropyl)-D-glucopyranose (3) was used directly for the next step.

Step 4, The preparation of methyl 3-0-3'-(Ν' ,N'dimethylaainopropyl)-6-deoxy-D-glucopyranoside, (V).

The 6-Deoxy-3-0-3'-(H' ,N'-dimethylaminopropyl)-0glucopyranose (3) (1.8 g) was dissolved in lOaL of anhydrous methanol containing approximately 5% of anhydrous hydrogen chloride by weight. After 2.5 hours at ambient temperature, the methanol was evaporated, the residue was neutralised with saturated potassium carbonate and «attracted several times with di chlorosis than·. The combined extract was dried over magnesium sulfate and evaporated to give the crude product, which was purified by flash chromatograph (100% ethyl acetate, then 20% methanol in ethyl acetate) to give 1.64 g (86.5%) of the methyl 3-0-3'(»' ,N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside compound. The NMR showed the presence of a mixture of a and ^-isomers in the ratio of 6:4. CIMS: 264 (M+1) Pharmacological Activity of Methyl 3-0-3'-(Ν',N'-dimethylamino-npropyl ) -6-deoxy-D-glucopyranoside, (V).

Methyl 3-0-3'-(Ν',N'-dimethylamino-n-propyl)-6-deoxy-Dglucopyranoside of this invention has demonstrated inhibitory effects on the proliferation of GS-109-V-20 human skin cell fibroblasts.

A compound that inhibits fibroblast proliferation has the potential to be utilised as a dermatological drug used to treat chronic dermatorae, such as psoriasis and autoimmune disorders which result in joint inflammation, such as rheumatoid arthritis. Also, an anti-proliferative effect may well be observed with other tissues, such as those that line the blood vessels, or joints, the uncontrolled proliferation of which produce disease, thereby broadening the scope of potential applications. 8p.oHle Method» Tlbroblatt MBIT Th· effect of methyl 3-0-3'-(Ν' -dimethylamino-n-propyl)-6. deoxy-D-glucopyranoside on the proliferative capacity of human GS109-V-20 ekin fibroblasts was measured with the use of 3Hthymidine incorporation assay. Cultured skin cells were detached from the surface of tissue culture flasks mechanically with a Teflon scraper. The cells were washed, resuspended in incubation medium and the viabilities determined. These cells were then plated in triplicate at a density of 1X103 cells/0. Iml/microtiter well. To these cells was added O.lmx incubation medium containing the methyl 3-0-3'-(Ν',N'-dimethylamino-n-propyl )-6deoxy-D-glucopyranoside compound.

After 3 days of culture, lfuci. 3H-thymidine was added in 50ul volume to each culture well of the microtiter plates. Eighteen hours later, each of the G8-109-V-20 cultures was examined morphologically for evidence of drugrinduced toxicity such as cell rounding or granularity. The 3H-thymidine-pulsed cells were then precipitated and the amount of 3H-thymidine incorporation was counted on a liquid scintillation counter.

Methyl 3-O-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-Dglucopyxano side was suspended directly into the medium by extensive sonication, without being filter-sterilised. A range of doses of this compound was used to measure effects of this compound upon GS-109-V-20 cell proliferation. The following doses were used: - 88 IE 913228 Group 1« 0 ug/al Group 2: 0.001 ug/ml Group 3s 0.01 ug/ml Group 4t 0.1 ug/ml Group 5» 1 ug/ml Group 6» 2.5 ug/al Group 7: 10 ug/ml Group 8: 25 ug/ml Group 9: 100 ug/ml Control Groups: Control: 2X1O’® M Indomethacin Control: 2X10® M NDGA £&2l£i£JS2£hs&_tnffgfretton asdlua The incubation medium used for culturing the GS-109-V-20 cells was RPMI-1640 medium containing 10» fetal bovine serum, 100 ug/ml streptomycin, 100 U/ml penicillin,. 0.2 X Hepes buffer solu- Soedfic method:_Roman skin cells The human skin cell fibroblast line, GS-109-V-20, was obtained from the American Type Culture Collection, This is a fibroblast-like cell line which was originally derived from the skin of an 18 year old Caucasian male with Gardner's syndrome, an autosomal dominant condition which predisposes to carcinoma and multiple polype of the colon (American Type Culture Collection, Catalogue of Cell Lines and Hybridomas, 6th 8d,, 150, 1988). These cells were selected for use because they are considered to exist in an initiated state, as opposed to being normal or transformed, and have a more extensive population doubling time and survival period in culture than do normal fibroblasts. In this regard, they would store closely reflect the biological characteristics of psoriatic or rheumatoid synovial fibroblasts which also proliferate more extensively than do normal - 89 IE 913228 fibroblasts, but not as extensively as immortalized transformed tumor cells.

The number of G8-109-V-20 cells was expanded for use in the 2 described assays by culture in 25 cm flasks at 37‘C in an atmosphere of 5% COj in air. At approximately 4-5 day intervals, or when confluence was reached, the cells were passaged. This was accomplished by detaching the cells by trypsin!cation, washing and reseeding the cells at a lower density into fresh tissue culture flasks.

Statistical analysis of data The Student's t test was used to determine the significance of the difference between values for skin cells cultured in the presence of experimental compounds versus in control medium alone.

The difference between the proliferative abilities of the skin cells cultured in the presence of methyl 3-O-3'-(H',H*dimethylamino-n-propyl )-6 -deoxy-D-glucopyranoside of the present invention versus that observed with the control cultures, can be seen in the data presented in Table XXIX below.

A significant inhibitory effect was observed in a non-dosedependent manner for cultures receiving the methyl 3-O-3'-(H',N'dimethylamino-n-propyl)-6-deoxy-D-glucopyranoslde. It is important to note that the inhibition occurred to the same degree, irrespective of the concentration employed without evidence of cytotoxicity, and may suggest that this compound exerts these effects through novel mechanisms.

Ui TABLE XXII Effect of Methyl 3-0-3'-(Ν',H’-dieethylawino-n-propyl)-6- I ι -Ο N I control. Sljolffconco of the effect of experlnenul corx>o«I»: *, PO.05 The compounds of th· present invention as shown by formulae I, II, III, IV and V are useful for treating mammals with inflammatory and/or autoimmune disorders such as psoriasis, atopic dermatitis, rheumatoid arthritis, oetearthritis, scleroderma and systemic lupus erythematosus. The proliferative activities of these compounds broaden the potential scope of their application as therapeutic agents for the treatment of uncontrolled proliferation of particular cell types. Due to their valuable pharmacological properties, the compounds of the present invention or their physiologically acceptable salts are particularly suitable for use as active compounds in pharmaceutical compositions for the treatment of, for example, chronic inflammatory rheumatic disorders.

The compounds can either be administered alone in the form of microcapsules, in mixtures with one another or in combination with acceptable pharmaceutical carriers. The invention, thus, also relates to pharmaceutical compositions which comprise an effective amount of at least one compound of the present invention with or without a pharmaceutically acceptable carrier. If appropriate, compounds containing an amino functionality may be in the form of an acid-addition salt. Preferred acid addition salts are hydrochloric acid salts.

The present invention also encompasses a method of treating antms 1« or humans suffering from inflammatory and/or autoimmune disorders which comprises administering to an animal or person an effective amount of at least one of the compounds of the invention - 93 IE 913228 or an acid-addition «alt thereof, with or without a pharmaceutically acceptable carrier· The compositions according to the invention can be administered orally, topically, rectally, internally, or, if desired, parenterally. Oral administration is preferred.

Suitable solid or liquid galenical formulations, for example are granules, powders, coated tablets, microcapsules, suppositories, syrups, elixirs, suspensions, emulsions, drops or injectable solutions. Preparations having a protracted release of the active compound may also be used. These formulations can also contain additives such as excipients, disintegrants, binders, coating agents, swelling agents, glldants, or lubricants, flavors, sweeteners or solubilisers. Frequently used additives are, for example, magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactoalbumin, gelatin, starch, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols, polysorbates and solvents, such as sterile water and monohydrio or polyhydric alcohols, i.e. glycerol.

The pharmaceutical compositions are preferably produced and administered in dosage units, each unit containing as active component a certain dose of at least one compound of the present invention and/or at least one of its physiologically acceptable acid-addition salts. The dose can range from about 1 to 100 mg per kilogram of body weight per day, preferably 10 - 200 mg. In the case of in vitro testing, the effective amount to achieve a 50% inhibition of the cultured cells range from about 1 - 200 ug/ ml of culture medium, preferably 10 -.100 ug/ml·

Claims (35)

1. Th· claimed invention is: X. A fruotofuranoee compound of formula (I)» 1 2 R and R are H, methyl, ethyl Cg-C^g alkenyl or together form an isopropylidene ring; R Is H, Cg—C^g alkyl, C 5“^*io alkenyl, Cg—C^g alkynyl, 2— oetyne, benzyl, or Cg-C 1Q ester; and R 4 is H, Cj-C 10 alkyl, Cg-C lQ alkenyl, Cg-C 10 alkynyl, 2octyne, benzyl,or Cj ^10 ester.

2. A f ruotof uranoee of formula (I) as claimed in claim 1, wherein A is methyl; 1 2 R and R are each ethyl, cis-2-octane, or together form an isopropylidene ring; R is H, C 7 H i5/ cis-2-octene, trans-2-octene, 2-oetyne, or octanoyl; and R 4 ie H, cls-2-octene, trans-2-octene, 2-oetyne, or octanoyl.

3. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier. - 95 IE 913228

4. λ method of treating an animal or human suffering from an inflammatory and/or autoimmune disorder comprising administering thereto an effective amount of the compound according to claim 1.

5. λ compound according to claim 1 selected from methyl 1,3-0-isopropylidene-6-0-heptyl-e-D-fructofuranoeide; methyl 1,3-di-O-ethyl-6-O»heptyl-a-D-f rue to furano side; methyl 1,3-0-isopropylidene-4-0-heptyl-e-D-f ructofuranoside j methyl 1,3-0-i«opropylidene-4,6-di-O- (2-Octynyl) -e-Dfructofuranoside ; methyl 1,3-0-isopropylidena-4,6-di-O- (trans, 2-octenyl) -a-Dfructofuranoside ; methyl 1,3-0-isopropylidone-4,6-di -0- (cis, 2-octenyl ) -a-Dfructofuranoeide? methyl 1,6 -di-0- (cis, 2 -octenyl) -a-D-fructofuranos ide ; methyl 1,3-0-isopropylidene-4 -O-octanoyl-a-Dfructofuranoside; methyl l,3-0-iaopropylidene-6-0-octanoyl-e-Dfruetofuranoside ; methyl 1,3-0-ieopropylidene-4,6-di-0-octanoyl-a-Dfructofuranoeide; and 2,3-o-ieopropylidene-4-0-heptyl-£, D-f ructof uranose.

6. λ pharmaceutical composition comprising a compound according to claim 5 and a pharmaceutically acceptable carrier.

7. λ method of treating an animal or human suffering from an inflammatory and/or autoimmune disorder comprising administering thereto an effective amount of the compound according to claim 5.

8. A comoouj (ID . . A----Z wherein* X 1 is 0 and R 5 is Cl2~ C 20 *1^1' or C A» T - wherein η» 1,2,3 or 4 and T ie selected from cyano, pyrrolyl, pyrrolidiny1, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrasolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperasinyl, morpholinyl, O(CH2) 3 N(CH 3 ) 2 , (Cj - ^*iq alkoxy), CH 2 CH(CH 3 )CS 2 N(CH 3 ) 2 , CH 2 CH 2 li(C 3 -C 10 alkyl) 2 , or (C 3 -C 7 alkenyl), or X^ is NH and R 5 is C 2 -C 10 alkyl, or C n H 2n T ’rf 1 · 1 · 1 ** n * x ' 2 ' 3 or * «Μ T I· selected from hydroxy, cyano, pyrrolyl, pyrrolidinyl methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyraeolidinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperacinyl, morpholinyl, O(CB 2 ) 3 N(CB 3 ) 2 , (Cj- C 1Q alkoxy), or phenyl; and and R 7 are hydrogen or form an isopropylideae ring. 97 IE 913228 £ a

9. λ compound of claim 8 wherein R and R together form an leopropylidene ring·

10. A compound of claim 9 wherein said compound ie a glucof uranose.

11. A compound of claim 9 wherein said compound is an allof uranose. fi 7

12. A compound of claim 8 wherein R and R each hydrogen.

13. A compound of claim 12 wherein said compound la a glucof uranose.

14. A compound of claim 12 wherein said compound is an allof uranose.

15. A compound according to claim 9 selected from 1 1 2-o-lsopropylidene-3-O-n-dodecyl-a-D-glucof uranose? 1.2- 0-Isopropylidene-3-0-n-pentadecyl-e-D-glucof uranose; 1.2- 0-Ieopropylidene-3-0-n-octodecyl-«-D-glacofuranoee; 1.2- 0-lsopropylidene-3-0-3 ' - (phenylpropyl) -e-D-glucof uranose t 1,2»5,6-Di-0-ieopropylidene-3-03 * - (morpholinylpropyl ) -e-Dglucof uranose ; 1,2:5,6-Di-0-isopropylidene-3-0-3 * -n-propoxy-n-heptyl-a-Dglucofuranose; 1.2- 0-Isopropylidene-3-0-3' -n-propoxy-n-heptyl-a-Dglucpf uranose; l,2»5,6-Di-0-ieopropylidene-3-0-2'-(ethylpyrrolidyl)-a-Dglucof uranose; 1 1 2-0-Isopropylidene-3-0-2' - (ethylpyrrolidyl) -a -Dglucof uranose; 1.2- 0~Ieopropylidene-3-0-3 * - (propan-1'-ol) -β-D-glucof uranose; 11215,6-Di-0-isopropylidene-3-deoxy-3-amino- (3 * -hydroxy-n-propyl) a-D-glucofuranose; 1.2- 0-ieopropylidene-3-0-3' - (H',N' -dimethylamine-n-propyl) α-D-allofuranose; 1,215, 6-Di-0-isopropylidene-3-0-3' - (phonylpropy 1) -a-Dallofuranose; 1.2- 0-Isopropyl idene-3-deoxy-3-N-3' (Ν', N' -dime thy lamino-npropyl -a-D-allofuranose; 1.2- 0-Isopropylidene-3-deoxy-3-N-3 ' -(phenylpropyl) -a-Dallofuranose; 1.2- 0-Isopropylidene-3-deoxy-3-amino-heptyl-e-D-allofuranoee; 1,2«0-ieopropylidene-3-deoxy-3-amino-2'-(propan-l *-ol)-a-Dglucofuranose; 1.2- 0-isopropylidene-3-deoxy-3-aaino-a-heptyl-a-D-glttCOfuranose; and 1.2- 0-Isopropylidene-3-deoxy-3-amino-3' - (phenylpropyl) -a-Dglucofuranose.

16. A pharmaceutical composition comprising a compound according to claim 9 and a pharmaceutically acceptable carrier.

17. A method of treating an animal or human suffering from an inflammatory and/or autoimmune disorder comprising administering thereto an affective amount of the compound according to claim 9.

18. A pharmaceutical composition comprising a compound according to claim 15 and a pharmaceutically acceptable carrier. - 99 IE 913228

19. A method of treating an animal or human suffering from an inflammatory and/or autoimmune disorder comprising administering thereto an effective amount of the compound according to claim 15.

20. A compound of formula III, a (III) wherein X is 0, .8 R w is C 8 -C 20 alkyl, or C n H 2n T, wherein n- 1,2,3, or 4 and Y is selected from phenyl, cyano, pyrrolyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyx&tolidinyl, oxazolyl, oxazolidinyl, isooxasolyl, isoosazolidinyl, imidasolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CHj)jN{CHj) 2 , (C 5 -C 1Q alkoxy), NH or N(CH 3 , 2 ; or X 2 is NH and R® is H, or C n H 2n Y, wherein n- 1,2,3 or 4 and Y is selected from OH, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidasolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoosazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CB 2 )jN(CH 3 ) 2 or (Cj-Cjq alkoxy) or phenyl; 100 ox X 2 is 8 and R® is C 5 -C 10 alkyl, ox C n H 2n* n “ 1/2,3 ox 4 and Y is ••looted from OR, phenyl, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolldinyl, oxazolyl, oxazolidinyl, isooxazolyl, isooxasolidinyl, imidazolidinyl, piperidinyl, piperasinyl, morpholinyl, Q(CH 2 ) 3 K(CH 3 ) 2 or (Cj-Cjq alkoxy); and R 9 and R 1 ® are hydrogen or form an isopropylidene group.

21. A compound of claim 20 wherein said compound is a glucofuranose.

22. A compound of claim 20 wherein said compound is an allofuranose.

23. A compound according to claim 13 selected from 1.2- 0-Isopropylidene-6-0-heptyl-a-D-glucof uranose; 1 1 2-o-isopropylidene-6-0-nonyl-e-D-glucof uranose; 1 1 2 -o- Isopropylidene-6 -O-dodecyl-o-D-glucofuranose; 1.2- 0-lsopropylidene-6-0-pentadecyl-e-D-glucofuranoee; 1.2- o-Isopropylidene-6-0-3 * - (phenylpropyl) -a-Dglucofuranose; 1.2- 0-Isopropylidene-6-0-3'-(»',S'-dimethylamino-n-propyl)-a-Dglucofuranose; 1,2:3,5-Di-0-isopropylidene-6-0-methoxyoctyl-a-Dglucofuranose; 1.2- O-Isopropylldene-6-O-propionitrile-e-D-glucof uranose; -101 1,2: 3,5-Di-0-Ieopropylidsne-6-deoxy-6-amiao-[ 2' -aminoethyl-2-(Ν' methy lpyrrolidyl)-«-D-glucofuranose; 1,2»3 , 5-Di-0-lsopropylidene-6-deoxy-6-amino-3' - (phenylpropyl) -«D-glucofuranose; 1.213.5- Di-0-Isopropyl±dene-6-deoxy-6-N-(N' -propylpipecolinyl) -aD-glucofuranose; 1,2» 3,5-DI-0-I»opropylidene-6-deoxy-6-amino-ethoxyethanol«-O-glucofuranose; 1.2- 0-X*opropylidene-$-deoxy-6-amino-3' - (propan-1 * -ol) -e-Dglucofuranose; 1.213.5- Di-0-Isopropylidene-6-deoxy-6-thio-n-heptyl-«-Dglucofuranose; 1,2 -Ο-1 sopropy1 idene- 6 -deoxy- 6 - thio-n-heptyl -« -Dglucofuranose; 1,2 > 3,5-Di-0-Isopropylidene-6-deoxy-6-thio-2 · - (ethylpyrrolidyl) α-D-glucofuranose; 1.2- 0-isopropylidene-6-deoxy-6-thio-2' - (ethylpyrrolidyl) a-D-glucofuranose; 1,2»3,5-Di-0-ieopropylidene-6-deoxy-6-thio-3'-(Ν' ,N' -dimethylamino-ieobutyl) -«-D-glucof uranose; 1,2»3, 5-Di-0-Isopropylidene-6-deoxy-6-thio-3 ' - (propan-1' -ol) «-D-glucofuranose; and 1.2- 0-Isopropylidene-6-deoxy-6-thio-3 ' - (phenylpropyl) -a-Dglucofuranose.

24. λ pharmaceutical composition comprising a compound according to claim 20 and a pharmaceutically acceptable carrier. 102 IE 913228

25. λ method of treating an animal or human suffering from an inflammatory and/or autoimmune disorder comprising administering thereto an effective amount of the compound according to claim 20.

26. λ pharmaceutical composition comprising a compound according to claim 23 and a pharmaceutically acceptable carrier.

27. λ method of treating an animal or human suffering from an inflammatory and/or autoimmune disorder comprising administering thereto an effective amount of the compound according to claim 23.

28. A compound selected from (38) 1,2-0-isopropylidene-a-D-ribo-hexos-3-uloee-l, 4:3,6 difuranoee; and Methyl 3-0-3' - (Ν' ,N'-dimethylamino-n-propyl)-6-deoxy-Dglucopyranoeide·

29. A pharmaceutical composition comprising a compound according to claim 28 and a pharmaceutically acceptable carrier.

30. A method of treating an animal or human suffering from an inflammatory and/or autoimmune disorder comprising administering thereto an effective amount of the compound according to claim 28. - 103 IE 913228

31. A fructofuranose compound of formula (I) given and defined in claim 1, substantially as hereinbefore described and exemplified.

32. A pharmaceutical composition according to any one of claims 3, 16, 18, 24, 26 or 29, substantially as hereinbefore described.

33. A compound according to claim 8 or 20, substantially as hereinbefore described and exemplified.

34. A process for preparing a fructofuranose compound of formula (I) given and defined in claim 1, substantially as hereinbefore described and exemplified.

35. A fructofuranose compound of formula (I) given and defined in claim 1, whenever prepared by a process claimed in claim 34.