CA2091587A1 - Monosaccharides having anti-proliferation and anti-inflammatory activity, compositions and uses thereof - Google Patents
Monosaccharides having anti-proliferation and anti-inflammatory activity, compositions and uses thereofInfo
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- CA2091587A1 CA2091587A1 CA002091587A CA2091587A CA2091587A1 CA 2091587 A1 CA2091587 A1 CA 2091587A1 CA 002091587 A CA002091587 A CA 002091587A CA 2091587 A CA2091587 A CA 2091587A CA 2091587 A1 CA2091587 A1 CA 2091587A1
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- Prior art keywords
- isopropylidene
- glucofuranose
- deoxy
- compound
- methyl
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H9/00—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical
- C07H9/02—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical the hetero ring containing only oxygen as ring hetero atoms
- C07H9/04—Cyclic acetals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
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- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
- C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/14—Acyclic radicals, not substituted by cyclic structures attached to a sulfur, selenium or tellurium atom of a saccharide radical
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/01—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing oxygen
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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
W092/04359 PCT/~S91/06~X
f ` - 1 -209~87 Dsscr_Dtion Monoszccharides Havina An;_;-Drolifer tion and ~ti-Inflammatorv Activl~v ComDcsitions and Uses Thereof Thls application is a continuation-~n-part of U.S.
P~tent Application serial number 07~581,542 -iled September 12, 1990, which is a continuat:ion-in-part of U.S.
Patent Application serial number 07/294,838 filed January , 1989.
Technlcal Field The compounds of this inven.ion a-e ~e_ ~2tives c simple monosac~harides which exhibit znti-~rolifer2_io. anc an-i-ir.'lammator~ ac~ivitv anc are usefl-' -c- --e~- n~
mammals having in'lammatory disorde-s and/c~ au~oimm~ne cisorders. This invention also encompasses pha_~2ce-tlca composi~ions containing these compounds anc me~hods of treating inflammatory and/or aut~immune d ser~ers Backaround Art Cer~ai-. ~onosaccharides and the-r de~ivatives are i:no~-.
to have .herapeu_ic value i.. the treatmen~ c- i..rla~ma o-;
and auto mmune clso ders. Derivatization c ~onosaccha~ir~e-at speciflc hyd~oxyl g~oups may be a-comDl sr.e~ hy syn~;ner:~
techniques which are known in the ar~. ~or example, _- i_ common to block o~ protec' one or more o' the hydroxyl aro~2s leaving one or more hydroxyl groups free to unde-go derivatization, such as formation of an ether ~roup. Various blocking groups and methods are described, for example, in U~S. Patent Nos. 2,715,121 and 4,056,32', ~ne r'lsclosurss c which are incorporated herein by refersnce.
Various derivatives of five and six c~r~on monosaccharides, as well as synthetlc methods, are describe^, for example, in U.S. Patent Nos. Re. 30,354, Re. 30,37, Re. 32,26B, 4,056,322, 4,735,934, 4,î38,953, 4,5C~j19, and 5,010,054. The therapeutic activi y of the vario~s subs~ituted monosaccharides is also disclosed ln the ar,ove .
SUBSTITUTE SHEET
: , :
:
W092/04359 PCT/-S91/061~8 2091~87 - 2 - ~
documents. The disclosures of these pa~ents are also incorporated herein by rererence.
A known derivative of ~-D-giucose having benef cial therapeutic properties is amip.-ilose, l,2-O-iso?ropvlidene 3-0-3'-(N,N'-dimethylamino-n-propyl)-~-D-glucofuranose, and its hydrochloric acid salt, amprilose HCl (THERAFECTIN~).
These two compounds are known to have anti-inflammatory activity and demons~rated utllitv in managins the signs and symptoms of rheumatoid arthri~is. More generally, these compounds have Lmmunomodulatorv activity, and therefore have a therapeutic effect on other autoimmune disorders such as psoriasis, eczema or systemic lupus erythematosus.
Ur.~ortunately, though some de_iva_-ves of ~he monosaccharldes have shown benefi_ial theraoeu~ic act-v~ v, high doses of these derivatives, such as TH_R~FECmIN~, are often needed to produce effectlve results. Because therapy for inflammatory or autoimmune disorders fs often mid-te-m o~
long-term, there is a need to àevelop more potent, non-~ox~c compounds which can be oraliy admin s~ered, and thereby promote patient compliance. mhis invent on descrlbes addi~ional monosaccharide deriva_ives wi~h nc-eased Fo_ency.
I~ is therefore an objec~ of ~he ~resent nven;icn ~o provide new compounàs and com~oslt cns wh c: exh-^il zn,_- _ prollferation and anti-inflammatory 2c~_vity.
It is also an object of the invention to ~_o;ide compounds and compositions which are useful in ~he ~_eatmen~
of mammals having inflammatory and/or autoimmune disorders.
It is a further object to provide new compounds that exhibit significantly increased potency over available compounds, such as THERAFECTIN~, in order to provide ease of oral administration.
Disclosure of the Invention To achieve the foregoing objec~s and in accordance ~ ,h the purposes of the invention as embodied ar.d broadly described herein, there is provided monosaacch2-ides, :r.2-;-ng the following formulae:
.
SUBSrlTUTE SHE~
' ' ' W092/04359 2 ~ 9 1 ~ 8 7 PCT/~S91/064~
A compound of formula I
~'~~/ \l ~r~O A
o~' (I) wherein A is H, methyl or ethyl;
Rl and R2 are ~, methyl, C~-C10 a-~keny' o- togethe~
an isopropylidene ring;
~ C5 C10 alkyl~ Cs-C10 alkenyl, C5-Clo alkynyl, benzyl, or C5-C10 ester; and R is H, Cj-C10 alkyl, C5-C10 alke~yl, C5~Clr alkynyl, benzyl, or Cj-C o ester;
A compound of formula II r a ( L I j wherein: \ / V
xl is O RS~X ~~o/\
_s C12 C20 alkyl~ or CnH2n~:, wherein n= 1,2,3 or a~d Y is selected from cyano, pyrrolyl, pyrrol diny_, methylpyrrolidinyl, pipecoliny', im dazol~l, pyrazc yl, pyrazo iny', pyrazo~i~_ny:, oxzz^l~
oxazolidinyl, 1sooxazolvl, isoozazol_d r.y , imidazolid nyl, ?ipe~dinyl, p per2z~ nyl, morpholinyl, O(CH2)3N(CH3)2, (C;-C10 aikoxy), CH2CH(CH3)CH2N(CH3)2/ CH2C 2N(~5 -10 Y )2 (C3-C7 alkenyl), or X is NH
and R5 is C2-C10 aLkyl, or CnH2nY wherein n= 1,2,3 or 4 and Y is selected from hydroxy, cyano, pyrrolyl !
pyrrolidinyl merhylpyrrolidinyl, ~ipecolir.yl, imidazolyi, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isoxazo~yl, isoozazolidiryl, im dazo idinv', piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (Cs- Clo alkoxy), o- phenvl;
.
SUBSrlTUTE SHEEr ~' , W092/04359 2 0 9 1 5 ~ 7 PCT/~'S91/~
and R6 and R7 are hydrogen or form an isopr^?ylidene -ing;
A compound of formula III: XZ~
Co ~
wnerein x2 is O, ~ `J ~ o X (I~I) R8 is C8-c20 alkyl~ or CnH2nY' whe-ein n= l~2~3~ or 4 and Y is selected from phenyl, cyano, pyrrclyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazoiinyl, pyrazolidiny , oxazolyi, oxazolidinyl, isooxazolyl, isooxazolidinyl, im.idazol'dlnyl, piperidinyl, ?'?e-~ 7 ' n-~morphclinyl, O(C~2)3N(C~3)2~ (C5 ~l0 N(CH3J2;
or x2 is NH
and R8 is H, or CnH2nY, whereln n= 1,2,3 or . and " is selec~ed from OH, cyanc, ~y-~-!yl, pyrrolidinyl, methylpyr~~ .Y-, ?~?eCc ~y_~
imidazolyl, ?yrazolyl, ?vraz~_ ~y', pyrazolidinyi, oxazo'y', cxazGlid r.yl, Lsooxazolyl, isoozazolidiny', midazcl d r.y`, piperidinyl, piperazinyl, morpholinyl, 2 OtCH2)3N(CH3)2 or (C;-Cl0 alkoxy) or pheny';
and R is C5-Cl0 alkyl~ or CnH2nY wherein n= l~2~3 or 4 and selected from O~, phenyl, cyano, pyrroly!, pyrrolidinyl, methylpyr-olidinyl, pipeccliny', imidazolyl, pyrazolyl, py-azo'inyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, im~i~dazolidinyl, piperidinyl, piperazinyl, mor?holinyl, 1 ( H2)3N~CH3)2 or (C5-ClQ alkoxy);
and R9 and ~' are hydrogen o~ form an iso?ropylidene g-ou~;
and SUBSTITUTE SHEET
. .
' ' W092/04359 2 ~ 7 PCT/~S91~0~;8 A compound selected from (3S) l,2-O-isopropylidene-~-D-~ibo-hexos-3-ulcse-l,4:3,6-difuranose; and Methyl 3-0-3'-(N',N'-dimethylamino-n-propyl)-~-deoxy-D-g~ucopyranoside.
Other emboaiments in accordance with the ~resent ~nvention are pharmaceutical compositions con'aining an effective amount of one or more or the ~bove compcunds, and a method of treating an inflammatory disorder and/o~ an autoimmune disorder comprlsing administering an effective amount of a compound described above.
Best 'ode for Carrvinc OUt the ~n~enrion Monosaccharides are known to exist in an equilib~i~m between hemiaceral cycl c strucl~-es and ~n open chain suaa~.
The preferred cyclic struc-ures are fur2noses (;-membered ring st-uctures) and pyranoses (6-me~ered rmqg st-uct~~es~.
Other _ing structures may be formed but are not as thermodynamically stable ar.d generally rearrange to fo ~., ne pyranose or furanose structures. When a cyc'ic hemiacetal fs reacted with a~. alcohol, an ace~al i= ~rmed.
A glyccside can be def~ned as a cycllzed de~a~f~.e --^ -monosaccharide ha~iing two ether (O-~ g_oupsj SU~St' -~en-- o-.
the acetal carbon o- the sugzr. One of thsse ethe~
substituents is the carbocyiic ~ing. The secor.d ethe-substituent is formed by the reac~ior. with the alcohol ana is termed the aglycon. Because of the second ether substi-uer._ t the resultant glycoside is stable and does not exis~ in an equilibrium with its open chain structure. Glycosides having a 5-membered ring are known as furanosides, those wi~h n_ membered ring as pyranosides.
One em~odiment of the presen~ invention relates _o ; derivatives of the simpie monosacchar de ,-uc~ose, n particular to fructofuranosides. ~ructof~ranosides can be in.
an ~-D or ~-D configuration. The f_uctof1lranosides o. -he present inventicn, shown below ~n formula I, encompass bot.r or d configurations and are substituted a_ one or more o-the free hydroxyl groups, but, preferably, ha~e a~ leas~ on-SU~SrlTUTE SHEET
..... .... .... . .
` . . .
. .
' ` .
' W092/04359 2 0 9 1 ~ 8 7 PCT/-S9]/064~
- 6 - ._ free hydroxyl group. As discussed above, the ~ech~iaues __ form these deri~-atives of the present inventlon are gener2i_y known in carbohyàrate chemistry.
The fructofuranosides of the oresen_ invent A_~ c_e represented by formula (I): ~ O~, o~
wherein the aglycon, A, is methyl or ethyl, preferaDly methyl;
R1 and R2 are r:, methyl, e~hy , ~ -C ~ alker.yl or ~oge~ner -orm an isopropyiidene ring;
R3 is ~, C5-C10 alkyl, C5-C10 alXenvl, _ -~10 alXynyl, 2-octyne, benzyl, or C5-C10 es~e_; anc R4 is H~ C;-C10 alky ~ Cs-C10 alXenyi, _5 _ 0 alkynyl, 2-octyne, benzyl, or C5-CiO ester.
Preferred f ructo f uranosLdes of f ormula _ where ~ is methyl are shown in Tabl- I.
SUBSrtTU~E SHEET
W092/04359 2 0 9 ~ 5 8 7 ` PCT/~S91/06458 TA~3LE I
Com~ound R1 R2 R~ R4 ~Ia) Rl & R2 = isopropylidene H C7H15 (Ib) C2H5 C2H5 H C7H15 (Ic) Rl & R2 = isopropylidene C7Hl5 H
(Id) ~1 ~ R2 = isopropylidene 2-oc~yl 2-oc~~y (Ie~ R1 & R2 = lsopropylidene trans-2- trans-2-octenyl octen~l (If) Rl & R2 = isopropylidene cis-2- cis-2-. oc~envl octenvl (Ig) cis-2-octene ~ . cis-,-o-~eny (Ih) Rl & R2 = isopropylidene ~C~.. ls H
C
(Ii) Rl & R2 = isopropylidene H ~C7~15 C O
(Ij) Rl & R2 = sopropyiider.e ~-? 1;
The compounds included in Tabie ~ zre:
methyl 1,3-O-isopropylidene-6-O-he?_~ -^a-D-fructofuranoside (Ia);
methyl 1,3-di-O-ethyl-6-O-hep~yi.-^a-~ uc~ofu~anos de (Ib);
methyl 1,3-O-isopropylidene-4-O-heptyl-^a-D-fructofuranoside (Ic);
methyl 1,3-O-isopropylidene-4,6-di-O-(2-octynyl)-^a-D-fructofuranoside (Id);
methyl l~3-o-isopropylidene-4~6-di-o-(trans~2-octen ^a-D-fructofuranoside (Ie);
methyl 1,3-O-isopropylidene-~,6-di-O-(cis,2-oc~enylj-^a-D-fructofuranoside (If);
methyl 1,6-di-O-(cis,2-octenyl)-^a-3---uc~ofuranosiàe tIg);
SUBSrlTUTE SHEr ; , :
.
~ '.. ,, '' ' ;
~092/04359 2 ~ 91 5 ~ 7 B - PCTt~S91/064~X
methyl 1,3-O-isopropylidene-~-0-octanoyi-~-D-fructofuranoside (Ih);
methyl 1,3-0-isopropylidene-6-0-octanoyl-~fructoruranoside (Ii); and methyl 1,3-O-isopropylidene-4,6-di--0-ocranoyl-~-D-fructofuranoside (Ij).
The present invention also rela~es tc f-uctofuranoses.
These compounds have the same subs~ituenrs as defined in ~ormula I where A is hydrogen. A particularly preferred compound is: 2,3-0-isopropylidene-4-0-hep~yl-~-D-fructofuranose (Ik).
The ~ollowing Examples 1-4 illus.rate r:ne prepar2_ on c-representative compounds o- formula - acc^r~ ng 3 ehis invention. The activity of these _om?our.ds s luc -~e~
Example S.
Examole 1 Preparation of methyl 1,3-0-isopropyLidene-6-0-n-heptyl-~fructofuranoside, (Ia).
Ste~ 1: The preparation of ~ethyl ~-D-fr c-c uranoside~
This compo~nd was prepared by ~ne merhc^ ~ua;.~ k~
Co_tez-Garcia, r~., L. Hough, and ~ icha_dscn (Jou-nc -the Chemicai Society, Per~in ~, pp. 3176-3~:, l9~1j exce~-tha~ a silica gel column was used, ellred ~it.~ chloro~or.~-methanol (9:1 to 8:2) to glve: methyl ~-~-f ucrofuranoside 23.8~; [~]D25 +81.6 (c 1.02, ethanol). {Cortez-Garcia, 22.5%; [~]D +90 (c 2, water)} methyl ~-D-fructofuranoside 28.2~ ]D25 -47.8 (c 1.23, ethanol).
~Cortez-Garcia, 35~; [~]D25 -49 (c 2, methanol)~.
Ste~ 2: The preparation of Methyl 1,3-O-isopropylidene-~-D-fructofuranoside.
~ ethod one: The reaction procedure was descrioed by Cortez-Garcia, excep~ the longer reaction time was used ~three days)~ The best yield was 50~. [x3D25 ~40.' ~s 1.79, chloroform)~ {Cortez-Garcia 33~, [X]D 42.5 (methanol)}. The compound had physical prope_ties in agreement with literaturP values.
.
SUBSTITUTE SHE~
.. , '" ~', ' ,~ ' ' ' , ;':
, ,-W092/043S9 2 ~ 7 PCT/-S91/064~8 g Method two: A mixture of me~hyl ~-D-fruc_ofuranoside (O.8g, 4.lmmole), dimethoxypropane (5g, 49mmole, 6ml) and p-toluenesulfonic acid (O.lg, 0.5mmole) in 20ml of D~ was stirred at 25C for 24 hours. Sodium bicarbonate was added to neutralize the solution. The solvent was lltered and ~he flltrate was evaporated to dryness. Column chromatograohy ~petroleum ether-ethyl acetate (8:2 to t': 3) then chloro-c ~.-methanol (9:1)} gave me~hyl 1,3-O-isopr~pylidene-~-D-fructofuranoside (o~38gl 39%).
Ste~ 3: The preparation of Methyl 1,3-O-isopropylidene-~-C-pivaloyl-~-D-fructofuranoside.
Me~hyl 1,3-O-isopropylidene-~-D-fruc_c-uranosi~- :.3c, 5.55mmole) was dissolved in methylene chloride (lOmll ~nd py~idine (7ml). To this sol~ion, ?ivaloyi ch.~eride ~0.68~, 5.68mmole, 0.7ml) was added at 0C. The solu~ion was s~1_-ec and the temperature rose to 25C. After 18 ho~-s, ano~her O.lml of pivaloyl chloride (o~8mmole) was added 2nd s~ir~lna was continued for 24 hours Water was added o the mix~ure and solvent was evaporated. The residue was ext~acted wi .
chloroform, washed with water, ~rine, criec ove~ sociu.-sulfate, and concent~ated. Column chrom2togr3-;~v ~pe~r~
ether-ethyl acetate (9:1 to 7:3)} gave me~hy' 1,3-C-isopro~ylidene-6-O-pivaloyl-~-D-fructo_-~anoside ('.3~, 83~ ]D25 30.9 (c 2.34, methylene chlo-ide!. -.i-N.m.r. (CDC13): i 4.4-3.9 (m, 8H), 3.3 (s, 3H, GCH3;, 1.~5, 1.38 (2 g, each 3H, C(CH3)2), 1.23 (sl 9H, CO(C~3)3) ppm-Steo 4: The preparation of Methyl 1,3-O-isopropylidene-~-O-benzyl-6-O-pivaloyl-~-D-fructofuranoside.
Methyl 1,3-O-isopropylidene-6-O-pivaloyl-~-D-fructofuranoside (1.3g, 4.08mmole) was dissolved in D~.F
(15ml) and 5.2ml of benzylbromide (7.4g, 43.7mmole) was added. To this solution, a total 5.2g of silver (I) oxide (22.4mmole) was added in three portions during one hour with stlrring. Stirring was contlnued at 25C for _wo davs. ~e silver salt was filtered and washed with DU.F and methylene chloride, and solvent was removed under reduced pressure.
The residue was extracted with methylene chlc-ide 2nc tn^
.
SUBSTITUTE SHEET
, W092/04359 PC~/~S91/n6~5~
2~91~87 -extract was washed with water, brine, dried and concentrated.
Column chromatoaraphy {petroleum ether-ethyl ace~ate (50~
gave methyl 1,3-0-isopropylidene-4-0-benzyl-6-0-Divaloyl-~-D-fructofuranoside [~]325 +50.70 ~_ 1.3;, chloroform)~
1H-N.m.r. (CDC13): ~ 7.33 (m, 5H, C6H;), 4.60 (dd, 2H, OCH2Ph), 4.3-4.1 (m, 4H), 3.9 (d, lH, J 12.1 Hz, H-1), 3.8 (m,lH), 3.7 (d, lH, J 12.1 Hz, ~-1), 3.' (s~ 3H, OC.H3), l.A.-1.3 (2 s, each 3H, C(CH3)2), 1.2 (s, 9H, CO(CH3)3) ppm.
Ste~ 5: The preparation of Methyl 1,3-0-isopropylidene-6-0-t-butyldimethylsilyl-~-D-fructofuranoside.
To a solution of methyl 1,3-0-isopropylidene-~fruc~oIuranoside (0.25~, 1.07mmole) ir. D~'F (,ml) was added imidazole (0.16g, 2.3mmole) and t-butyldlmethylsilyl chlcride (0.2~ '.32mmole). The m~.Yture was s~ --ed 2- 2~C o~ Q8 hours. Water was added and solven was evapora~ed under reduced pressure. Column chromatography {petroleum ether-ethyl acetate (9:1 to 8:2)~ gave methyl 1,3-0-isopropylidene-6-0-t-butyldimethylsilyl-~-D-fruct~fu-anoside (0.26g, 97~), then using chloroform-methanol (100:1) gave startins materia!
methyl 1,3-0-isopropylidene-~-3-f-~__ofuranoside ~omg) Methyl ',3-0-isopropylidene-6-0-t-butyldimethy's_lyl-~fructo.uranoside: ~]~2_ 21.3 (c 1.0 , chlcroro ~..~.
~-N-m-- (CDC13): ~ 4 '-3., (m. 7.~)/ 3.3 (s~ 3~, CC~
(d, lH, OH), 1.45-1.35 (2 s, each 'H, C(C_3)2), C.~ (s~ CH, t-Bu~, 0.1 (s, 6H, Si(CH3)2) ppm-Ste~ 6: The preparation of Methyl 1,3-0-isopropylidene- A _O_ benzyl-6-0-t-butyldmethylsilyl-~-D-fructofuranoside.
To a solution of methyl 1,3-0-isopropylidene-6-0-t-butyldimethylsilyl-~-D-f-uctofuranoside (0.26g, 0.75mmole) and benzyl ~romide (0.15g, 0.9mmo7e, O.lml) Ln DMr '2ml) was added 80~ sodium hydride (25mg, 0.83mmole) slowl~. ~he mixture was stirred a~ 25C under n trogen ~o- 40 m nutes.
Methanol was added to destroy the excess sodium h~d_ide anc solvent was removed under _educed pressure at 25C. The residue was extracted with chlorororm, washe~ with ~ate~, brine, dried and concentrated. Column chroma~ogra~n~
SU8S~lTUrE SHEEI-. . -, ' '. ' W092/043~9 2 ~ 9 1 ~ 8 7 PCT/~!S91/06~8 It. li --{(petroleum ether-ethyl acetate (20:1 _o 8:2)} gave methvl 1,3-O-isopropyiidene-4-O-benzyl-~-G-t-butyldimethylsiiyl-~-D-fructofuranoside (0.28g, 85.6~) and methyl 1,3-O-isopropylidene-'-O-benzyl-~-~-fruc-o-u-anoside (10mg, Methyl 1,3-O-isopropylidene-~-O-benzyl-6~O-t-butyldimethvlsilyl-~-D-fruc'ofuranoside: !~]r25 +48.7 (c 7.5~, chlorofo~m). H-N.m.r. (CDC13): ~ .8 (m~ 5U, C6U,), 4.5 (s~ 2H, OCU~Ph), 4.1-3.7 (m,7H), 3.3 (sr 3H, OCH3), 1.~- -1.3 (2 s, each 3H, C(CH3)2), 0.9 (sl 9H, t-Bu), 0.C6 (s, 6~, Si(cH3)2) pp~n.
Ste~ 7: The ~re~aration of Methyl 1,3-O-isoprc~Ylidene-~-O-benzvl-Q-~-fruc~oruranoside.
Method one: .~ethyl 1,3-O-isopropylidene-~-G-benzv~ G-pivz7Oyl-~-~-f-~cto_u-anoside was dissolved ~ e~hAn~
(20ml) anà sodium methyiate (0.2g) was added and s~i-red a~
25C for 20 hours. The reaction solution was neutrallzed with Ambe-lite IR-120 ~H ) ion sxchange resir., f_ltered and concentrated. Column chromatography ~perr~lelm e~her-etr.~
acetate (9:1 ~o 8:2)} gave methyl 1,3-O-isopreDv7idene-'-O-benzyl-~-D-fruc~ rAnoside (l.l5gr 86.8~ yield).
.~ethod two: A solution o~ me~hyl ~ ^- so~ y'_~en--4-O-benzyl-~-O-t-~utyldimethvlsilyl-~-3---_~ -2-.osi~e (0.2g, 0.45mmole) and 1~. t-8u N~-T~7- (0.5.m_, ~..~.mole~
(5ml) was stirred z_ 25C for 1~ hours. Solven~ was removec and the residue was chromatographed {petroleum e~her-ethyl ace~ate (8:2)} to give methyl 1,3-O-isoF_opylidene-~-O-benzyl-~-D-fructofuranoside (0.14g, 5~).
Method three: To a solution of methyl 1,3-O-isopropylidene-6-O-pivaloyl-~-D-fructofuranoside (0.2g, 0.63mmole) and benzyl bromide (0.14g, 0.81mmole, 0~lml) in DMF (2ml) was added 80~ sodium hydride !27m . 0.9mmole) slowly. The mixt-lre was st-rred at 25C under n t-ogen for 15 minutes. ~ethanol was added ~o dest~ov the excess sodium hydride and solver.~ was evaporated under -eauced pressure 2_ 25C. The residue was dissolved in methano_ (5ml) and sodium methylate (0.lg) was added. The mixture was s~-~red at 2;
for 10 hours and neutralized with Amberli~e .R-i20 (~:
SUBSTITUTE SHEET
W092/04359 ~ ~ 9 1 5 8 7 12 - PCT/~S91/06~5 resin, filtered and concentrared. Columr. ch_omatogr~phy {petroleum ether-ethyl acetate (9:1 to 8:2)} gave merhyl ',~-O-isopropylidene-4-0-benzyl-~-3-fructofuranoslde (0.13g, 64%). [~]D25 -83.~ (c 0.98, methylene chlo-ide). 1-~_ N.m.r. ~CDC13): ~ 7.3 (m, SH, C6.i5), 4-6 (dd, 2H, OCH2ph!~
4.1 ~m, 2H), 3.9-3.8 ~m, 4H), 3.6 (dd, 1~), 3.3 (s, 3H, OCH3), 2.1 (s, lH, OH), 1.4-1.3 (2s, each 3-., C(CH3)~) ppm.
Ste~ 8: The preparation of ~ethyl 1,3-0--sopropylidene- -o-benzyl-6-0-n-heptyl-~-D-fructoturznoside.
~ o a solution of methyl 1,3-0-isopropylidene-4-0-benzyl-~-D-fructofuranoside (1.63g, 5.03mmole) and bromohepcane (4.1g, 23~mole, 3.6ml) in D?~.F (35~1! was added 80~ sodium hydride (0.6g, ,Ommole) slowly. The mix_ure was Sr m ~red 2-25C for 1.5 hours. Methanol was added ~- _es__o, zhe exces~
sodium hydr-de and solvent was evaporzred. ~he res due W2S
extracted wi~h chloroform, washed with ware-, ~ e, d-ied over anhydrous sodium sulfate and concentra~ed. Collmn chromatogra~hy {petroieum ether-ethyl ace~a~e (5:1)} gave methyl 1,3-0-isopropylidene-~-0-benzyl-6-v-n-neptyl-~fructofuranoside (2~, 94% ! [~]D25 -51.?' ~- 1.2?, methylene chlor de). ~~-N.m~. (C3~
C6H5), 4-6 (c, 2~, OCH2Ph), ~.1 (ml -~ d, lH), 3.~ ~, lH), 3.6-3.~ (m, 4H), 3.3 (s, 3:-, C~U3), :.~ 3~., 2., cH2cH2c5u~ -1-3 (2 s, each 3Y, _(C~ , !.2 (b-, 8.
0.9 (t, 3~, C~3) ppm.
Ste~_9: ~he preparation of Methyl !,3-G-isopropylidene-6~G-n-heptyl-~-D-fructofuranoside.
Method one: A mixture of methyl 1,3-0-isopropylidene- -O-benzyl-6-G-n-heptyl-~-D-fructofuranoslde (0.13g, 0.3mmole), 10% palladium on activated carbon (25mg) in methanol (Sml) was shaken under hydrogen (2; lbs. per sa.
inch) for 8 hours. Catalyst was f ltereà and solven~ was removed. Column chromatography {petrole--~ ether-erhyl acetate (9:1)} gave me~hyl 1,3-0-lsopropy_ ~^ne-6-0-~.-hep~v -~-D-fructofu~anoside (Ia) (70mg, 70%).
Method ~wo: A mixture of methy~ 1,3-0-iso?ro?y idene--.-O-benzyl-6-0-n-hep~yl-~-D-fruc~ofuranoside (0.22g, SUBSrlTlJTE~ SHEET
., ,. . . .
~V092/04359 2 ~ 7 PCT/~S91/~58 l~ - 13 -O.52mmole), 10% palladium on ac~ivated carbon (O.2g, ~OOmgf mmole per Bn) and ammonium formate (0.3g) in methanol ~10ml) was refluxed for 2 hours. Another 0.25g of ammonium forma~e was added and refluxed for 3 hours. A final Dart of ~.2~ c-ammonium formate was added and refluxing was continued for another 2.5 hours. After cooling, catalvst was filtered and washed with methanol, filtrate was evaporated to d-vness.
Column chromatocraphy ~petroleum ether-ethyl aceta~e ( a gave methyl 1,3-O-isopropylidene-6-O-n-heptyl-~-D
f_uctofuranoside ~Ia) (0.14g, 81%). [~]D25 +27.4 (c 1.01, methylene chloride). 1H-N.m.r. (CDC13): o 4.2-3.S (~., 5H), 3.4-3., (m, 4H), 3.3 (s, 3H, OCH3), ~-~ (d, `H), 1-~1.~-1.4 (2 s, each 3H, C(CH3)2), 1,3 (~~ S, 8H), o,c !-~C~3) ppm- 3C-NMR (CDCl,): ~i 102.0 (Ç-2), 98.6 (~(C.- ,2), 85.9, 79.7, 78., (C-3, C-~, C-5), 71.7, :._ (C-6, -~-2-C6H13), 61 .7 ~C-l), 48., tor~3j~ 31.&, 29.5, 29.1, 2l.6, 2s.a, 22.6~ 19. ' 14.1 ppm. m.s.: m/z 333 (~_uj)l 301 (~-Y.-CH~O-), 260 (M-CH2OC(CH3)2).
Exam~le 2 ~reparation of methvl 1,3-O-isopropylidene- ,6-di-G- ~-ocrynyl)-~-D-fru~~o~u.anoside, (Id).
~ ethvl 1,3-O-isopropylidene-~-D-fru-rofu-anoside ~as prepared by the procedure cf Cortez-Garcla et al. zs described in Example 1. mO a solution of methyl 1,3-O-isopropylidene-~-D-fructofuranoside (0.3 g, 1.28 mmole) and 1-bromo,2-octyne (0.73g, 3.85 mmole) in DMF (4 mL) was added 80% NaH (0.12g, 4.0 mmole) slowly and the mixture was s.irre-at 25 under nitrogen for 40 min. Methanol was addeà to destroy the excess sodium hydride and solvent was removed.
The residue was extracted with methylene chloride and this solution washed with water, brine and sodi~m bicarbona~e.
This solution was then dried with anhydrous sodium sulfa~e, filtered and evaporated to a resiàue. This residue was dissoived in eluant and chromatagrapned on a silicaael ^~
using pelroleum ether-acetone (24:1) which gave methvl :,3-i^-isopropylidene-4r6-di-o-(2-octyny~ -D-fruc~ofuranoside SUBi~lTUTE SHEET
W092/04359 2 ~ 9 1 ~ 8 7 PCT/~S91/061~X
(Id) (0.41 g, 71~) as a colorless oil. The com~ound was treated wlth sodium bicarbonate and kept in freeze~. ~he compound was identified by its spe~ fic ro~atlon, N~.R znc mass spectral analysis.
Exam~le 3 Preparation of methyl 1,3-O-isopropylidene-~,6-di-O-!tr3ns,2-octenyl)-~-D-fructofuranoside, (Ie).
The procedure of Example 2 was followed excep~ that 1-bromo-trans-2-octe~e was substituted for i-bromo-2-oczvne.
Methyl 1,3-O-isopropylidene-4,6-di-O-(trans,2-octenyl)-~-D-fruc~ofuranoside was obtained n 70.0% yield 2S 2 c_lc-less oil and identified by ts specifi~ rota~ior., N~R 2~ ZSS
spec-ral analvsis.
Exam~le Preparation of 2,3-O-isopropylidene-~-O-hep fruc~ofuranose (Ik);
Ste~ 1: Preparation of ,6-Di-O-r_ityl-5-f-u-lose.
To a solution of D-fruc-ose (10 g, 55.; ~ole) 1.. ?v~id ne (40 mL) and chloroform (25 mL) was added ~ c:-lc~
g, 126.3 mmole) and the mixture was s-irred at 25~ or ~
days, another 5 g Gf .- yl ch!oride was addeà and s~ nc was continued for 2 days. Solvent was removed under -educe~
pressure and the residue was extracted with chloroform, washed with aqueous saturated cupric sulfa~e soiu~ior~, brine, dried over anhydrous sodium sulfate and concentrated. Column chromatography [chlorofm-methanol (100:1)] gave 1,6-Di-O~
trityl-D-fructose (11 g, 30%). mhis compound was identified by its specific rotation and by NMR and Mass spec~rometry.
Ste~ 2: Preparation of 1,6-Di-O-trityl-2,3-O-isopropylidene-~-D-fructosfuranose.
A mixture of 1,6-Di-O-trityl-D-fructose (2 g, 3 mmole),.
dimethoxypropane (5 mL) and p-toluenesulfon ~ acid t.- S) ~~
D~F (20 mL) was stirred at 25 for 3 h. sodium bic2rbona~e was added to neutrali~e the solution. Afte- remov2! c ~;~e salt and solvent, the residue was extr2cted wit.- c;.lorc~orm, 5U13SrlTlJTE SHEET
, ' , W092/043s9 2~91 ~87 PCT/~S9]/06~8 ~- - 15 -washed with water and brine, dried anà concentrated. Columr.
chromotagreaphy [petroleum ether-ethyl acetate] gave l,6-Dl O-trityl-2,3-O-isopropylidene-3-D-fruc'osfuranose (0~9 5, 42%). This compound was identified by its specific rota~ on and by N.MR and Mass spectrometry.
S~e~ 3: Preparation of 2,3-O-Isopropv' Aene-~-O-heptyl-l,5-di-O-trityl-~3-D-f-uc~ofu-ano~e.
l,6-Di-O-~rityl-2,3-O-isopropylidene~
fructosfuranose (0.5 g, 0.71 mmole) was dissolved in DMF (lO
mL) and sodium hydride (60~l 0.12 g) was added. The mix_ure was sti~red at 25C for 20 min. ~hen l-bromoheptane (0.62 g, 3.5 mmole) was added ana s~irrec fc~ ethanol was added to destroy the excess sodium hvc.-ide and solver.t was evaporated. mhe resld~e was e.~:t-~--e~ ^hlorc~o~
washed with water, brine, dried over anhydrous sod u.., s~lfa~s and concentrated. Column chromatog-ap;~ petroleum ethe~-ethyl aceta~e (lOO:l)] gave 2,3-0-Isopropylidene-~-0-hepty -1,6-di-0-trityl-~-D-fructofuranose, (0.~ g, 82.5~). T~is compound was identified by its spec_f - -~tation and by N~R
and Mass spectrometry.
Ste~ 4: ~reparation of 2,3-O-_so?ropyil ene--.-G-he~-y`-;-D-fructofuranose.
Ammonia (about lO0 mL) was passed th~ough _ po_ass_u~.
hydroxide tower and cooied i-.to liquid w~h drv ice to a solution of 2,~-O-Isopropylidene-4-C-ne?tyl-',6-di-O-t~itvl-~-D-fructofuranose (l.35 g, l.68 mmoi.e) -. dry THF (30 mL) cooled with dry ice. The solution was s~ ~-ed and pieces of lithium was added until the blue color persisted. The solution was stirred until the reaction was finished (checked with TLC). ~thanol was added to discharge the reaction and ammonia was allowed to evaporate overnigh~. The the residue, chloroform was added and salt was removed. Column chromatography [petroleum ether-ace~one (8:2)] aave ~,3-G-Isopropylidene-,-O-heptvl-~-D-fru-r-turanose ( Ik ) ( O .
84.1%). This compound was identified bv _-_ spec f -rotation and by NMR and Mass spect~omet~y.
SU8STITUTE~ SHEEl W092/04359 2091587 16 - PCT/~S91/0645~
Exam~le 5 Phzrmacological Activity of compounds o. Formula I.
The pharmacologic assays performed te dete~mine the immunomodulatory effects of the experimen-21 compeunds in vitro include the Mixed Lymphocyte Reac~ion (MLR) and the ConA blastogenesis assav. These assays were used to determine the inhibitory effects of the com?ounds of the invention on T lymphocyle activation and p.olifer2tion.
Since inflammation at the cellular level s characterized ~y T lymphocyte recruitment, activation and proliferation, these assavs are appropriate to use as screens for no~rel compoun.~C
having therapeutic poten~ial in ~he treatmen- o- cisorders _ n which inflamm2tory mechanisms are ir.~olve~.
The .M~R is a classicai assay used t~ measure m cell func~ion b~ studying the proliferative response of m ce'l 1 S
which are activated Ln ~.ritro by gene~ically d sparate stimulator cells. This is accomplished by co-culturing spleen cells from two different strains of mice. Splenic ~
cell proliferation occu-s as a resul~ ellula- activat on signals generated by the onaolng ce ul 2~ eraC,_OI;C.
S~ecific M.ethod: ~R ASSAY
Balb/c mice were euthanised by ce-~; cal disloca~~cn a-.-their spleens removed. Single cel` susper.s ~ns o- ~he spleens were prepared in culture med um (hepes buffered RP~--1640 supplemented with 10% fetal cal serum, 2m~. glutamLne, 500 units penicillin/streptomycin, and 4 x 10 5 ~ 2-msrcaptoethanol) using a Teflon pestle. The cells were centrifuged at 1500 rpm and the pellets resuspended in ACm (0.l5 M tris, 0.14 M ammonium chloride, pH 7.2) Ln order to lyse the red blood cells. After a 5 minute incubation in a 37 C waterba~h, the cells were resuspendeà in culture meaiu~
and counted. C57Bl/5 spleen cells which were used as stimulator cells, were aiso prepared bv this method. mhe stimulator cells were treated with 100 ug/ml cf mi~-ycin for 1 hour at 37 C (to inhibit stimulatorv cel' proliferatior.) and were then washed 5 times in cll-lre .
SUBSTlTUTE SHEET
~ .
, :
.
, ' . ,.~ .
Wo92/04359 2~ 8 7 PCT/~S91/0645R
~ - 17 -medium. The proliferatlve responses were measured bv cult~-ing 2.5 x 105 responder cells with 5 x 105 st m~i2~c-y cells in 96 well microtiter plates in the presence or absence of various doses of test compounds or vehicle ~DMSO).
Syngeneic control cultures using mitomvcin c ;realed Balb~c spleen cells as stimulato- cells were also _un. All ^u'~ures were run in tri?licate.
Solutions oî compounds of the present invention i n, DM'S~^
were ~repared at a stock concentration of 120 mM. Diiutions were made in culture medium to the following concensr2~ ons:
3, lC, 30, 100, and 300 uM. The vehlcle (DMSO) was use~ as a negatlve control.
A'_er incubation of fo_ 5 days at ', C with ~ , the amour.~ o, cell prol ^e~a~ion was meas_-ea bv adài~ ` c-MTT (3-[4,5-dimethy'thiazol-2-yl]-2,5diphenyl-tet~azG um bromide) (10 mg/ml i.~ phosphate buffered sailne) to e~c~
well. Plates were Lncubated for 4 hours at 3? C, af~e~ w~ h 180 ui of 10% sodium dodecyi sulphate in p~.osoha~e bu~-sre~
saline was added. After an overnigh- incub2tion ! the ootic2`
densitv (OD) of eac-. well was read on a ~olecul2- ~e~i-es mic-^?late reader 2- 570 - 650 rm. ~he resul-s were dete-~ined bv calculating the c f f erence be~ween ~he r.~an.s _-the allogeneic cul ures and the means o~ the svngen~c cultures for each test ar~icle concentration. Dif-erences --the test article groups were compared to the con'ro' group and the percent change from the control was determined. Test articles which were found to inhibit proliferation are ~resented as -()% of control with inhibitions greater ~han -20~ of control being considered active.
Mouse Seleen Cell ConA Blastosenesis Assa~
Tt is well known that several plan~ lect ns, when cultured in vitro with lymphocytes, s.imula~e cellula-activation and proliferation. Concanavolin, (ConA) selectively stimulates the blastoaenic res?onse of ~
lymphocytes. Therefore, the ConA blastogenesis assay is SUBSrlTUTE SHET
..
. . . .
W092/04359 2 ~ 9 ~ ~ ~ 7 18 - PCT/~S91/06~5~
useful for screening the i~munomodulato_y 2nd an~ -proliferative activities of experimentzl compounds.
S~ecific Method: ConA Assa~
Six to 8 week old male C57Bl/6 mice were purchased 'rom Harlan Sprague Dawley (Indianapoiis, IN!.
Spleens were removed and were homo~enized to obtain a single cell suspension. Erythrocytes were lysed by hypotonic shock. UDon determlnation of the viability and concentra ~on of the lymphoid cells, they were adjusted to . x lo6 cells/ml in culture medium (~MI-1640) suppiemente~ wi_h 0% fetal bovine serum; 100 ug/ml strep~omycin; 100 ~/ml penici'li.., 0.2 u hepes buffe~; 5 x 10 5 ~ ^-me--zptoethancl and ~
glu~amine). Spleen cells were seeded ir._o mi~-otite_ pl2~e wells at 2 x 105 cells/O.OiO ml/wel'. To these cul~res were added various doses of expe~fmen~al compounds and ConA 2- -final concen~ration of 4 o- 1 ug/ml. Contro` cultu_es consisted of cells, ConA and cul~ure medium contain ng ~he vehicle, DMSO only. In some assays the pos tive controis cyclosporin A (CSP) and ~ZT were also run. ~or -he -es- ng o' ~he methyl 1,3-O-isopropylidene-5-O-n-hep~yl~
- f-uc;ofu-anoside compounc. indome~ha_:r. znd NDGA w--- us_- ~_ ; cont~ols. All cul~ures were run in _-ip ic--e.
Solutions of compounds o_ the inven- on in DU~S~ ~er-prepared and dilutior.s were made in cu ~u~e medium~ .~ssa~
; concent_ations were either: 1, 2.5, 10, 25, 100, 300 and 750 ug/ml or 0.001, 0.01, 0.1, 1, 2.;, 10, 25 and 100 ug/ml.
Cultllres were incubated for 3 days at 37C in a humidified atmosphere o- 5~ of CO2 in ai-. 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 3H-thvmidine incor?orated by the cuLtures, as a measure o. cell proliferztion, was me2sured r.
a liouid sc~ntillation counter. The amount c r radloa~~ive incor?oration is proportional _o the zmour.~ o- cellu a-proliferation ir. individual wells. The studer,ts ~ tes_ was ` SU8STITUTE~ SHEEr .. .. . . .
.
~092/04359 2 0 915 8 7 PCT/~591/064~X
used to determine the significance of the difference be~ween experimental and control alues.
IL-l Assav Interleukir. 1 (IL-1) is a potent immunomodulatory cytokine that has a broad range of pro-Lnfl2mmatory activities. IL-1 is known to be produced bv activated accessory cells such as macrophages. ~ccesso_y cell production of T, L-1 results in the ac_ivztior. and proliferation of T and B lymphocytes. Therefo~e, by inhibiting macrophage activation and production of _~ he activation and proliferation of the cellular effectors of inflammation car. be modulated. Compounds o_ the invent cn were screened for inhibitory activity i~ a ciasslca` -;-1 - assay.
SPecific Method~ l Assay Six to 8 week old male C57Bl/6 mice were pu~chase~ rom Harlan Sprague Dawley. Peritoneal macroohaaes were ellcited by the adminLstration to mice of a single -~traoeritonea!
injection of 0.2 ml of complete Freund's adjuvant. Af ter ~ a hours, elicited mac-ophages were removed frcm the mice bv lavage with the use of ~ank's baiance~ sal~ soiu_ier.~
~ acrophages were washed and seeded lr.to m c-o- ~e- ~e'`s at a density of 2 x 105 cells/well n cu'ture meàium (zs described in the ConA blastogenesis assav)~ To the mac-ophage cul~ures were added variou, doses of ~he compounds of the invention and 10 ug/ml of the macrophage act vator Lipopolysaccharide (to stimulate IL-1 production). Control cultures consisted of macrophages, lipopolysaccAaride and culture medium containing various doses Oc DMSO only.
Cultures were incubated for 24 hours at 37C in a humidified atmosphere of 5% CO2 in air. Compounds of the invention were prepared in DMSO and diluted to the following concentrations in culture med ums 0.001, 0.01, 1, 2.5, '0, ,~ and 100 ug/ml.
The amount of I~-i produced in the nàiviàual wellc ~as determined in a bioassay for I~-1 This invclved the remova of thvmuses f-om mice less than 8 weeks cf age. Th~ocvtes were isolated bV passing each thymus t~.rough a s~ain'ess SUBSTITUTE SHEET
W092/04359 PCT/~S91/06 2 ~9l 5 ~ 7 20 - f steel mesh screen. Thymocytes were placed in culture at 1.5 x lO6 cells~well in RP~I-1640 medium containing 5~ feta bovine serum and l ug/ml ~HA in microtiter plates.
Triplicate cultures were cultured with dilutions o_ macrophage supernatants in the same medium at 37~C in a humldified atmosphere of 5'~ C02 in air.
After ~.8 hours, wells were pulsed ~ith C.5 uCi of 3H-thymidine. After 18 hours cells were harvested and radioactive incorporation (as a measure of cell proliferation) was quantitated in z liquid scintillation counter. ~he students t test was used to de~ermine the significance of the difference between experimental and control values.
~esults c C_~e=rmnc .~SS2~'S ? ~_c~.e~ cn ~e~_esent2~f~.~e c-Formula I.
A summary of the inhibitory e,fects of compounds (Ib! -(I~) on T lvmphocyte proliferation as measured in the ConA
blastogenesis assay is glven in Table II. As demonstrated by these data, the fructofuranosldes inhibit dose dependent inhibition of ConA stimulated ~ lvmphocvte ~rol~feration ~71'-strong inhibitlon (creat ~han -sa~ OI cor._-c~) Dy these compounds cf the present inventior. z- lO0 ug/m1 with the exception of (Ii) wnich W2S found --. be a less ~oter.t inhibitor of the mLtogen induce~ T ym?hocyte ~roiirerative 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 III, compound (Ik) is inhibitory in a dose dependent manner wit:-significant inhibition of ~ lymphocyte proliferation obser~ed at concen~rations ranging 'rom ~ ua/ml ~o lO0 ug/ml. Tabl=
IV demonstrates that compound (Ti) exerts signiricant, non dose dependent modulation of ~ lvmphocyte ?roliferaticn ~it.-significant inhibition observed 2t 2' 1 cose ievel_ on t~e :
ug/ml ConA cultures. The results sno~n in Table V indicate that methyl l,3-O-isopropvlidene-6-O-n-he~tvl-r~-3-fructofuranoside produced a dose-àependen~, sisnif can~, SUBSrlT~lTE SHEEl , ., , W092/04359 2 0 9 1 ~ 8 7 PCT/~S91/0~
( - 21 -inhibitory effect upon the abillty of normal, splenic21lv-derived, mouse T-cells to prcliferate ln response to mitogenic stimulation. There were less T-cells ~n _ne treated c~ltures at the end cf the assav in compa_ son ~ ~r.e untreated control cultures.
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2 ~ g 7 W092/0~35~ PCT/~S91/06~5g : - 29 -Compounds of formula I were found to also be inhib~ ~0~7 to T lymphocyte activation, and proliferation when assessed for activity in mixed lvmpnocyte reactions. As is seen in Table VI, when compared to controis, fructoruranosides of formula I exhibit non dose dependent reductions in k~I~
generated proliCeration with st-ong inhibition de~ined 25 greater than -50~ c- control cul_~res and moderale inhibi~lor defined as -20% of controls.
Further immunomodulatory efiects of compounds ~Ia), (Id), and (Ie) were demonstrated by testing ;or inhibi~or~.~
effects of these compounds on macrophage I~-1 production. In results given ir. Table V-I, the compounds were obse-ved ~o inhibit macrophage IL-1 production in a dose dependen manner with signi~icznt invento-y effec ~ obse-ved ir. ~1' com~o~r.As ar concentrations ranging between 2.; to 100 ug/ml.
In summary, compounds of formula I have been founc to have immunomodulatory and anti-Dro!iferative effects wh'ch predict that these compounds or the present invention have utility, from a therapeutic standpoint, in the treatmer.r c~ 2 variety of infl2mma~3rv and/c~ autoimmune diseases SVBSrl'rVTF SHFET
WO 92/04359 2 ~ 7 PCl/~S91/1)6~X
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SUBSrlTUTE S3-lEET
W0~2/04~59 2 0 9 1 5 8 7 PCT/~'S91/0~5X
A second embodiment of this invention are derivatives of glucose and allose. Glucose and allose are six carbon monosaccharides which differ rrom each ^ther by the configuration of the 3-carbon in the sugar. More particularly, these compounds include mono-substituted ethe-derivatives of ~-D or ~-D glucofuranoses and ~-D or ~-D
allofuranoses as well as analogs thereof. The generic formulae II and III, below, encompasses bolh ~ or ~
configurations. The compounds are substi~uted at the 3- or 6-position of the monosaccharide. The analogs of these compounds are those where the oxygen at .he 3- or 6-positon has been replaced by an amino grou~ or bv sulfur.
As will be apparent from the follo~in~ discussion, ~he compounds of this embodiment can be broadly classifie ir.to two groups: fully blocked mono-substituted compounds, .e., those having two isopropylidene protect ng groups, and partially bloc~ed mono-substi~uted compounds, .e., those having only one isopropylidene protecting group. Applicanrs have found that fully blocked and partially ~locked de-_va ives of ciucofuranose and allofuranose are e--ec_ ve in the treatment of autoimmune and~or ir.flammatory diso-cers.
The 3-substituted deriva~ives of glucofuranose anà
allofuranose a_e shown by the following general formula ~_:
~VO ~l~o wherein: R~X ~ X (II) xl is O
and R5 is C12-C~0 alkyl, or CrH2nY, wherein n= 1,2,3 or 4 and Y is selected from cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecol~ny', imi~azolyi, pyrazolyl, pyrazolinyi, pyraz-lid r.y`, oxazolyl, oxazolidiryl, isooxazolyl, SUBSTITI~TE SHEFr W092/04359 -2 a ~ PCT/~S91/0645~
! - 3~ - ~
isooxazolldinyl, imidazoli~lnyl, piperidinyl, piperazinyl, morpholinyl, O(C~2)3N(C~3)~, (C5-ClO alkoxy), C~2CH(CH3)CH2N(CH3)2, C~2CU N(CC-Clo 21kyl)2, or (C3-C_ alkenyl;
or ~~ is NH
and R5 is C2-ClO alkyl, or CnH2nY wherein n= ',2,3 or 4 and '~ is selected f_om hydroxy, cyano, pyrrolyl, pyrrolidinyl methylpyrrolidinyl, pipecoI~nvl, imidazolyl, pvrazoly1, pyrzzolinyl, pyrazolidinyl, oxazolyl, oxazolLdinyi, soxazolyl, soczzz^l ~iny', i.~ 2zo'i~in~ , piperidinyl, piperazinyl, mo_phollnyl, G(CH2)3N(CH3)2, (C;- Cl~ alkoxy " and ?henyl;
and R6 and R7 are hydrogen or form an isopropy'~dene rina.
Selecti~e removal o4 the pro~ec-~ng qroup ar the ',o-position by the following general procedurs yields par~ i2~
blocked compounds of formula ;I where ?.6 and R' are hvdrcgen.
Preferred co~?ounds of _^nmula L are:
l,2-0-~sopropylidene-3-0-n-dodecyl-c-~-g~o_-~_2nose ~ Ia~;
l,2-Q-Isopropylidene-3-0-n-pen~adecyl-,~-~-gluco_u-anose (IIb);
l,2-0-Isopropylidene-3-0-n-octodecyl-~-3-glu^o-uranose (IIc);
l,2-0-Isopropylidene-3-0-3'-(phenylpropyl)-~-D-gluco4uranose (IId);
l,2:5,6-Di-O-isopropylidene-3-0-3'-(morphoiinyl?_opyl)-~-D-glucofuranose (IIe);
l,2:5,6-Di-Q-isopropylidene-3-0-3'-n-Dropoxy-n-heptyl-~-D-glucofuranose (IIf);
l,2-0-Isopropylidene-3-0-3r-n-propoxy-n-hep~yl-~-D-glucofuranose (IIg);
l,2:5,6-Di-0-isopropylidene-3-0-2'-(etAylpvr-oliàyl)-~-5-glucofuranose (_Ih);
:
SUBSTITUTE SHE~T
~ . . . . ~
:, , .
,, . .. : , . .
i, i~
. . . . . .
, .
W092tO~3ss ~ ~9 ~ PCT/~S91/064 1_ 36 -l,2-0-Isopropylidene-3-0-2'-(ethylpyrrolidyl)-~-D-glucofuranose (-Ii);
l,2-0-isopropylidene-3-0-3'-(propan-l'-ol)-~-D-alucoruranose (;Ij); _ 1,2:5,6-~i-0-isopropylidene-3-deoxy-3-amino-l'-(3'~hyd_oxv-r-?ropyl)-~-D-glucof1~ranose (IIk);
',2-0-isopropylidene-3-0-3'-(N',.''-di~ethylamine-~-~_o~vl\-~-D-allofuranose (IIl);
l,2:5,6-Di-0-isopropylidene-3-0-3'-(phenylpropyl)-~-D-allofuranose (IIm);
',2-0-Isopropyl dene-3-deoxy-3-N-3'!N',~'-dimethylamino-n-~-opyi-~-D-allofuranose (-In);
i,2-0-Isopropylidene-3-deoxv-3-amino-~-n--.e?~yl-~z lofuTanose (I-o);
l~2-0-isopropyiidene-3-deoxy-3-amino-3~-(?roo2n-l O`~ -L~
glucofuranose (IIp);
l,2-0-Isopropylidene-3-deoxy-3-N-3'-(phenyl?rcpyl)-~aLlofuranose ~IIq);
1,2-0-isopropylidene-3-deoxy-3-amino-n-he?ty'-C!-~-5 ucofuranose ( T T_ ); and l,~-0-;sopropy'_dene-3-deoxy-3-amino-.-~ phen.y ?rcsi_ -n-~-alu^ofurznose ~ TIs).
Representat ve examples c- ~he above-~en-~onec --m?our.~_ o- r_-mula I-~ were ?repared by one o- the following procedures in Examples 6-ll. The pharmacclosical acrlv ~ c-the compounds of formula I- is illustrated in Examole 12.
Examole 5 General Procedure for the Selective Y.ydrolysis of 5,6-positions.
The fully blocked monosaccharide (l.Og) 's dissolved n tetranydrofuran (l ml) and to this was aàded dropwise a perchloric acid sclution (30%, l ml~, ~ith sti-rins, 2-0-5~. The reaclion ic monlro-ed b~ C and GC. The no=mz~
reaction time varies from 20 minutes to l hou-. Afte~ ~he completion of the reactior., ~ is neutralizea wi_;. z saturated solution of potassium carbonate to a pH c- 9.C.
SUBSrITUTE Sl lEEr .
W092/04359 o 9 ~ ~ g 7 PCT/~S91/06~8 The solid salt formed is filtered through Celite and wa~ne~
well with tetrahydrofuran (50 ml) in several por~ions. ~he combined filtrate is subjected to rotary e~aporation and Ihe residue obtained is purified by flash chroma~ography using silica gel and appropriate solvents. ~he yield of the final products varies from 75-98~.
Exam~le 7 Preparation of l,2:5,6-di-0-isopropylidene-3-0-n-dodecyl-~-D-glucofuranose and ',2-G-isopropylidene-3-0-n-dodecyl-~-D-glucofuranose, (IIa).
Ste~ 1: l,2:5,6-v_-0-isopropylidene-3-0-n-dodecyl-~
glucofuranose (R =~l2H25)-A mixture of l,2:5,6-Di-0-Isopropylidene-~glucofuranose (5.2 g; 0.02 mol) and dry powdered sodium hydroxide (3 equLvalents) was heated togethe- at '2~-:'^ n an cll bath. ThLs reaction ~as conducted under diminisned pressure so as to get -_d of ~ater formed du~~ng the reaction. ThLs reaction takes abou~ 30-45 minutes dependir.c upon the batch size. The vacuum lir.e ~ac ~:~en disconne- e-and '-bromododecane (i.2 eq.) was added ln one po-- o,.. ~h~
reacrion mixture was stirred at the same tempe-~tu-e _- _^
minutes to 2 hours. ~he reaction flask was ~hen cooied, dichloromethane (lO0 ml) was added and the mixture was filtered through Celite and washed with lO0 ml more or rh~
solvent. Solvent was removed using rotary evaporator an~ the residue purified by flash chromatography using ether: hexane (10:90).
Ste~ 2: Hydrolysis according to the general procedure described in Example 6 gave the partially blockec compound l,2-0-isop_opylidene-3-0-n-dodecyl-~glucofuranose, (IIa).
Exam~le 8 Preparation of l,2:5,6-~l-0-isopropylidene-3-0-3'(n-propoxyhepty~ -D-glucofuranose~ (ITf) and ',2-G-Isopropylidene-3-0-3~-(n-propoxy-heptyl)-~-~-gluco-ur2n.ose, (IIg).
SUE3STlTl)TE SHEEl W092/04359 2 0 9 ~ ~ 8 7 PCT/~S91/06458 SteP 1: Preparation of 1,2:5,6-Di-0-isopropyiidene-3-0-3'(n-propoxyhe~tyl)-~-D-glu~o~uranose (R5 = -(CH2)30C7H15) 1,2:5,6-Di-0-isopropylidene-3-0-3'-propanol-~-3-glucofuranose was prepared by reac ing 1,2:5,~-di-v-isopropylidene-~-D-glucofuranose Wit~ soàium hydroxide (equivalents) at 120 - 130C under vacuum. After 30 minutes, the vacuum line was disconnected and 1-bromopropanol (3 e~!
was added. The reaction mixture was heated at the same temperature fo 4; minu~es. The -'lask was then cooled and ether added (lOC mL). The solution was ~'l-ered th_ough Celite, washed ~i tA 100 ml, more o_ e-he- and ~he soi~ent removed. The residue was puri~ie_ bv ~'ac;~ -A--m2~to-raphv using 10~ ethe~ in hexane to afford the title compound in 84 yield.
A mixture of 1,2:5,6-Di-0-iscpropylidene-3-0-3'-propanol-~-D-glucofuranose(Q.o2 mole) and -~ ?owdered sodium hydroxide (3 equivalents) was heated at 120-130 unde-vacuum. When ~he evolution of water had cease_, Ae ~-acuum line was disconnected and heptyl bromide ~'.2 ec~) ~as acded in one portion ~nd the mixture heaLea 2- 1 ;Ae same ~empe_a-~-e for 1 hour. mhe reaction flask was cooied and ether ~130 m was added. The solution was 'iltered through Celi~e 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 S~ ether in hexane.
1,2:5,6-Di-0-isopropylidene-3-0-3'(n-propoxyheptyl)-~-D-glucofuranose was obtained and characterized by NMR and mass spectral analysis.
Steo 2: Hydrolysis according to the general procedure described in Example 6 gave ,Ae parlially ~ __ke~
compound 1,2-0-Isopropylidene-3-0-3'-(n-propoxyheptyl)-~-D-glucoIuranose.
Exam~le 9 r~reparation of 1,2-0-Isopropylidene-3-deoxy-3-amino o-substituted amino-~-D-glucoruranoses.
SUBSrlTUTE SHEFr .
` ~, :
W092/04359 2 0 9 1 5 87 pcr/~s9l/0645x SteD 1: Preparation of ',2:5,6-di-O-iso~ropyliden.e-3-deoxy-3-azido-~-D-giucofuranose;
A mixture of 1,2:5,6-di-O-isopropylidene-3 tosyl-~-D-allofuranose (prepared as described in Methods in Carbohydrate Chemistry, vol. ~, p. 197 (20g) and sodium azide (2.5-equivalents) in anhydrous D~r (100 ml! was heared 2-80C for 3 hours. The progress of the reac~ on was mon~tored by TLC and GC. When the reaction W2S complete, D~ was removed under reduced pressure and the residue was extracted' with ether (150 ml), and washed with wa~e- (~x25 ml), saturated solution of sodium bica-bona-e (2x20 m'~ znd -he~.
with brine (lx25 ml). The organic laye- was d-~e~ e-MgS04, filtered and solvent was removed using a -_-a-y evaporator. This compound tg5% yield) was found t~ be suf-ficiently pure by TLC and GC and hence was used in he ~ex-step.
Ste~ 2: P-eparation of i,2:~,6-di-3-'sopropylidene-;-deoxv-3-amino-~-D-glucofu~anose.
The azido compoun~ (5~) obtained '-, s~e? ~ waC -educG-catalytically using ~2~ Palladium-c:a_co2' (1o~/ ,0 mg) an~
methanol (100 ml) in a Parr-hydrogen2t-- a- 2 ~ress_-e c- '_ psL for 6 hours. The reaction mixture was ~hen ' lle-ed using Celite, washed with methanol ~100 ml) and the solver.~
removed using a rotary evapora~or. The residue obtained showed a single homogenous spot on TLC and complete disappearance of azido 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 pzrtially blocked compound 1,2-di-O-isopropylidene-3-deoxy-3-amino-~-D-giucofuranose.
Ste~ 4: Preparation of 1,2:5,6-Di-O-isopropylidene-3-deox~-3-amino-n-heptyl-~-D-gluco.--anose.
3-Deoxy-3-amino compound obtained in step 3 was heater with 1-bromoheptane at 70-80C in the ratio c. ':2.~ fc- 3-' hours. The progress of the reac~ion was followed bv TLr and .
SUBSTITUTE SHEET
.
, .
.
.
.
W092/043~9 2 Q ~ 7 pCT/~S91/06~8_ 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 or~ani_ layer dried over anhydrous .~gSO~. The xemoval of ~he sol~e~.-gave the crude compound which was purified by flash chromztography. Other amino substituents can be synthesizec by the same procedure usin~ an appropria~e zlkyl halide compound. The alkyl halide itself can be further functionalized.
Ste~ 5: Hydrolysis accc_ding to the general pro-edure described in Example 6 gave the par~ially blocked compound ',2-Di-O-iso~ropylidene-3-deoxv-3-ami.ro~
heptyl-~-D-glucofuranose, (II-).
~ xam~le '~
Prepara~ion of i,2:5,6-Di-O-isopropylidene-3-O-subst_-_ e-~-D--allofuranoses and 1,2-O-Isopropylidene-3-O-subs~i~u-e_ ~-D-allofuranose.
Ste~ ': 1,2:5,6-di-O-isopropylidene-,D-allo_uranose was treated with d-y powde-ed sodium hyd-oxide ar.
suit2bie alkyi halide c- substi~uted a 1~y -.~
in the same manner and rat 3 as desc-ibed --- h-glucofuranose derivative in Example I, step Ste~ 2: Hydrolysis according to the general 2rocedure described in Example 6 gave the par_ially biocked compounds.
Example 11 Preparation of 1,2:5,6-Di-O-isopropylidene-3-deoxy-3-amino-n-heptyl-~-D-allofuranose and 1,2-O-Isopropropvlidene-3-deoxy-3-amino-n-heptyl-~-D-allofuranose, (IIo).
Ste~ 1: Preparation of 1,2:5,6-Di-O-isopropylidene-3-deoxy-3-amir.o-n-heptyl ~-D-allofuranose.
1,2:5,6-Di-O-isopropylidene-~-D-ribo-hexo,u_anose-3-ulose (prepared ac_ording to the l ~eratu-_ procedure "~ethods in Carbonydrate Chemistry, Vol. VI ?~- 125! an~
heptylamine, in the ratio of 1:', were mixed an~ heared z~
SUElSTlTuTE SHEE~T
..
~V092/04359 2 0 9 1 ~ g 7 41 - PCT/~S91/0~58 50-80~ for 30 minutes to 2 hours under dim nlshed pressure.
When the evolu~ion of water had ceased (the ~roaress o- the reaction was monitored by TLC and G~), the vacuum line was disconnected. The product was dissolved in anhvdrous THF anc added dropwise to a stirred suspension cf lithium aluminum hydride (LAH, 2 equivalents) in anhydrous TH~. The reaction was carried out 2t 5-10C with r-gorous sti-_ing and wzs complete in 2 to 3 hours. The excess LAH W2S ~hen decom~osec by careful addition of water and lS~ sodium hydroxide solution (1 ml cf each per gram of L~H used). The reactior.
mixturs was then filtered throush Celite, washed with THF anc solvent removed. ~he residue was dissolved in ethylace~a~-, washed with water, dried and solven_ removed. '~ was purified by ~lash chromatography using ap?ro~_ ate ssl.rer. s Other amino substi~uted compounds can be prepa ed by substituting other appropriate amine compounds f or heptylamine in this procedure.
Ste~ 2: Hydroiysis according to the genera1 procedure described in ~xample 6 gave the pa_tially blocked compo~nd ',2-0-isopropv'Ldene-3-deoxy-3-amin--r-nep~yi-~-D-allofuranose, ~ Tq), Exam~le '2 Pharmacclogical Ac_i~ity of Com~ounds of Fo~.ula ~-.
Compounds were tested for immunomodulatory, ant -proliferative and anti-inflammatory activities in screenin~
assays which measure T-cell proliferation, activation and macrophage I~-l production as described in Example 5.
Compounds of the present inventLon were also tes~ed fo-effects on f-~roblast proliferation and production of ~-o-inflammatory mediators.
Since the early l970~s it has been kno~ ~hat impcr_an-mediators o~ the inflammatory process are the leukotrienes and prostaglandins wnich are synthesized by tissue cel s anc macrophages at the site of inflamma~ion (Flo~er et al., ~Analgesics-antipyretics and Anti-inflammatory Agents; Druas Employed in the Treatment of Gout,~ he Pharmacoloaical ~asis W092t04359 P~T/~S91/0645X
2 0 9 ~5 87 _ 42 -of Thera~eutlcs, New Yorkt 1985). In inflammatory disorders damage to mammaiian cells occurs by phvsical 'rauma or the combination of an antigen with antiDody and this is though~
to initiate the the biosynthesis oî these mediators of inflammation, which are, in turn, responsible for the physiological and visible signs of inflamma~ion. This correlates with the recruitment, activation and prolifera~ion of T-lymphocytes to the localized area of inflammation. In psoriasis, there is an increase in the formation of arachidonic acid in the psoriatic skin that results in mildly increased production of prosta~landins, a~d a several-~old increase in the concentration of leuko~-ienes, princ ?ally LTB4. LT~4 ls the principal biological media~c~ ~hish is responsible fo- the promotion of the inflamma~ory p_ocess that exacerbates the disease (Anderson, T.F., ~INew Re~sons for Using Tlme-Honored Empir - Therapy,'~ Consul~ant, 1985.
In the au~oimmune diseases with ar~hritic components, proliferating synovial Cibroblasts are responsible ~or t~e production of inflammation mediators. The data below demonstrates th~t the com~ounds cc _he presen~ lnver.~ o~. have pharmacolosic21 activity in reducins brcbizs~ ~oduc~ion of LTB4 and PGE2, ~nich have an effec~ n regulatlna _he activity of the infiltrating T-lymphocy~es, and are antiproliferative agents in skin fibroklast c~'tures.
Moreover, this activity indica~es that physiologically acceptable doses of these claimed compounds can be used, either topically or systemically, to inhibi~ T-cell 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 ribroblasts and to modulate the productlon cf PGE2 and LTB4 Svecific ~ethod: Fib-blast Assav The human skin cell fibroblast line, BUD-8 W2S ob;ainea prior to each assay from the American Type Cul~ure Collection. This ïs a fibroblast-iike cell line whieh was ,~' ., SUBS~ITUTE SHEi~T
`. `
' . ; :
:, W092/04359 2 ~ PCT/~S91/06~58 _ 43 -originally derived from the normal skin cf a 56 year old white female.
BUD-8 cell cultures were expanded for use in 25 cm flasks at 37C in an atmosphere of 5% C02 in air. At approximately 4-5 five day intervals, or ~hen confluence was reached, the cells were passaged. This was acco~plisAed by detaching the cells with a Teflon sc-aper, ~ashing ar.d reseeding the cells at a lower densit~; into f~esh tissue culture flasks.
The effect of the compound of the presen~ invention on the proliferative ca~acity of human 8UD-8 s~in f-b-obias~s was measureà with the use o~ a ~H-thvmidine lncorpora~ior.
assay using culture conditions which were simila_ ~o those used for a Con-A blastogenesis assay, desc_ibed ~reviously.
Cultured skin cells were detached from -he surface cf tissue culture flasks mechanically wilh a ~erlon sc-aper. mhe ce`is were washed, resuspended ln incuba~ion medi~m and the viabilLties were determined. These cells we~e then plared n triplicate at a density of 2X103 cells/0.1 ~l/microtiter well fo- the proliferation assay and a densitv o :XlO~ ceils, O.lml/microtiter well for .he assays t~ quan~ ~ate ~G_~ anc LTB~. To these cells was added incuba~ on me~lum cor.tai-. s.
indomethacin to inhibit prostaglandir. ?roduclion, o-nordihydroguaiaretic.acld ~NDGA) to inhibi leukotriene production (positive controls).
After 18 hours of culture, samples of ~he BUD-8 skin cell superna~ants were collected from one set of microtiter plates and frozen until assayed for PGE2 or for LTB4 content using the radioimmunoassays described below.
After 3 days of cultùre, l uCi 3H-thymidine was added in a 50 ul volume to each culture well of the microtiter plates.
Eighteen hours iater, each of the BUD-8 cul.u-es was examined morphologically for evidence of compound-induced toxicitv such as cell rounding or granularity. mhe thymidine-pulsed ; cells were then precipitated and the amount of 3H-thvm dine incorporated was counted in a liquid scintillat_on co~n-e~.
SUBSTITUTE SHEET
, , , :, . :
. . :;
WO92/043s9 PCT/~'S9l/06458 2 0 91 .S ~ ~ i The concentrations of the compounds of the inven~ion which were used in these assays ~ere:
Group 1. 0 ugiml ~Group ': 100 uaiml Group 2: 1 ug/ml Group 6: 300 ugiml Group 3: 10 ug/ml Group 7: 750 ugiml Group 4: 2~ ug/ml The incubation medium useà fo- culturing the ~'D-8 cel's was RPMI-1540 medium containing 10~ fetal bovine serur., lOG
ug/ml streptomycin/ 100 U/ml penicillln, 0.2 ~ Hepes bu_fe~
solut~on, 5x10-5 M 2-mercaptoethanol and 2 mM glutamine.
A radioimmunoassay (New England ~luclear) was used ~-quantitate PGE2 le~els in BUD-8 s~ . ce`l cul~ure supernatants. Briefly, into each polypropylene tube werc mixed 0.1 ml anti-PGE2, 0.1 mi 125;-?G~2 and 0.1 m~_ ~G~
standard or PGEA containing sample. The BUD-8 skin ~ultu-e supernatants were diluted 1:2 prior to adci~ior. to the radioimmunoassay. Tubes were ref-iaerated overr.iqh~~ .
polyethylene glycol solution (16~, 6,000 m.w.) was hen adde~
to precipitate immune complexes and the radioactl~ity in the precipitate was counted in a gamma counte~
Levels of LTB4 in al quots of BUD-8 skin cell c~ rec supernatants were quantitated by radloimmunoassay (Ne~
England Nuclear). Briefly, in~o each polyproplyene tube ~ere mixed 0.1 ml anti-LTB4, 0.1. ml 3H-LTBI and 0.1 ml LTB
standard or LTB4 containing sample. The BUD-8 skin cell culture supernatants were used directly in the radioimmunoassay. Tubes were refrigerated overnighl. A
charcoal solution (0.5 ml of 0.5~ charcoal Norit A) 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 ~-lymDhocytes or skin cells cultured in the presence c' expe_imental compounds versus ln control medium alone.
The inhibitory effe-cts of compounàs (IIa), (IIg;, a~.~
(Ilk) on the T lymphocyte blastogen-- response ~o ConA are SUBSrlTl.JTE S-EFr ~,. ' ' :
. ' , : ' W092/04359 2 0 9 1 ~ 8 7 45 - PCT/~S91/064~8 illustrated in Table VIII. These compounds were found ~o exhibit non-dose dependent inhibition of T lymphocyte proliferation with all compounds tested being st~ongly inhibitory (greater than -50% of control responsê! â~ ~ he 'Cu ug/ml concentration of compound. A speci'ic example o- 2 compound of the present invention mediateA modul2tion c the T lymphocy~e proliferative -es?onse ~o~~ConA s siven lr. T2b e IX. Statistically significant inhibitïon o T cell proliferatlon was exerted by compound (I Tg ) a~ concen-~a __OA.S
ranging between 100 and 750 ug/ml.
~ u~ther immunomodulatory effec~s of these compounds were demonstrated in the M~R assay (Table X). S~rong non-cose dependent inhibition ~greater ~han -50~ o. con~-ol~ was observed zt seve~21 concent-~~ or.s oL cc~.-ou-.-c ~ ar.^
~IIa), This indicates that the compounds lr.rib__ T
lymphocyte .unc~ion in acti~a~ed C~ U~êS.
Sl)BSrll~TE SHEET
', WO 9~/04359 2 ~ 9 1 ~ 8 7 PCI`/I~'S91/06~
A .
~ _ c E ~, ~ co C C _ ~ ~ ~ C~ C, ~ ~ C .~ ~-oi O ,~
., ~ C __ Z
C
I u 6 c C ~ ~ E- ~ ~ U
o ~ V
~ o C~ ~ _ . _ ^ r~
. ~ ~ aJ ~ i '3 ~ ~ ~ ~ ~ ~ ~r~ G a~
Z U ~
c r 1~ D ~ O~ C.
O , O
O . ~ _ C' _ ~1 3 C~
f ` - 1 -209~87 Dsscr_Dtion Monoszccharides Havina An;_;-Drolifer tion and ~ti-Inflammatorv Activl~v ComDcsitions and Uses Thereof Thls application is a continuation-~n-part of U.S.
P~tent Application serial number 07~581,542 -iled September 12, 1990, which is a continuat:ion-in-part of U.S.
Patent Application serial number 07/294,838 filed January , 1989.
Technlcal Field The compounds of this inven.ion a-e ~e_ ~2tives c simple monosac~harides which exhibit znti-~rolifer2_io. anc an-i-ir.'lammator~ ac~ivitv anc are usefl-' -c- --e~- n~
mammals having in'lammatory disorde-s and/c~ au~oimm~ne cisorders. This invention also encompasses pha_~2ce-tlca composi~ions containing these compounds anc me~hods of treating inflammatory and/or aut~immune d ser~ers Backaround Art Cer~ai-. ~onosaccharides and the-r de~ivatives are i:no~-.
to have .herapeu_ic value i.. the treatmen~ c- i..rla~ma o-;
and auto mmune clso ders. Derivatization c ~onosaccha~ir~e-at speciflc hyd~oxyl g~oups may be a-comDl sr.e~ hy syn~;ner:~
techniques which are known in the ar~. ~or example, _- i_ common to block o~ protec' one or more o' the hydroxyl aro~2s leaving one or more hydroxyl groups free to unde-go derivatization, such as formation of an ether ~roup. Various blocking groups and methods are described, for example, in U~S. Patent Nos. 2,715,121 and 4,056,32', ~ne r'lsclosurss c which are incorporated herein by refersnce.
Various derivatives of five and six c~r~on monosaccharides, as well as synthetlc methods, are describe^, for example, in U.S. Patent Nos. Re. 30,354, Re. 30,37, Re. 32,26B, 4,056,322, 4,735,934, 4,î38,953, 4,5C~j19, and 5,010,054. The therapeutic activi y of the vario~s subs~ituted monosaccharides is also disclosed ln the ar,ove .
SUBSTITUTE SHEET
: , :
:
W092/04359 PCT/-S91/061~8 2091~87 - 2 - ~
documents. The disclosures of these pa~ents are also incorporated herein by rererence.
A known derivative of ~-D-giucose having benef cial therapeutic properties is amip.-ilose, l,2-O-iso?ropvlidene 3-0-3'-(N,N'-dimethylamino-n-propyl)-~-D-glucofuranose, and its hydrochloric acid salt, amprilose HCl (THERAFECTIN~).
These two compounds are known to have anti-inflammatory activity and demons~rated utllitv in managins the signs and symptoms of rheumatoid arthri~is. More generally, these compounds have Lmmunomodulatorv activity, and therefore have a therapeutic effect on other autoimmune disorders such as psoriasis, eczema or systemic lupus erythematosus.
Ur.~ortunately, though some de_iva_-ves of ~he monosaccharldes have shown benefi_ial theraoeu~ic act-v~ v, high doses of these derivatives, such as TH_R~FECmIN~, are often needed to produce effectlve results. Because therapy for inflammatory or autoimmune disorders fs often mid-te-m o~
long-term, there is a need to àevelop more potent, non-~ox~c compounds which can be oraliy admin s~ered, and thereby promote patient compliance. mhis invent on descrlbes addi~ional monosaccharide deriva_ives wi~h nc-eased Fo_ency.
I~ is therefore an objec~ of ~he ~resent nven;icn ~o provide new compounàs and com~oslt cns wh c: exh-^il zn,_- _ prollferation and anti-inflammatory 2c~_vity.
It is also an object of the invention to ~_o;ide compounds and compositions which are useful in ~he ~_eatmen~
of mammals having inflammatory and/or autoimmune disorders.
It is a further object to provide new compounds that exhibit significantly increased potency over available compounds, such as THERAFECTIN~, in order to provide ease of oral administration.
Disclosure of the Invention To achieve the foregoing objec~s and in accordance ~ ,h the purposes of the invention as embodied ar.d broadly described herein, there is provided monosaacch2-ides, :r.2-;-ng the following formulae:
.
SUBSrlTUTE SHE~
' ' ' W092/04359 2 ~ 9 1 ~ 8 7 PCT/~S91/064~
A compound of formula I
~'~~/ \l ~r~O A
o~' (I) wherein A is H, methyl or ethyl;
Rl and R2 are ~, methyl, C~-C10 a-~keny' o- togethe~
an isopropylidene ring;
~ C5 C10 alkyl~ Cs-C10 alkenyl, C5-Clo alkynyl, benzyl, or C5-C10 ester; and R is H, Cj-C10 alkyl, C5-C10 alke~yl, C5~Clr alkynyl, benzyl, or Cj-C o ester;
A compound of formula II r a ( L I j wherein: \ / V
xl is O RS~X ~~o/\
_s C12 C20 alkyl~ or CnH2n~:, wherein n= 1,2,3 or a~d Y is selected from cyano, pyrrolyl, pyrrol diny_, methylpyrrolidinyl, pipecoliny', im dazol~l, pyrazc yl, pyrazo iny', pyrazo~i~_ny:, oxzz^l~
oxazolidinyl, 1sooxazolvl, isoozazol_d r.y , imidazolid nyl, ?ipe~dinyl, p per2z~ nyl, morpholinyl, O(CH2)3N(CH3)2, (C;-C10 aikoxy), CH2CH(CH3)CH2N(CH3)2/ CH2C 2N(~5 -10 Y )2 (C3-C7 alkenyl), or X is NH
and R5 is C2-C10 aLkyl, or CnH2nY wherein n= 1,2,3 or 4 and Y is selected from hydroxy, cyano, pyrrolyl !
pyrrolidinyl merhylpyrrolidinyl, ~ipecolir.yl, imidazolyi, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isoxazo~yl, isoozazolidiryl, im dazo idinv', piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (Cs- Clo alkoxy), o- phenvl;
.
SUBSrlTUTE SHEEr ~' , W092/04359 2 0 9 1 5 ~ 7 PCT/~'S91/~
and R6 and R7 are hydrogen or form an isopr^?ylidene -ing;
A compound of formula III: XZ~
Co ~
wnerein x2 is O, ~ `J ~ o X (I~I) R8 is C8-c20 alkyl~ or CnH2nY' whe-ein n= l~2~3~ or 4 and Y is selected from phenyl, cyano, pyrrclyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazoiinyl, pyrazolidiny , oxazolyi, oxazolidinyl, isooxazolyl, isooxazolidinyl, im.idazol'dlnyl, piperidinyl, ?'?e-~ 7 ' n-~morphclinyl, O(C~2)3N(C~3)2~ (C5 ~l0 N(CH3J2;
or x2 is NH
and R8 is H, or CnH2nY, whereln n= 1,2,3 or . and " is selec~ed from OH, cyanc, ~y-~-!yl, pyrrolidinyl, methylpyr~~ .Y-, ?~?eCc ~y_~
imidazolyl, ?yrazolyl, ?vraz~_ ~y', pyrazolidinyi, oxazo'y', cxazGlid r.yl, Lsooxazolyl, isoozazolidiny', midazcl d r.y`, piperidinyl, piperazinyl, morpholinyl, 2 OtCH2)3N(CH3)2 or (C;-Cl0 alkoxy) or pheny';
and R is C5-Cl0 alkyl~ or CnH2nY wherein n= l~2~3 or 4 and selected from O~, phenyl, cyano, pyrroly!, pyrrolidinyl, methylpyr-olidinyl, pipeccliny', imidazolyl, pyrazolyl, py-azo'inyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, im~i~dazolidinyl, piperidinyl, piperazinyl, mor?holinyl, 1 ( H2)3N~CH3)2 or (C5-ClQ alkoxy);
and R9 and ~' are hydrogen o~ form an iso?ropylidene g-ou~;
and SUBSTITUTE SHEET
. .
' ' W092/04359 2 ~ 7 PCT/~S91~0~;8 A compound selected from (3S) l,2-O-isopropylidene-~-D-~ibo-hexos-3-ulcse-l,4:3,6-difuranose; and Methyl 3-0-3'-(N',N'-dimethylamino-n-propyl)-~-deoxy-D-g~ucopyranoside.
Other emboaiments in accordance with the ~resent ~nvention are pharmaceutical compositions con'aining an effective amount of one or more or the ~bove compcunds, and a method of treating an inflammatory disorder and/o~ an autoimmune disorder comprlsing administering an effective amount of a compound described above.
Best 'ode for Carrvinc OUt the ~n~enrion Monosaccharides are known to exist in an equilib~i~m between hemiaceral cycl c strucl~-es and ~n open chain suaa~.
The preferred cyclic struc-ures are fur2noses (;-membered ring st-uctures) and pyranoses (6-me~ered rmqg st-uct~~es~.
Other _ing structures may be formed but are not as thermodynamically stable ar.d generally rearrange to fo ~., ne pyranose or furanose structures. When a cyc'ic hemiacetal fs reacted with a~. alcohol, an ace~al i= ~rmed.
A glyccside can be def~ned as a cycllzed de~a~f~.e --^ -monosaccharide ha~iing two ether (O-~ g_oupsj SU~St' -~en-- o-.
the acetal carbon o- the sugzr. One of thsse ethe~
substituents is the carbocyiic ~ing. The secor.d ethe-substituent is formed by the reac~ior. with the alcohol ana is termed the aglycon. Because of the second ether substi-uer._ t the resultant glycoside is stable and does not exis~ in an equilibrium with its open chain structure. Glycosides having a 5-membered ring are known as furanosides, those wi~h n_ membered ring as pyranosides.
One em~odiment of the presen~ invention relates _o ; derivatives of the simpie monosacchar de ,-uc~ose, n particular to fructofuranosides. ~ructof~ranosides can be in.
an ~-D or ~-D configuration. The f_uctof1lranosides o. -he present inventicn, shown below ~n formula I, encompass bot.r or d configurations and are substituted a_ one or more o-the free hydroxyl groups, but, preferably, ha~e a~ leas~ on-SU~SrlTUTE SHEET
..... .... .... . .
` . . .
. .
' ` .
' W092/04359 2 0 9 1 ~ 8 7 PCT/-S9]/064~
- 6 - ._ free hydroxyl group. As discussed above, the ~ech~iaues __ form these deri~-atives of the present inventlon are gener2i_y known in carbohyàrate chemistry.
The fructofuranosides of the oresen_ invent A_~ c_e represented by formula (I): ~ O~, o~
wherein the aglycon, A, is methyl or ethyl, preferaDly methyl;
R1 and R2 are r:, methyl, e~hy , ~ -C ~ alker.yl or ~oge~ner -orm an isopropyiidene ring;
R3 is ~, C5-C10 alkyl, C5-C10 alXenvl, _ -~10 alXynyl, 2-octyne, benzyl, or C5-C10 es~e_; anc R4 is H~ C;-C10 alky ~ Cs-C10 alXenyi, _5 _ 0 alkynyl, 2-octyne, benzyl, or C5-CiO ester.
Preferred f ructo f uranosLdes of f ormula _ where ~ is methyl are shown in Tabl- I.
SUBSrtTU~E SHEET
W092/04359 2 0 9 ~ 5 8 7 ` PCT/~S91/06458 TA~3LE I
Com~ound R1 R2 R~ R4 ~Ia) Rl & R2 = isopropylidene H C7H15 (Ib) C2H5 C2H5 H C7H15 (Ic) Rl & R2 = isopropylidene C7Hl5 H
(Id) ~1 ~ R2 = isopropylidene 2-oc~yl 2-oc~~y (Ie~ R1 & R2 = lsopropylidene trans-2- trans-2-octenyl octen~l (If) Rl & R2 = isopropylidene cis-2- cis-2-. oc~envl octenvl (Ig) cis-2-octene ~ . cis-,-o-~eny (Ih) Rl & R2 = isopropylidene ~C~.. ls H
C
(Ii) Rl & R2 = isopropylidene H ~C7~15 C O
(Ij) Rl & R2 = sopropyiider.e ~-? 1;
The compounds included in Tabie ~ zre:
methyl 1,3-O-isopropylidene-6-O-he?_~ -^a-D-fructofuranoside (Ia);
methyl 1,3-di-O-ethyl-6-O-hep~yi.-^a-~ uc~ofu~anos de (Ib);
methyl 1,3-O-isopropylidene-4-O-heptyl-^a-D-fructofuranoside (Ic);
methyl 1,3-O-isopropylidene-4,6-di-O-(2-octynyl)-^a-D-fructofuranoside (Id);
methyl l~3-o-isopropylidene-4~6-di-o-(trans~2-octen ^a-D-fructofuranoside (Ie);
methyl 1,3-O-isopropylidene-~,6-di-O-(cis,2-oc~enylj-^a-D-fructofuranoside (If);
methyl 1,6-di-O-(cis,2-octenyl)-^a-3---uc~ofuranosiàe tIg);
SUBSrlTUTE SHEr ; , :
.
~ '.. ,, '' ' ;
~092/04359 2 ~ 91 5 ~ 7 B - PCTt~S91/064~X
methyl 1,3-O-isopropylidene-~-0-octanoyi-~-D-fructofuranoside (Ih);
methyl 1,3-0-isopropylidene-6-0-octanoyl-~fructoruranoside (Ii); and methyl 1,3-O-isopropylidene-4,6-di--0-ocranoyl-~-D-fructofuranoside (Ij).
The present invention also rela~es tc f-uctofuranoses.
These compounds have the same subs~ituenrs as defined in ~ormula I where A is hydrogen. A particularly preferred compound is: 2,3-0-isopropylidene-4-0-hep~yl-~-D-fructofuranose (Ik).
The ~ollowing Examples 1-4 illus.rate r:ne prepar2_ on c-representative compounds o- formula - acc^r~ ng 3 ehis invention. The activity of these _om?our.ds s luc -~e~
Example S.
Examole 1 Preparation of methyl 1,3-0-isopropyLidene-6-0-n-heptyl-~fructofuranoside, (Ia).
Ste~ 1: The preparation of ~ethyl ~-D-fr c-c uranoside~
This compo~nd was prepared by ~ne merhc^ ~ua;.~ k~
Co_tez-Garcia, r~., L. Hough, and ~ icha_dscn (Jou-nc -the Chemicai Society, Per~in ~, pp. 3176-3~:, l9~1j exce~-tha~ a silica gel column was used, ellred ~it.~ chloro~or.~-methanol (9:1 to 8:2) to glve: methyl ~-~-f ucrofuranoside 23.8~; [~]D25 +81.6 (c 1.02, ethanol). {Cortez-Garcia, 22.5%; [~]D +90 (c 2, water)} methyl ~-D-fructofuranoside 28.2~ ]D25 -47.8 (c 1.23, ethanol).
~Cortez-Garcia, 35~; [~]D25 -49 (c 2, methanol)~.
Ste~ 2: The preparation of Methyl 1,3-O-isopropylidene-~-D-fructofuranoside.
~ ethod one: The reaction procedure was descrioed by Cortez-Garcia, excep~ the longer reaction time was used ~three days)~ The best yield was 50~. [x3D25 ~40.' ~s 1.79, chloroform)~ {Cortez-Garcia 33~, [X]D 42.5 (methanol)}. The compound had physical prope_ties in agreement with literaturP values.
.
SUBSTITUTE SHE~
.. , '" ~', ' ,~ ' ' ' , ;':
, ,-W092/043S9 2 ~ 7 PCT/-S91/064~8 g Method two: A mixture of me~hyl ~-D-fruc_ofuranoside (O.8g, 4.lmmole), dimethoxypropane (5g, 49mmole, 6ml) and p-toluenesulfonic acid (O.lg, 0.5mmole) in 20ml of D~ was stirred at 25C for 24 hours. Sodium bicarbonate was added to neutralize the solution. The solvent was lltered and ~he flltrate was evaporated to dryness. Column chromatograohy ~petroleum ether-ethyl acetate (8:2 to t': 3) then chloro-c ~.-methanol (9:1)} gave me~hyl 1,3-O-isopr~pylidene-~-D-fructofuranoside (o~38gl 39%).
Ste~ 3: The preparation of Methyl 1,3-O-isopropylidene-~-C-pivaloyl-~-D-fructofuranoside.
Me~hyl 1,3-O-isopropylidene-~-D-fruc_c-uranosi~- :.3c, 5.55mmole) was dissolved in methylene chloride (lOmll ~nd py~idine (7ml). To this sol~ion, ?ivaloyi ch.~eride ~0.68~, 5.68mmole, 0.7ml) was added at 0C. The solu~ion was s~1_-ec and the temperature rose to 25C. After 18 ho~-s, ano~her O.lml of pivaloyl chloride (o~8mmole) was added 2nd s~ir~lna was continued for 24 hours Water was added o the mix~ure and solvent was evaporated. The residue was ext~acted wi .
chloroform, washed with water, ~rine, criec ove~ sociu.-sulfate, and concent~ated. Column chrom2togr3-;~v ~pe~r~
ether-ethyl acetate (9:1 to 7:3)} gave me~hy' 1,3-C-isopro~ylidene-6-O-pivaloyl-~-D-fructo_-~anoside ('.3~, 83~ ]D25 30.9 (c 2.34, methylene chlo-ide!. -.i-N.m.r. (CDC13): i 4.4-3.9 (m, 8H), 3.3 (s, 3H, GCH3;, 1.~5, 1.38 (2 g, each 3H, C(CH3)2), 1.23 (sl 9H, CO(C~3)3) ppm-Steo 4: The preparation of Methyl 1,3-O-isopropylidene-~-O-benzyl-6-O-pivaloyl-~-D-fructofuranoside.
Methyl 1,3-O-isopropylidene-6-O-pivaloyl-~-D-fructofuranoside (1.3g, 4.08mmole) was dissolved in D~.F
(15ml) and 5.2ml of benzylbromide (7.4g, 43.7mmole) was added. To this solution, a total 5.2g of silver (I) oxide (22.4mmole) was added in three portions during one hour with stlrring. Stirring was contlnued at 25C for _wo davs. ~e silver salt was filtered and washed with DU.F and methylene chloride, and solvent was removed under reduced pressure.
The residue was extracted with methylene chlc-ide 2nc tn^
.
SUBSTITUTE SHEET
, W092/04359 PC~/~S91/n6~5~
2~91~87 -extract was washed with water, brine, dried and concentrated.
Column chromatoaraphy {petroleum ether-ethyl ace~ate (50~
gave methyl 1,3-0-isopropylidene-4-0-benzyl-6-0-Divaloyl-~-D-fructofuranoside [~]325 +50.70 ~_ 1.3;, chloroform)~
1H-N.m.r. (CDC13): ~ 7.33 (m, 5H, C6H;), 4.60 (dd, 2H, OCH2Ph), 4.3-4.1 (m, 4H), 3.9 (d, lH, J 12.1 Hz, H-1), 3.8 (m,lH), 3.7 (d, lH, J 12.1 Hz, ~-1), 3.' (s~ 3H, OC.H3), l.A.-1.3 (2 s, each 3H, C(CH3)2), 1.2 (s, 9H, CO(CH3)3) ppm.
Ste~ 5: The preparation of Methyl 1,3-0-isopropylidene-6-0-t-butyldimethylsilyl-~-D-fructofuranoside.
To a solution of methyl 1,3-0-isopropylidene-~fruc~oIuranoside (0.25~, 1.07mmole) ir. D~'F (,ml) was added imidazole (0.16g, 2.3mmole) and t-butyldlmethylsilyl chlcride (0.2~ '.32mmole). The m~.Yture was s~ --ed 2- 2~C o~ Q8 hours. Water was added and solven was evapora~ed under reduced pressure. Column chromatography {petroleum ether-ethyl acetate (9:1 to 8:2)~ gave methyl 1,3-0-isopropylidene-6-0-t-butyldimethylsilyl-~-D-fruct~fu-anoside (0.26g, 97~), then using chloroform-methanol (100:1) gave startins materia!
methyl 1,3-0-isopropylidene-~-3-f-~__ofuranoside ~omg) Methyl ',3-0-isopropylidene-6-0-t-butyldimethy's_lyl-~fructo.uranoside: ~]~2_ 21.3 (c 1.0 , chlcroro ~..~.
~-N-m-- (CDC13): ~ 4 '-3., (m. 7.~)/ 3.3 (s~ 3~, CC~
(d, lH, OH), 1.45-1.35 (2 s, each 'H, C(C_3)2), C.~ (s~ CH, t-Bu~, 0.1 (s, 6H, Si(CH3)2) ppm-Ste~ 6: The preparation of Methyl 1,3-0-isopropylidene- A _O_ benzyl-6-0-t-butyldmethylsilyl-~-D-fructofuranoside.
To a solution of methyl 1,3-0-isopropylidene-6-0-t-butyldimethylsilyl-~-D-f-uctofuranoside (0.26g, 0.75mmole) and benzyl ~romide (0.15g, 0.9mmo7e, O.lml) Ln DMr '2ml) was added 80~ sodium hydride (25mg, 0.83mmole) slowl~. ~he mixture was stirred a~ 25C under n trogen ~o- 40 m nutes.
Methanol was added to destroy the excess sodium h~d_ide anc solvent was removed under _educed pressure at 25C. The residue was extracted with chlorororm, washe~ with ~ate~, brine, dried and concentrated. Column chroma~ogra~n~
SU8S~lTUrE SHEEI-. . -, ' '. ' W092/043~9 2 ~ 9 1 ~ 8 7 PCT/~!S91/06~8 It. li --{(petroleum ether-ethyl acetate (20:1 _o 8:2)} gave methvl 1,3-O-isopropyiidene-4-O-benzyl-~-G-t-butyldimethylsiiyl-~-D-fructofuranoside (0.28g, 85.6~) and methyl 1,3-O-isopropylidene-'-O-benzyl-~-~-fruc-o-u-anoside (10mg, Methyl 1,3-O-isopropylidene-~-O-benzyl-6~O-t-butyldimethvlsilyl-~-D-fruc'ofuranoside: !~]r25 +48.7 (c 7.5~, chlorofo~m). H-N.m.r. (CDC13): ~ .8 (m~ 5U, C6U,), 4.5 (s~ 2H, OCU~Ph), 4.1-3.7 (m,7H), 3.3 (sr 3H, OCH3), 1.~- -1.3 (2 s, each 3H, C(CH3)2), 0.9 (sl 9H, t-Bu), 0.C6 (s, 6~, Si(cH3)2) pp~n.
Ste~ 7: The ~re~aration of Methyl 1,3-O-isoprc~Ylidene-~-O-benzvl-Q-~-fruc~oruranoside.
Method one: .~ethyl 1,3-O-isopropylidene-~-G-benzv~ G-pivz7Oyl-~-~-f-~cto_u-anoside was dissolved ~ e~hAn~
(20ml) anà sodium methyiate (0.2g) was added and s~i-red a~
25C for 20 hours. The reaction solution was neutrallzed with Ambe-lite IR-120 ~H ) ion sxchange resir., f_ltered and concentrated. Column chromatography ~perr~lelm e~her-etr.~
acetate (9:1 ~o 8:2)} gave methyl 1,3-O-isopreDv7idene-'-O-benzyl-~-D-fruc~ rAnoside (l.l5gr 86.8~ yield).
.~ethod two: A solution o~ me~hyl ~ ^- so~ y'_~en--4-O-benzyl-~-O-t-~utyldimethvlsilyl-~-3---_~ -2-.osi~e (0.2g, 0.45mmole) and 1~. t-8u N~-T~7- (0.5.m_, ~..~.mole~
(5ml) was stirred z_ 25C for 1~ hours. Solven~ was removec and the residue was chromatographed {petroleum e~her-ethyl ace~ate (8:2)} to give methyl 1,3-O-isoF_opylidene-~-O-benzyl-~-D-fructofuranoside (0.14g, 5~).
Method three: To a solution of methyl 1,3-O-isopropylidene-6-O-pivaloyl-~-D-fructofuranoside (0.2g, 0.63mmole) and benzyl bromide (0.14g, 0.81mmole, 0~lml) in DMF (2ml) was added 80~ sodium hydride !27m . 0.9mmole) slowly. The mixt-lre was st-rred at 25C under n t-ogen for 15 minutes. ~ethanol was added ~o dest~ov the excess sodium hydride and solver.~ was evaporated under -eauced pressure 2_ 25C. The residue was dissolved in methano_ (5ml) and sodium methylate (0.lg) was added. The mixture was s~-~red at 2;
for 10 hours and neutralized with Amberli~e .R-i20 (~:
SUBSTITUTE SHEET
W092/04359 ~ ~ 9 1 5 8 7 12 - PCT/~S91/06~5 resin, filtered and concentrared. Columr. ch_omatogr~phy {petroleum ether-ethyl acetate (9:1 to 8:2)} gave merhyl ',~-O-isopropylidene-4-0-benzyl-~-3-fructofuranoslde (0.13g, 64%). [~]D25 -83.~ (c 0.98, methylene chlo-ide). 1-~_ N.m.r. ~CDC13): ~ 7.3 (m, SH, C6.i5), 4-6 (dd, 2H, OCH2ph!~
4.1 ~m, 2H), 3.9-3.8 ~m, 4H), 3.6 (dd, 1~), 3.3 (s, 3H, OCH3), 2.1 (s, lH, OH), 1.4-1.3 (2s, each 3-., C(CH3)~) ppm.
Ste~ 8: The preparation of ~ethyl 1,3-0--sopropylidene- -o-benzyl-6-0-n-heptyl-~-D-fructoturznoside.
~ o a solution of methyl 1,3-0-isopropylidene-4-0-benzyl-~-D-fructofuranoside (1.63g, 5.03mmole) and bromohepcane (4.1g, 23~mole, 3.6ml) in D?~.F (35~1! was added 80~ sodium hydride (0.6g, ,Ommole) slowly. The mix_ure was Sr m ~red 2-25C for 1.5 hours. Methanol was added ~- _es__o, zhe exces~
sodium hydr-de and solvent was evaporzred. ~he res due W2S
extracted wi~h chloroform, washed with ware-, ~ e, d-ied over anhydrous sodium sulfate and concentra~ed. Collmn chromatogra~hy {petroieum ether-ethyl ace~a~e (5:1)} gave methyl 1,3-0-isopropylidene-~-0-benzyl-6-v-n-neptyl-~fructofuranoside (2~, 94% ! [~]D25 -51.?' ~- 1.2?, methylene chlor de). ~~-N.m~. (C3~
C6H5), 4-6 (c, 2~, OCH2Ph), ~.1 (ml -~ d, lH), 3.~ ~, lH), 3.6-3.~ (m, 4H), 3.3 (s, 3:-, C~U3), :.~ 3~., 2., cH2cH2c5u~ -1-3 (2 s, each 3Y, _(C~ , !.2 (b-, 8.
0.9 (t, 3~, C~3) ppm.
Ste~_9: ~he preparation of Methyl !,3-G-isopropylidene-6~G-n-heptyl-~-D-fructofuranoside.
Method one: A mixture of methyl 1,3-0-isopropylidene- -O-benzyl-6-G-n-heptyl-~-D-fructofuranoslde (0.13g, 0.3mmole), 10% palladium on activated carbon (25mg) in methanol (Sml) was shaken under hydrogen (2; lbs. per sa.
inch) for 8 hours. Catalyst was f ltereà and solven~ was removed. Column chromatography {petrole--~ ether-erhyl acetate (9:1)} gave me~hyl 1,3-0-lsopropy_ ~^ne-6-0-~.-hep~v -~-D-fructofu~anoside (Ia) (70mg, 70%).
Method ~wo: A mixture of methy~ 1,3-0-iso?ro?y idene--.-O-benzyl-6-0-n-hep~yl-~-D-fruc~ofuranoside (0.22g, SUBSrlTlJTE~ SHEET
., ,. . . .
~V092/04359 2 ~ 7 PCT/~S91/~58 l~ - 13 -O.52mmole), 10% palladium on ac~ivated carbon (O.2g, ~OOmgf mmole per Bn) and ammonium formate (0.3g) in methanol ~10ml) was refluxed for 2 hours. Another 0.25g of ammonium forma~e was added and refluxed for 3 hours. A final Dart of ~.2~ c-ammonium formate was added and refluxing was continued for another 2.5 hours. After cooling, catalvst was filtered and washed with methanol, filtrate was evaporated to d-vness.
Column chromatocraphy ~petroleum ether-ethyl aceta~e ( a gave methyl 1,3-O-isopropylidene-6-O-n-heptyl-~-D
f_uctofuranoside ~Ia) (0.14g, 81%). [~]D25 +27.4 (c 1.01, methylene chloride). 1H-N.m.r. (CDC13): o 4.2-3.S (~., 5H), 3.4-3., (m, 4H), 3.3 (s, 3H, OCH3), ~-~ (d, `H), 1-~1.~-1.4 (2 s, each 3H, C(CH3)2), 1,3 (~~ S, 8H), o,c !-~C~3) ppm- 3C-NMR (CDCl,): ~i 102.0 (Ç-2), 98.6 (~(C.- ,2), 85.9, 79.7, 78., (C-3, C-~, C-5), 71.7, :._ (C-6, -~-2-C6H13), 61 .7 ~C-l), 48., tor~3j~ 31.&, 29.5, 29.1, 2l.6, 2s.a, 22.6~ 19. ' 14.1 ppm. m.s.: m/z 333 (~_uj)l 301 (~-Y.-CH~O-), 260 (M-CH2OC(CH3)2).
Exam~le 2 ~reparation of methvl 1,3-O-isopropylidene- ,6-di-G- ~-ocrynyl)-~-D-fru~~o~u.anoside, (Id).
~ ethvl 1,3-O-isopropylidene-~-D-fru-rofu-anoside ~as prepared by the procedure cf Cortez-Garcla et al. zs described in Example 1. mO a solution of methyl 1,3-O-isopropylidene-~-D-fructofuranoside (0.3 g, 1.28 mmole) and 1-bromo,2-octyne (0.73g, 3.85 mmole) in DMF (4 mL) was added 80% NaH (0.12g, 4.0 mmole) slowly and the mixture was s.irre-at 25 under nitrogen for 40 min. Methanol was addeà to destroy the excess sodium hydride and solvent was removed.
The residue was extracted with methylene chloride and this solution washed with water, brine and sodi~m bicarbona~e.
This solution was then dried with anhydrous sodium sulfa~e, filtered and evaporated to a resiàue. This residue was dissoived in eluant and chromatagrapned on a silicaael ^~
using pelroleum ether-acetone (24:1) which gave methvl :,3-i^-isopropylidene-4r6-di-o-(2-octyny~ -D-fruc~ofuranoside SUBi~lTUTE SHEET
W092/04359 2 ~ 9 1 ~ 8 7 PCT/~S91/061~X
(Id) (0.41 g, 71~) as a colorless oil. The com~ound was treated wlth sodium bicarbonate and kept in freeze~. ~he compound was identified by its spe~ fic ro~atlon, N~.R znc mass spectral analysis.
Exam~le 3 Preparation of methyl 1,3-O-isopropylidene-~,6-di-O-!tr3ns,2-octenyl)-~-D-fructofuranoside, (Ie).
The procedure of Example 2 was followed excep~ that 1-bromo-trans-2-octe~e was substituted for i-bromo-2-oczvne.
Methyl 1,3-O-isopropylidene-4,6-di-O-(trans,2-octenyl)-~-D-fruc~ofuranoside was obtained n 70.0% yield 2S 2 c_lc-less oil and identified by ts specifi~ rota~ior., N~R 2~ ZSS
spec-ral analvsis.
Exam~le Preparation of 2,3-O-isopropylidene-~-O-hep fruc~ofuranose (Ik);
Ste~ 1: Preparation of ,6-Di-O-r_ityl-5-f-u-lose.
To a solution of D-fruc-ose (10 g, 55.; ~ole) 1.. ?v~id ne (40 mL) and chloroform (25 mL) was added ~ c:-lc~
g, 126.3 mmole) and the mixture was s-irred at 25~ or ~
days, another 5 g Gf .- yl ch!oride was addeà and s~ nc was continued for 2 days. Solvent was removed under -educe~
pressure and the residue was extracted with chloroform, washed with aqueous saturated cupric sulfa~e soiu~ior~, brine, dried over anhydrous sodium sulfate and concentrated. Column chromatography [chlorofm-methanol (100:1)] gave 1,6-Di-O~
trityl-D-fructose (11 g, 30%). mhis compound was identified by its specific rotation and by NMR and Mass spec~rometry.
Ste~ 2: Preparation of 1,6-Di-O-trityl-2,3-O-isopropylidene-~-D-fructosfuranose.
A mixture of 1,6-Di-O-trityl-D-fructose (2 g, 3 mmole),.
dimethoxypropane (5 mL) and p-toluenesulfon ~ acid t.- S) ~~
D~F (20 mL) was stirred at 25 for 3 h. sodium bic2rbona~e was added to neutrali~e the solution. Afte- remov2! c ~;~e salt and solvent, the residue was extr2cted wit.- c;.lorc~orm, 5U13SrlTlJTE SHEET
, ' , W092/043s9 2~91 ~87 PCT/~S9]/06~8 ~- - 15 -washed with water and brine, dried anà concentrated. Columr.
chromotagreaphy [petroleum ether-ethyl acetate] gave l,6-Dl O-trityl-2,3-O-isopropylidene-3-D-fruc'osfuranose (0~9 5, 42%). This compound was identified by its specific rota~ on and by N.MR and Mass spectrometry.
S~e~ 3: Preparation of 2,3-O-Isopropv' Aene-~-O-heptyl-l,5-di-O-trityl-~3-D-f-uc~ofu-ano~e.
l,6-Di-O-~rityl-2,3-O-isopropylidene~
fructosfuranose (0.5 g, 0.71 mmole) was dissolved in DMF (lO
mL) and sodium hydride (60~l 0.12 g) was added. The mix_ure was sti~red at 25C for 20 min. ~hen l-bromoheptane (0.62 g, 3.5 mmole) was added ana s~irrec fc~ ethanol was added to destroy the excess sodium hvc.-ide and solver.t was evaporated. mhe resld~e was e.~:t-~--e~ ^hlorc~o~
washed with water, brine, dried over anhydrous sod u.., s~lfa~s and concentrated. Column chromatog-ap;~ petroleum ethe~-ethyl aceta~e (lOO:l)] gave 2,3-0-Isopropylidene-~-0-hepty -1,6-di-0-trityl-~-D-fructofuranose, (0.~ g, 82.5~). T~is compound was identified by its spec_f - -~tation and by N~R
and Mass spectrometry.
Ste~ 4: ~reparation of 2,3-O-_so?ropyil ene--.-G-he~-y`-;-D-fructofuranose.
Ammonia (about lO0 mL) was passed th~ough _ po_ass_u~.
hydroxide tower and cooied i-.to liquid w~h drv ice to a solution of 2,~-O-Isopropylidene-4-C-ne?tyl-',6-di-O-t~itvl-~-D-fructofuranose (l.35 g, l.68 mmoi.e) -. dry THF (30 mL) cooled with dry ice. The solution was s~ ~-ed and pieces of lithium was added until the blue color persisted. The solution was stirred until the reaction was finished (checked with TLC). ~thanol was added to discharge the reaction and ammonia was allowed to evaporate overnigh~. The the residue, chloroform was added and salt was removed. Column chromatography [petroleum ether-ace~one (8:2)] aave ~,3-G-Isopropylidene-,-O-heptvl-~-D-fru-r-turanose ( Ik ) ( O .
84.1%). This compound was identified bv _-_ spec f -rotation and by NMR and Mass spect~omet~y.
SU8STITUTE~ SHEEl W092/04359 2091587 16 - PCT/~S91/0645~
Exam~le 5 Phzrmacological Activity of compounds o. Formula I.
The pharmacologic assays performed te dete~mine the immunomodulatory effects of the experimen-21 compeunds in vitro include the Mixed Lymphocyte Reac~ion (MLR) and the ConA blastogenesis assav. These assays were used to determine the inhibitory effects of the com?ounds of the invention on T lymphocyle activation and p.olifer2tion.
Since inflammation at the cellular level s characterized ~y T lymphocyte recruitment, activation and proliferation, these assavs are appropriate to use as screens for no~rel compoun.~C
having therapeutic poten~ial in ~he treatmen- o- cisorders _ n which inflamm2tory mechanisms are ir.~olve~.
The .M~R is a classicai assay used t~ measure m cell func~ion b~ studying the proliferative response of m ce'l 1 S
which are activated Ln ~.ritro by gene~ically d sparate stimulator cells. This is accomplished by co-culturing spleen cells from two different strains of mice. Splenic ~
cell proliferation occu-s as a resul~ ellula- activat on signals generated by the onaolng ce ul 2~ eraC,_OI;C.
S~ecific M.ethod: ~R ASSAY
Balb/c mice were euthanised by ce-~; cal disloca~~cn a-.-their spleens removed. Single cel` susper.s ~ns o- ~he spleens were prepared in culture med um (hepes buffered RP~--1640 supplemented with 10% fetal cal serum, 2m~. glutamLne, 500 units penicillin/streptomycin, and 4 x 10 5 ~ 2-msrcaptoethanol) using a Teflon pestle. The cells were centrifuged at 1500 rpm and the pellets resuspended in ACm (0.l5 M tris, 0.14 M ammonium chloride, pH 7.2) Ln order to lyse the red blood cells. After a 5 minute incubation in a 37 C waterba~h, the cells were resuspendeà in culture meaiu~
and counted. C57Bl/5 spleen cells which were used as stimulator cells, were aiso prepared bv this method. mhe stimulator cells were treated with 100 ug/ml cf mi~-ycin for 1 hour at 37 C (to inhibit stimulatorv cel' proliferatior.) and were then washed 5 times in cll-lre .
SUBSTlTUTE SHEET
~ .
, :
.
, ' . ,.~ .
Wo92/04359 2~ 8 7 PCT/~S91/0645R
~ - 17 -medium. The proliferatlve responses were measured bv cult~-ing 2.5 x 105 responder cells with 5 x 105 st m~i2~c-y cells in 96 well microtiter plates in the presence or absence of various doses of test compounds or vehicle ~DMSO).
Syngeneic control cultures using mitomvcin c ;realed Balb~c spleen cells as stimulato- cells were also _un. All ^u'~ures were run in tri?licate.
Solutions oî compounds of the present invention i n, DM'S~^
were ~repared at a stock concentration of 120 mM. Diiutions were made in culture medium to the following concensr2~ ons:
3, lC, 30, 100, and 300 uM. The vehlcle (DMSO) was use~ as a negatlve control.
A'_er incubation of fo_ 5 days at ', C with ~ , the amour.~ o, cell prol ^e~a~ion was meas_-ea bv adài~ ` c-MTT (3-[4,5-dimethy'thiazol-2-yl]-2,5diphenyl-tet~azG um bromide) (10 mg/ml i.~ phosphate buffered sailne) to e~c~
well. Plates were Lncubated for 4 hours at 3? C, af~e~ w~ h 180 ui of 10% sodium dodecyi sulphate in p~.osoha~e bu~-sre~
saline was added. After an overnigh- incub2tion ! the ootic2`
densitv (OD) of eac-. well was read on a ~olecul2- ~e~i-es mic-^?late reader 2- 570 - 650 rm. ~he resul-s were dete-~ined bv calculating the c f f erence be~ween ~he r.~an.s _-the allogeneic cul ures and the means o~ the svngen~c cultures for each test ar~icle concentration. Dif-erences --the test article groups were compared to the con'ro' group and the percent change from the control was determined. Test articles which were found to inhibit proliferation are ~resented as -()% of control with inhibitions greater ~han -20~ of control being considered active.
Mouse Seleen Cell ConA Blastosenesis Assa~
Tt is well known that several plan~ lect ns, when cultured in vitro with lymphocytes, s.imula~e cellula-activation and proliferation. Concanavolin, (ConA) selectively stimulates the blastoaenic res?onse of ~
lymphocytes. Therefore, the ConA blastogenesis assay is SUBSrlTUTE SHET
..
. . . .
W092/04359 2 ~ 9 ~ ~ ~ 7 18 - PCT/~S91/06~5~
useful for screening the i~munomodulato_y 2nd an~ -proliferative activities of experimentzl compounds.
S~ecific Method: ConA Assa~
Six to 8 week old male C57Bl/6 mice were purchased 'rom Harlan Sprague Dawley (Indianapoiis, IN!.
Spleens were removed and were homo~enized to obtain a single cell suspension. Erythrocytes were lysed by hypotonic shock. UDon determlnation of the viability and concentra ~on of the lymphoid cells, they were adjusted to . x lo6 cells/ml in culture medium (~MI-1640) suppiemente~ wi_h 0% fetal bovine serum; 100 ug/ml strep~omycin; 100 ~/ml penici'li.., 0.2 u hepes buffe~; 5 x 10 5 ~ ^-me--zptoethancl and ~
glu~amine). Spleen cells were seeded ir._o mi~-otite_ pl2~e wells at 2 x 105 cells/O.OiO ml/wel'. To these cul~res were added various doses of expe~fmen~al compounds and ConA 2- -final concen~ration of 4 o- 1 ug/ml. Contro` cultu_es consisted of cells, ConA and cul~ure medium contain ng ~he vehicle, DMSO only. In some assays the pos tive controis cyclosporin A (CSP) and ~ZT were also run. ~or -he -es- ng o' ~he methyl 1,3-O-isopropylidene-5-O-n-hep~yl~
- f-uc;ofu-anoside compounc. indome~ha_:r. znd NDGA w--- us_- ~_ ; cont~ols. All cul~ures were run in _-ip ic--e.
Solutions of compounds o_ the inven- on in DU~S~ ~er-prepared and dilutior.s were made in cu ~u~e medium~ .~ssa~
; concent_ations were either: 1, 2.5, 10, 25, 100, 300 and 750 ug/ml or 0.001, 0.01, 0.1, 1, 2.;, 10, 25 and 100 ug/ml.
Cultllres were incubated for 3 days at 37C in a humidified atmosphere o- 5~ of CO2 in ai-. 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 3H-thvmidine incor?orated by the cuLtures, as a measure o. cell proliferztion, was me2sured r.
a liouid sc~ntillation counter. The amount c r radloa~~ive incor?oration is proportional _o the zmour.~ o- cellu a-proliferation ir. individual wells. The studer,ts ~ tes_ was ` SU8STITUTE~ SHEEr .. .. . . .
.
~092/04359 2 0 915 8 7 PCT/~591/064~X
used to determine the significance of the difference be~ween experimental and control alues.
IL-l Assav Interleukir. 1 (IL-1) is a potent immunomodulatory cytokine that has a broad range of pro-Lnfl2mmatory activities. IL-1 is known to be produced bv activated accessory cells such as macrophages. ~ccesso_y cell production of T, L-1 results in the ac_ivztior. and proliferation of T and B lymphocytes. Therefo~e, by inhibiting macrophage activation and production of _~ he activation and proliferation of the cellular effectors of inflammation car. be modulated. Compounds o_ the invent cn were screened for inhibitory activity i~ a ciasslca` -;-1 - assay.
SPecific Method~ l Assay Six to 8 week old male C57Bl/6 mice were pu~chase~ rom Harlan Sprague Dawley. Peritoneal macroohaaes were ellcited by the adminLstration to mice of a single -~traoeritonea!
injection of 0.2 ml of complete Freund's adjuvant. Af ter ~ a hours, elicited mac-ophages were removed frcm the mice bv lavage with the use of ~ank's baiance~ sal~ soiu_ier.~
~ acrophages were washed and seeded lr.to m c-o- ~e- ~e'`s at a density of 2 x 105 cells/well n cu'ture meàium (zs described in the ConA blastogenesis assav)~ To the mac-ophage cul~ures were added variou, doses of ~he compounds of the invention and 10 ug/ml of the macrophage act vator Lipopolysaccharide (to stimulate IL-1 production). Control cultures consisted of macrophages, lipopolysaccAaride and culture medium containing various doses Oc DMSO only.
Cultures were incubated for 24 hours at 37C in a humidified atmosphere of 5% CO2 in air. Compounds of the invention were prepared in DMSO and diluted to the following concentrations in culture med ums 0.001, 0.01, 1, 2.5, '0, ,~ and 100 ug/ml.
The amount of I~-i produced in the nàiviàual wellc ~as determined in a bioassay for I~-1 This invclved the remova of thvmuses f-om mice less than 8 weeks cf age. Th~ocvtes were isolated bV passing each thymus t~.rough a s~ain'ess SUBSTITUTE SHEET
W092/04359 PCT/~S91/06 2 ~9l 5 ~ 7 20 - f steel mesh screen. Thymocytes were placed in culture at 1.5 x lO6 cells~well in RP~I-1640 medium containing 5~ feta bovine serum and l ug/ml ~HA in microtiter plates.
Triplicate cultures were cultured with dilutions o_ macrophage supernatants in the same medium at 37~C in a humldified atmosphere of 5'~ C02 in air.
After ~.8 hours, wells were pulsed ~ith C.5 uCi of 3H-thymidine. After 18 hours cells were harvested and radioactive incorporation (as a measure of cell proliferation) was quantitated in z liquid scintillation counter. ~he students t test was used to de~ermine the significance of the difference between experimental and control values.
~esults c C_~e=rmnc .~SS2~'S ? ~_c~.e~ cn ~e~_esent2~f~.~e c-Formula I.
A summary of the inhibitory e,fects of compounds (Ib! -(I~) on T lvmphocyte proliferation as measured in the ConA
blastogenesis assay is glven in Table II. As demonstrated by these data, the fructofuranosldes inhibit dose dependent inhibition of ConA stimulated ~ lvmphocvte ~rol~feration ~71'-strong inhibitlon (creat ~han -sa~ OI cor._-c~) Dy these compounds cf the present inventior. z- lO0 ug/m1 with the exception of (Ii) wnich W2S found --. be a less ~oter.t inhibitor of the mLtogen induce~ T ym?hocyte ~roiirerative 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 III, compound (Ik) is inhibitory in a dose dependent manner wit:-significant inhibition of ~ lymphocyte proliferation obser~ed at concen~rations ranging 'rom ~ ua/ml ~o lO0 ug/ml. Tabl=
IV demonstrates that compound (Ti) exerts signiricant, non dose dependent modulation of ~ lvmphocyte ?roliferaticn ~it.-significant inhibition observed 2t 2' 1 cose ievel_ on t~e :
ug/ml ConA cultures. The results sno~n in Table V indicate that methyl l,3-O-isopropvlidene-6-O-n-he~tvl-r~-3-fructofuranoside produced a dose-àependen~, sisnif can~, SUBSrlT~lTE SHEEl , ., , W092/04359 2 0 9 1 ~ 8 7 PCT/~S91/0~
( - 21 -inhibitory effect upon the abillty of normal, splenic21lv-derived, mouse T-cells to prcliferate ln response to mitogenic stimulation. There were less T-cells ~n _ne treated c~ltures at the end cf the assav in compa_ son ~ ~r.e untreated control cultures.
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WO 92/04359 2 0 9 1 ~ ~ 7 PCT/~ Sg, //)64~X
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2 ~ g 7 W092/0~35~ PCT/~S91/06~5g : - 29 -Compounds of formula I were found to also be inhib~ ~0~7 to T lymphocyte activation, and proliferation when assessed for activity in mixed lvmpnocyte reactions. As is seen in Table VI, when compared to controis, fructoruranosides of formula I exhibit non dose dependent reductions in k~I~
generated proliCeration with st-ong inhibition de~ined 25 greater than -50~ c- control cul_~res and moderale inhibi~lor defined as -20% of controls.
Further immunomodulatory efiects of compounds ~Ia), (Id), and (Ie) were demonstrated by testing ;or inhibi~or~.~
effects of these compounds on macrophage I~-1 production. In results given ir. Table V-I, the compounds were obse-ved ~o inhibit macrophage IL-1 production in a dose dependen manner with signi~icznt invento-y effec ~ obse-ved ir. ~1' com~o~r.As ar concentrations ranging between 2.; to 100 ug/ml.
In summary, compounds of formula I have been founc to have immunomodulatory and anti-Dro!iferative effects wh'ch predict that these compounds or the present invention have utility, from a therapeutic standpoint, in the treatmer.r c~ 2 variety of infl2mma~3rv and/c~ autoimmune diseases SVBSrl'rVTF SHFET
WO 92/04359 2 ~ 7 PCl/~S91/1)6~X
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SUBSrlTUTE S3-lEET
W0~2/04~59 2 0 9 1 5 8 7 PCT/~'S91/0~5X
A second embodiment of this invention are derivatives of glucose and allose. Glucose and allose are six carbon monosaccharides which differ rrom each ^ther by the configuration of the 3-carbon in the sugar. More particularly, these compounds include mono-substituted ethe-derivatives of ~-D or ~-D glucofuranoses and ~-D or ~-D
allofuranoses as well as analogs thereof. The generic formulae II and III, below, encompasses bolh ~ or ~
configurations. The compounds are substi~uted at the 3- or 6-position of the monosaccharide. The analogs of these compounds are those where the oxygen at .he 3- or 6-positon has been replaced by an amino grou~ or bv sulfur.
As will be apparent from the follo~in~ discussion, ~he compounds of this embodiment can be broadly classifie ir.to two groups: fully blocked mono-substituted compounds, .e., those having two isopropylidene protect ng groups, and partially bloc~ed mono-substi~uted compounds, .e., those having only one isopropylidene protecting group. Applicanrs have found that fully blocked and partially ~locked de-_va ives of ciucofuranose and allofuranose are e--ec_ ve in the treatment of autoimmune and~or ir.flammatory diso-cers.
The 3-substituted deriva~ives of glucofuranose anà
allofuranose a_e shown by the following general formula ~_:
~VO ~l~o wherein: R~X ~ X (II) xl is O
and R5 is C12-C~0 alkyl, or CrH2nY, wherein n= 1,2,3 or 4 and Y is selected from cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecol~ny', imi~azolyi, pyrazolyl, pyrazolinyi, pyraz-lid r.y`, oxazolyl, oxazolidiryl, isooxazolyl, SUBSTITI~TE SHEFr W092/04359 -2 a ~ PCT/~S91/0645~
! - 3~ - ~
isooxazolldinyl, imidazoli~lnyl, piperidinyl, piperazinyl, morpholinyl, O(C~2)3N(C~3)~, (C5-ClO alkoxy), C~2CH(CH3)CH2N(CH3)2, C~2CU N(CC-Clo 21kyl)2, or (C3-C_ alkenyl;
or ~~ is NH
and R5 is C2-ClO alkyl, or CnH2nY wherein n= ',2,3 or 4 and '~ is selected f_om hydroxy, cyano, pyrrolyl, pyrrolidinyl methylpyrrolidinyl, pipecoI~nvl, imidazolyl, pvrazoly1, pyrzzolinyl, pyrazolidinyl, oxazolyl, oxazolLdinyi, soxazolyl, soczzz^l ~iny', i.~ 2zo'i~in~ , piperidinyl, piperazinyl, mo_phollnyl, G(CH2)3N(CH3)2, (C;- Cl~ alkoxy " and ?henyl;
and R6 and R7 are hydrogen or form an isopropy'~dene rina.
Selecti~e removal o4 the pro~ec-~ng qroup ar the ',o-position by the following general procedurs yields par~ i2~
blocked compounds of formula ;I where ?.6 and R' are hvdrcgen.
Preferred co~?ounds of _^nmula L are:
l,2-0-~sopropylidene-3-0-n-dodecyl-c-~-g~o_-~_2nose ~ Ia~;
l,2-Q-Isopropylidene-3-0-n-pen~adecyl-,~-~-gluco_u-anose (IIb);
l,2-0-Isopropylidene-3-0-n-octodecyl-~-3-glu^o-uranose (IIc);
l,2-0-Isopropylidene-3-0-3'-(phenylpropyl)-~-D-gluco4uranose (IId);
l,2:5,6-Di-O-isopropylidene-3-0-3'-(morphoiinyl?_opyl)-~-D-glucofuranose (IIe);
l,2:5,6-Di-Q-isopropylidene-3-0-3'-n-Dropoxy-n-heptyl-~-D-glucofuranose (IIf);
l,2-0-Isopropylidene-3-0-3r-n-propoxy-n-hep~yl-~-D-glucofuranose (IIg);
l,2:5,6-Di-0-isopropylidene-3-0-2'-(etAylpvr-oliàyl)-~-5-glucofuranose (_Ih);
:
SUBSTITUTE SHE~T
~ . . . . ~
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,, . .. : , . .
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. . . . . .
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W092tO~3ss ~ ~9 ~ PCT/~S91/064 1_ 36 -l,2-0-Isopropylidene-3-0-2'-(ethylpyrrolidyl)-~-D-glucofuranose (-Ii);
l,2-0-isopropylidene-3-0-3'-(propan-l'-ol)-~-D-alucoruranose (;Ij); _ 1,2:5,6-~i-0-isopropylidene-3-deoxy-3-amino-l'-(3'~hyd_oxv-r-?ropyl)-~-D-glucof1~ranose (IIk);
',2-0-isopropylidene-3-0-3'-(N',.''-di~ethylamine-~-~_o~vl\-~-D-allofuranose (IIl);
l,2:5,6-Di-0-isopropylidene-3-0-3'-(phenylpropyl)-~-D-allofuranose (IIm);
',2-0-Isopropyl dene-3-deoxy-3-N-3'!N',~'-dimethylamino-n-~-opyi-~-D-allofuranose (-In);
i,2-0-Isopropylidene-3-deoxv-3-amino-~-n--.e?~yl-~z lofuTanose (I-o);
l~2-0-isopropyiidene-3-deoxy-3-amino-3~-(?roo2n-l O`~ -L~
glucofuranose (IIp);
l,2-0-Isopropylidene-3-deoxy-3-N-3'-(phenyl?rcpyl)-~aLlofuranose ~IIq);
1,2-0-isopropylidene-3-deoxy-3-amino-n-he?ty'-C!-~-5 ucofuranose ( T T_ ); and l,~-0-;sopropy'_dene-3-deoxy-3-amino-.-~ phen.y ?rcsi_ -n-~-alu^ofurznose ~ TIs).
Representat ve examples c- ~he above-~en-~onec --m?our.~_ o- r_-mula I-~ were ?repared by one o- the following procedures in Examples 6-ll. The pharmacclosical acrlv ~ c-the compounds of formula I- is illustrated in Examole 12.
Examole 5 General Procedure for the Selective Y.ydrolysis of 5,6-positions.
The fully blocked monosaccharide (l.Og) 's dissolved n tetranydrofuran (l ml) and to this was aàded dropwise a perchloric acid sclution (30%, l ml~, ~ith sti-rins, 2-0-5~. The reaclion ic monlro-ed b~ C and GC. The no=mz~
reaction time varies from 20 minutes to l hou-. Afte~ ~he completion of the reactior., ~ is neutralizea wi_;. z saturated solution of potassium carbonate to a pH c- 9.C.
SUBSrITUTE Sl lEEr .
W092/04359 o 9 ~ ~ g 7 PCT/~S91/06~8 The solid salt formed is filtered through Celite and wa~ne~
well with tetrahydrofuran (50 ml) in several por~ions. ~he combined filtrate is subjected to rotary e~aporation and Ihe residue obtained is purified by flash chroma~ography using silica gel and appropriate solvents. ~he yield of the final products varies from 75-98~.
Exam~le 7 Preparation of l,2:5,6-di-0-isopropylidene-3-0-n-dodecyl-~-D-glucofuranose and ',2-G-isopropylidene-3-0-n-dodecyl-~-D-glucofuranose, (IIa).
Ste~ 1: l,2:5,6-v_-0-isopropylidene-3-0-n-dodecyl-~
glucofuranose (R =~l2H25)-A mixture of l,2:5,6-Di-0-Isopropylidene-~glucofuranose (5.2 g; 0.02 mol) and dry powdered sodium hydroxide (3 equLvalents) was heated togethe- at '2~-:'^ n an cll bath. ThLs reaction ~as conducted under diminisned pressure so as to get -_d of ~ater formed du~~ng the reaction. ThLs reaction takes abou~ 30-45 minutes dependir.c upon the batch size. The vacuum lir.e ~ac ~:~en disconne- e-and '-bromododecane (i.2 eq.) was added ln one po-- o,.. ~h~
reacrion mixture was stirred at the same tempe-~tu-e _- _^
minutes to 2 hours. ~he reaction flask was ~hen cooied, dichloromethane (lO0 ml) was added and the mixture was filtered through Celite and washed with lO0 ml more or rh~
solvent. Solvent was removed using rotary evaporator an~ the residue purified by flash chromatography using ether: hexane (10:90).
Ste~ 2: Hydrolysis according to the general procedure described in Example 6 gave the partially blockec compound l,2-0-isop_opylidene-3-0-n-dodecyl-~glucofuranose, (IIa).
Exam~le 8 Preparation of l,2:5,6-~l-0-isopropylidene-3-0-3'(n-propoxyhepty~ -D-glucofuranose~ (ITf) and ',2-G-Isopropylidene-3-0-3~-(n-propoxy-heptyl)-~-~-gluco-ur2n.ose, (IIg).
SUE3STlTl)TE SHEEl W092/04359 2 0 9 ~ ~ 8 7 PCT/~S91/06458 SteP 1: Preparation of 1,2:5,6-Di-0-isopropyiidene-3-0-3'(n-propoxyhe~tyl)-~-D-glu~o~uranose (R5 = -(CH2)30C7H15) 1,2:5,6-Di-0-isopropylidene-3-0-3'-propanol-~-3-glucofuranose was prepared by reac ing 1,2:5,~-di-v-isopropylidene-~-D-glucofuranose Wit~ soàium hydroxide (equivalents) at 120 - 130C under vacuum. After 30 minutes, the vacuum line was disconnected and 1-bromopropanol (3 e~!
was added. The reaction mixture was heated at the same temperature fo 4; minu~es. The -'lask was then cooled and ether added (lOC mL). The solution was ~'l-ered th_ough Celite, washed ~i tA 100 ml, more o_ e-he- and ~he soi~ent removed. The residue was puri~ie_ bv ~'ac;~ -A--m2~to-raphv using 10~ ethe~ in hexane to afford the title compound in 84 yield.
A mixture of 1,2:5,6-Di-0-iscpropylidene-3-0-3'-propanol-~-D-glucofuranose(Q.o2 mole) and -~ ?owdered sodium hydroxide (3 equivalents) was heated at 120-130 unde-vacuum. When ~he evolution of water had cease_, Ae ~-acuum line was disconnected and heptyl bromide ~'.2 ec~) ~as acded in one portion ~nd the mixture heaLea 2- 1 ;Ae same ~empe_a-~-e for 1 hour. mhe reaction flask was cooied and ether ~130 m was added. The solution was 'iltered through Celi~e 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 S~ ether in hexane.
1,2:5,6-Di-0-isopropylidene-3-0-3'(n-propoxyheptyl)-~-D-glucofuranose was obtained and characterized by NMR and mass spectral analysis.
Steo 2: Hydrolysis according to the general procedure described in Example 6 gave ,Ae parlially ~ __ke~
compound 1,2-0-Isopropylidene-3-0-3'-(n-propoxyheptyl)-~-D-glucoIuranose.
Exam~le 9 r~reparation of 1,2-0-Isopropylidene-3-deoxy-3-amino o-substituted amino-~-D-glucoruranoses.
SUBSrlTUTE SHEFr .
` ~, :
W092/04359 2 0 9 1 5 87 pcr/~s9l/0645x SteD 1: Preparation of ',2:5,6-di-O-iso~ropyliden.e-3-deoxy-3-azido-~-D-giucofuranose;
A mixture of 1,2:5,6-di-O-isopropylidene-3 tosyl-~-D-allofuranose (prepared as described in Methods in Carbohydrate Chemistry, vol. ~, p. 197 (20g) and sodium azide (2.5-equivalents) in anhydrous D~r (100 ml! was heared 2-80C for 3 hours. The progress of the reac~ on was mon~tored by TLC and GC. When the reaction W2S complete, D~ was removed under reduced pressure and the residue was extracted' with ether (150 ml), and washed with wa~e- (~x25 ml), saturated solution of sodium bica-bona-e (2x20 m'~ znd -he~.
with brine (lx25 ml). The organic laye- was d-~e~ e-MgS04, filtered and solvent was removed using a -_-a-y evaporator. This compound tg5% yield) was found t~ be suf-ficiently pure by TLC and GC and hence was used in he ~ex-step.
Ste~ 2: P-eparation of i,2:~,6-di-3-'sopropylidene-;-deoxv-3-amino-~-D-glucofu~anose.
The azido compoun~ (5~) obtained '-, s~e? ~ waC -educG-catalytically using ~2~ Palladium-c:a_co2' (1o~/ ,0 mg) an~
methanol (100 ml) in a Parr-hydrogen2t-- a- 2 ~ress_-e c- '_ psL for 6 hours. The reaction mixture was ~hen ' lle-ed using Celite, washed with methanol ~100 ml) and the solver.~
removed using a rotary evapora~or. The residue obtained showed a single homogenous spot on TLC and complete disappearance of azido 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 pzrtially blocked compound 1,2-di-O-isopropylidene-3-deoxy-3-amino-~-D-giucofuranose.
Ste~ 4: Preparation of 1,2:5,6-Di-O-isopropylidene-3-deox~-3-amino-n-heptyl-~-D-gluco.--anose.
3-Deoxy-3-amino compound obtained in step 3 was heater with 1-bromoheptane at 70-80C in the ratio c. ':2.~ fc- 3-' hours. The progress of the reac~ion was followed bv TLr and .
SUBSTITUTE SHEET
.
, .
.
.
.
W092/043~9 2 Q ~ 7 pCT/~S91/06~8_ 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 or~ani_ layer dried over anhydrous .~gSO~. The xemoval of ~he sol~e~.-gave the crude compound which was purified by flash chromztography. Other amino substituents can be synthesizec by the same procedure usin~ an appropria~e zlkyl halide compound. The alkyl halide itself can be further functionalized.
Ste~ 5: Hydrolysis accc_ding to the general pro-edure described in Example 6 gave the par~ially blocked compound ',2-Di-O-iso~ropylidene-3-deoxv-3-ami.ro~
heptyl-~-D-glucofuranose, (II-).
~ xam~le '~
Prepara~ion of i,2:5,6-Di-O-isopropylidene-3-O-subst_-_ e-~-D--allofuranoses and 1,2-O-Isopropylidene-3-O-subs~i~u-e_ ~-D-allofuranose.
Ste~ ': 1,2:5,6-di-O-isopropylidene-,D-allo_uranose was treated with d-y powde-ed sodium hyd-oxide ar.
suit2bie alkyi halide c- substi~uted a 1~y -.~
in the same manner and rat 3 as desc-ibed --- h-glucofuranose derivative in Example I, step Ste~ 2: Hydrolysis according to the general 2rocedure described in Example 6 gave the par_ially biocked compounds.
Example 11 Preparation of 1,2:5,6-Di-O-isopropylidene-3-deoxy-3-amino-n-heptyl-~-D-allofuranose and 1,2-O-Isopropropvlidene-3-deoxy-3-amino-n-heptyl-~-D-allofuranose, (IIo).
Ste~ 1: Preparation of 1,2:5,6-Di-O-isopropylidene-3-deoxy-3-amir.o-n-heptyl ~-D-allofuranose.
1,2:5,6-Di-O-isopropylidene-~-D-ribo-hexo,u_anose-3-ulose (prepared ac_ording to the l ~eratu-_ procedure "~ethods in Carbonydrate Chemistry, Vol. VI ?~- 125! an~
heptylamine, in the ratio of 1:', were mixed an~ heared z~
SUElSTlTuTE SHEE~T
..
~V092/04359 2 0 9 1 ~ g 7 41 - PCT/~S91/0~58 50-80~ for 30 minutes to 2 hours under dim nlshed pressure.
When the evolu~ion of water had ceased (the ~roaress o- the reaction was monitored by TLC and G~), the vacuum line was disconnected. The product was dissolved in anhvdrous THF anc added dropwise to a stirred suspension cf lithium aluminum hydride (LAH, 2 equivalents) in anhydrous TH~. The reaction was carried out 2t 5-10C with r-gorous sti-_ing and wzs complete in 2 to 3 hours. The excess LAH W2S ~hen decom~osec by careful addition of water and lS~ sodium hydroxide solution (1 ml cf each per gram of L~H used). The reactior.
mixturs was then filtered throush Celite, washed with THF anc solvent removed. ~he residue was dissolved in ethylace~a~-, washed with water, dried and solven_ removed. '~ was purified by ~lash chromatography using ap?ro~_ ate ssl.rer. s Other amino substi~uted compounds can be prepa ed by substituting other appropriate amine compounds f or heptylamine in this procedure.
Ste~ 2: Hydroiysis according to the genera1 procedure described in ~xample 6 gave the pa_tially blocked compo~nd ',2-0-isopropv'Ldene-3-deoxy-3-amin--r-nep~yi-~-D-allofuranose, ~ Tq), Exam~le '2 Pharmacclogical Ac_i~ity of Com~ounds of Fo~.ula ~-.
Compounds were tested for immunomodulatory, ant -proliferative and anti-inflammatory activities in screenin~
assays which measure T-cell proliferation, activation and macrophage I~-l production as described in Example 5.
Compounds of the present inventLon were also tes~ed fo-effects on f-~roblast proliferation and production of ~-o-inflammatory mediators.
Since the early l970~s it has been kno~ ~hat impcr_an-mediators o~ the inflammatory process are the leukotrienes and prostaglandins wnich are synthesized by tissue cel s anc macrophages at the site of inflamma~ion (Flo~er et al., ~Analgesics-antipyretics and Anti-inflammatory Agents; Druas Employed in the Treatment of Gout,~ he Pharmacoloaical ~asis W092t04359 P~T/~S91/0645X
2 0 9 ~5 87 _ 42 -of Thera~eutlcs, New Yorkt 1985). In inflammatory disorders damage to mammaiian cells occurs by phvsical 'rauma or the combination of an antigen with antiDody and this is though~
to initiate the the biosynthesis oî these mediators of inflammation, which are, in turn, responsible for the physiological and visible signs of inflamma~ion. This correlates with the recruitment, activation and prolifera~ion of T-lymphocytes to the localized area of inflammation. In psoriasis, there is an increase in the formation of arachidonic acid in the psoriatic skin that results in mildly increased production of prosta~landins, a~d a several-~old increase in the concentration of leuko~-ienes, princ ?ally LTB4. LT~4 ls the principal biological media~c~ ~hish is responsible fo- the promotion of the inflamma~ory p_ocess that exacerbates the disease (Anderson, T.F., ~INew Re~sons for Using Tlme-Honored Empir - Therapy,'~ Consul~ant, 1985.
In the au~oimmune diseases with ar~hritic components, proliferating synovial Cibroblasts are responsible ~or t~e production of inflammation mediators. The data below demonstrates th~t the com~ounds cc _he presen~ lnver.~ o~. have pharmacolosic21 activity in reducins brcbizs~ ~oduc~ion of LTB4 and PGE2, ~nich have an effec~ n regulatlna _he activity of the infiltrating T-lymphocy~es, and are antiproliferative agents in skin fibroklast c~'tures.
Moreover, this activity indica~es that physiologically acceptable doses of these claimed compounds can be used, either topically or systemically, to inhibi~ T-cell 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 ribroblasts and to modulate the productlon cf PGE2 and LTB4 Svecific ~ethod: Fib-blast Assav The human skin cell fibroblast line, BUD-8 W2S ob;ainea prior to each assay from the American Type Cul~ure Collection. This ïs a fibroblast-iike cell line whieh was ,~' ., SUBS~ITUTE SHEi~T
`. `
' . ; :
:, W092/04359 2 ~ PCT/~S91/06~58 _ 43 -originally derived from the normal skin cf a 56 year old white female.
BUD-8 cell cultures were expanded for use in 25 cm flasks at 37C in an atmosphere of 5% C02 in air. At approximately 4-5 five day intervals, or ~hen confluence was reached, the cells were passaged. This was acco~plisAed by detaching the cells with a Teflon sc-aper, ~ashing ar.d reseeding the cells at a lower densit~; into f~esh tissue culture flasks.
The effect of the compound of the presen~ invention on the proliferative ca~acity of human 8UD-8 s~in f-b-obias~s was measureà with the use o~ a ~H-thvmidine lncorpora~ior.
assay using culture conditions which were simila_ ~o those used for a Con-A blastogenesis assay, desc_ibed ~reviously.
Cultured skin cells were detached from -he surface cf tissue culture flasks mechanically wilh a ~erlon sc-aper. mhe ce`is were washed, resuspended ln incuba~ion medi~m and the viabilLties were determined. These cells we~e then plared n triplicate at a density of 2X103 cells/0.1 ~l/microtiter well fo- the proliferation assay and a densitv o :XlO~ ceils, O.lml/microtiter well for .he assays t~ quan~ ~ate ~G_~ anc LTB~. To these cells was added incuba~ on me~lum cor.tai-. s.
indomethacin to inhibit prostaglandir. ?roduclion, o-nordihydroguaiaretic.acld ~NDGA) to inhibi leukotriene production (positive controls).
After 18 hours of culture, samples of ~he BUD-8 skin cell superna~ants were collected from one set of microtiter plates and frozen until assayed for PGE2 or for LTB4 content using the radioimmunoassays described below.
After 3 days of cultùre, l uCi 3H-thymidine was added in a 50 ul volume to each culture well of the microtiter plates.
Eighteen hours iater, each of the BUD-8 cul.u-es was examined morphologically for evidence of compound-induced toxicitv such as cell rounding or granularity. mhe thymidine-pulsed ; cells were then precipitated and the amount of 3H-thvm dine incorporated was counted in a liquid scintillat_on co~n-e~.
SUBSTITUTE SHEET
, , , :, . :
. . :;
WO92/043s9 PCT/~'S9l/06458 2 0 91 .S ~ ~ i The concentrations of the compounds of the inven~ion which were used in these assays ~ere:
Group 1. 0 ugiml ~Group ': 100 uaiml Group 2: 1 ug/ml Group 6: 300 ugiml Group 3: 10 ug/ml Group 7: 750 ugiml Group 4: 2~ ug/ml The incubation medium useà fo- culturing the ~'D-8 cel's was RPMI-1540 medium containing 10~ fetal bovine serur., lOG
ug/ml streptomycin/ 100 U/ml penicillln, 0.2 ~ Hepes bu_fe~
solut~on, 5x10-5 M 2-mercaptoethanol and 2 mM glutamine.
A radioimmunoassay (New England ~luclear) was used ~-quantitate PGE2 le~els in BUD-8 s~ . ce`l cul~ure supernatants. Briefly, into each polypropylene tube werc mixed 0.1 ml anti-PGE2, 0.1 mi 125;-?G~2 and 0.1 m~_ ~G~
standard or PGEA containing sample. The BUD-8 skin ~ultu-e supernatants were diluted 1:2 prior to adci~ior. to the radioimmunoassay. Tubes were ref-iaerated overr.iqh~~ .
polyethylene glycol solution (16~, 6,000 m.w.) was hen adde~
to precipitate immune complexes and the radioactl~ity in the precipitate was counted in a gamma counte~
Levels of LTB4 in al quots of BUD-8 skin cell c~ rec supernatants were quantitated by radloimmunoassay (Ne~
England Nuclear). Briefly, in~o each polyproplyene tube ~ere mixed 0.1 ml anti-LTB4, 0.1. ml 3H-LTBI and 0.1 ml LTB
standard or LTB4 containing sample. The BUD-8 skin cell culture supernatants were used directly in the radioimmunoassay. Tubes were refrigerated overnighl. A
charcoal solution (0.5 ml of 0.5~ charcoal Norit A) 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 ~-lymDhocytes or skin cells cultured in the presence c' expe_imental compounds versus ln control medium alone.
The inhibitory effe-cts of compounàs (IIa), (IIg;, a~.~
(Ilk) on the T lymphocyte blastogen-- response ~o ConA are SUBSrlTl.JTE S-EFr ~,. ' ' :
. ' , : ' W092/04359 2 0 9 1 ~ 8 7 45 - PCT/~S91/064~8 illustrated in Table VIII. These compounds were found ~o exhibit non-dose dependent inhibition of T lymphocyte proliferation with all compounds tested being st~ongly inhibitory (greater than -50% of control responsê! â~ ~ he 'Cu ug/ml concentration of compound. A speci'ic example o- 2 compound of the present invention mediateA modul2tion c the T lymphocy~e proliferative -es?onse ~o~~ConA s siven lr. T2b e IX. Statistically significant inhibitïon o T cell proliferatlon was exerted by compound (I Tg ) a~ concen-~a __OA.S
ranging between 100 and 750 ug/ml.
~ u~ther immunomodulatory effec~s of these compounds were demonstrated in the M~R assay (Table X). S~rong non-cose dependent inhibition ~greater ~han -50~ o. con~-ol~ was observed zt seve~21 concent-~~ or.s oL cc~.-ou-.-c ~ ar.^
~IIa), This indicates that the compounds lr.rib__ T
lymphocyte .unc~ion in acti~a~ed C~ U~êS.
Sl)BSrll~TE SHEET
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20915~7 ~8-~ C ` Co C
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SUSSTlTtJTE SHEEr .
W092t04359 ~ PCT/~S91/0~58 Mouqe peritoneal macrophage I~-1 production was also found to be inhibited by compounds of the present invention as described in Table XI, Compound (IIg) exerted dose dependent inhibition of macrophage IL-l production with significant decreases in L-l activity observed at 10, 25 and 100 ug/ml of compound. Since IL-l is a potent stimulator c-B and T lymphoc~tes, bo~h of ~-nich are acti~e in inflammat^~v and autoimmune diseases, these data are indicative of the extensive immunomodulatory effects cf he compounds of the present invention. Because uncontrolled fibroblast proliferation and biosynthetic activity are hallmarks c-inflammatory diseases in which joint damags is obser~ed !suc-.
as rheumatoid arthritis)~ compounds of ~he present inver. ior.
were teste~ f^~ activity fn fi~roblast cul~ es. Studies were performed to de~ermine the anti-p~c iCerat~ve effec~s Oc the compounds, and these effec~s were cG__elated with 's~tels of the pro-infla~matory mediators PGr~ and ~TB~ in the fibroblast cultures.
Compounds of the present invent~on were aiso testec 'cr potential effects on fibroblast prcliferative and biosvn~hetic ac'ivity. Com?our.a !--5~ ~as observed ~- ex--=
non dose deDendent anti-proliferat ve efrec~s on Bud-8 skin cell fibroblasts with significant an~i-proliferative ac__~
seen at 100, 25 and 10 ug/ml o compounc (Table XLI).
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 decreaseà in Bud 8 skin cell cultures at concen~rations of 1-100 ug/ml OI comvound (TIg) (Table XIII). Cell toxicity was again observed in cultures which included 300 and 750 ug/ml o_ the compound. Non dose dependent inhibitory effects on fibroblas~ PGE2 produc~ion are exhibited i-. Table XIV where concentrations o. ', '~ ana 25 ug/ml OI compound are seen to correla~e ~i-h significar._ decreases in PGE2 levels in Bud 8 skin cell f brcblas~s SUE~ UTE SREEI`
.. . . ...... . ..
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W092/04359 2 0 9 ~ ~ 8 7 PCT/~:S91tO6458 ~
cultures. Compound induced cell ~oxicity was observed on y at the highest dose level tested. .
Since uncontrolled fibroblast proliferation and activation are dominant features in ir.~lammatory diseases such as rheumatoid arthritis and psoriasis, ~hese data strongly suggest the potentizl for _he use of the compounàs of the present invention as therapeutic agents for ~he treatment of these diseases.
SUBSTITUTE SHEET
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W092/04359 2 0 9 1 ~ 8 7 PCT/US91/0~S8 i _ 5; _ The fully blocked 6-substituted derivatlves of glucofuranose and allofuranose are shown by the followina general formula III: ~s~
~0 ~o 2 ~'~ ~ ( III) wherein X is O, R is C8-C20 alkyl~ or CnH2nY' wherein n= 1~2~3~ o and Y is selected from phenyl, cyano, pyrrolyl, methy'pyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyi, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidiny_, imidazolidinyl, piperidinyi, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (C5 Cl0 N(CH3)2;
or X is NH
and R8 is H, or CnH2nY, wherein n= 1,2,3 or 4 and Y is selec~ed from OH, cyano, pyr-olyl, pyrrolidinyl, methylpyr-olidinyl, pipecol_~
imidazolyl, pyrazolyl, pyrazcl ny:, pyrazolidinyl, oxazolyl, oxazolidinyl isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, 2 (C~2)3N~CH3)2 or (C5-C10 alkoxy) or phenyl;
or X is S
and R8 is C5-Cl0 alkyl, or CnH2nY wherein n= 1,2,3 or 4 and Y
is selected from OH, phenyl, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazol-.~!, oxazo!idinyl, isooxazolyl, isooxazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N~CH3)2 or ~ Fs cl o alkoxy);
SUBSTITUTE SHEEI' . , ~. .
~u~la~ ~
W092/043s9 PCT/~'S91/0~5~
- 56 - ~-and R9 and RlO are hydrogen or form an isopropylidene group.
Selective removal of the protecting group at the 3,5-positlon is achieved by the general procedure described in Example 6 to yield partially blocked compounds of formula III where R-and RlO are hydrogen. Preferred compounds of formula III
are:
l,2-O-Isopropylidene-6-O-h2ptyl-~-D-glucofuranose (~IIa!;
l,2-O-Isopropylidene-6-O-nonyl-~-3-glucofuranose (IIIb);
l,2-O-Isopropylidene-6-O-dodecyl-~-D-glucofuranose (IIIc);
l,2-O-Isopropylidene-6-O-pentadecyl-~-D-glucofuranose (IIId);
l,2-O-Isopropylidene-6-0-3'-(pher.yl?-opyl)-~-D-glucofuranose (IIIe);
l,2-O-Isopropylidene-6-0-3'-(N',N'-dime~r.ylamino-n-propy1!-~-D-glucofuranose (IIIf);
l,2:3,5-Di-0-isopropylidene-6-0-met.~oxyoctyl-~-C-glucofuranose (IIIg);
1,2-0-Isopropylidene-6-0-propionitrlle-~-D-g!uco~uranose (IIIh);
l,2:3,5-Di-0-Isopropylidene-5-deoxy-o-amino-[2'-~minoeth~
2"-(N'-methylpyrrolidyl)-~-3-s1_cof~_-2ncse (II-~);
l,2:3,5-Di-0-Isopropylidene-6-àeoxy-5-zmino-3'-(phenylpropyl)-~-D-glucofuranose ( TT-, );
l,2:3,5-Di-Q-Isopropylidene-6-deoxy-6-N-(N'-propylpipecolinyl)-a-D-glucofuranose (IIIk);
l,2:3,5-Di-0-Isopropylidene-6-deoxy-o-amino-ethoxyerhanol-~-D-glucofuranose (IIIl);
l,2-O-Isopropylidene-6-deoxy-6-amino-3~-(propan-l~-ol~ D-glucofuranose (IIIm);
l,2:3,5-Di-0-Isopropylidene-6-deoxy-6-thio-n-heptyl-~-D-glucofuranose (IIIn);
l,2-O-Isopropylidene-6-deoxy-6-thio-n-heptyl-~-3-glucofuranose (IIIo);
l,2:3,5-Di-0-Isopropylidene-6-deoxy-6-thio-2r-(ethyl-N~-pyrrolidyl)-~-D-glucofuranose (IIIp);
l,2-O-Isopropylidene-6-deoxy-6-thio-2~-(ethyipyrrolidyl)-~-D-glucofuranose ~IIIq);
:
SUBSTIT~)TE SHEET
. .
W092/04359 2 0 91 ~ 8 7 PCT/~'S91/O~SB
- S7 - _ l~2:3~5-Di-o-Isopropylidene-6-deoxy-6-thio-3~-(N/~N
dimethyl-amino-isobutyl)-~-D-glucofuranose (IIIr);
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-~hio-}'-(propan-1'-ol)-~-D-glucofuranose (IIIs); and 1,2-O-Isopropylidene-6-deoxy-6-thio-3'-(phenylpropyl)-~-D-glucofuranose (IIIt).
Synthetic procedures ror represenbative compounds according to this embodiment are shown in the Fxamples wnl-h follow. The activity of selected compounds is shown in Example 17.
Exam~le 13 Preparation of _,2:3,5-di-O-isop-opyiidene-o-deoxy-O-_hio-n-heptyl-~-D-glucofuranose and 1,2-O-Isopro?yl dene-5-deoxv-5 thio-n-heptyl-~-D-glucofuranose, (IIIo).
Ste~ 1: The preparation of 1,2:3,5-di-O-isopropylidene-o-deoxy-5-thio-~-D-glucofuranose has been dsscr~bed in U.S. Paten~ No. ~,996,195, the disclosure o which is incorporated by reference.
Ste~ 2: mhe preparation of :,2:3,5-ai-O-isop~o?yli~en.e-~-deoxv-6-thio-n-heptyl-~,D-glucofuranose:
mixture o~ 1,2:3,5-di-O-isopropylidene-5-dec~.~-6-_n o-~-D-glucofuranose (2.76g; 0.C1 mol) and dry, powdered soc~um hydroxide (1.20g) were mixed togethe- and heated at 90-95C, in an oil bath, under diminished pressure (o~lmm Hg).
When the formation of water bubbles in the flask ceased (40 minutes)l the vacuum line was disconnected and l-bromohep~ane (2.15g, 0.012 mol) was added. The mixture was hea~ed at the same temperature for 45 minutes (the progress of the reac~ion as followed by TLC) and then the flask was cooled to ambient temperature. Dichloromethane (75mL) was added anà stl--ed well. The resultant mixture was filtered and the residue was washed with 75mL more dlchloromethane in small po-. ons. ~he solvent was removed and the residue was purlfied by 'lash chromatography using ether:hexane = 30:70. mhe yield o' ~he pure compound was 3.33g (89.2~). NMR (CD~13): ~ 6.0' (d/
SUBSTlTlJTE SHEET
-~U~15~ ~
W092/043ss PCT/~:S9l/06458 lH, H1), 4.59 (c, lH, H~), 1.34 (m, 12H) 0.89 (~r 3H, CH2CH3)- CIMS: 375 (~
Ste~ 3: One gram of 1,2:3,5-di-0-isoprooyiidene-6-deoxv-o-thio-n heptyl-~-D-glucofuranose was dissolved in tetrahydrofuran (lm~) and hydrochloric acld (3~, lmL) was dropwise-added, over a period of 10 minutes, at O-10C. The reaction mixlure was stirred at the same temperaturA for 1.5 hours, ar.à
then neutralized with saturated potassium carbona~e solution to pH = 9Ø The mixt--re was extracted with ethylacetate (lOOmL) a~.d the solven~ removed using a rotary evaporator. ~2p~-a-ion c- the solvent gave the crude produc- ~h~ch W2S ?u_ - eA
by flash chromatography uslng E 20:hexane = 50:~0.
The yield of the pure compound wa_ 0.77g (86.2~`.
NMR (CDC13): ~ 5-96 (d, lH, H~ -54 (d~ H~ ~2) 0-87 (t, 3H, CH2CH3)- CIMS: :'35 (~ I 1), 277 (~ -C4Hg).
Examole l~
~reparation of ,2:3,5-Di-O-~sop-_?ylidene-~ n-nor.~
glucofuranose ar.d 1,2-0-lsooropv' dene-o-û-n-nenyl-~-D-giucofuranose, (IIIb).
Ste~ 1: 1,2:3,5-Di-O-Isopropylidene-6-0-n-nonyl-~-D-glucofuranose (R8 = C9~18) The synthesis of 1,2:3,5-Di-O-isopropylidene-~-D-glucofuranose (as described in U.S. Patent No. 4,996,195) was achieved by reacting pivaloyl chloride with 1,2-0-isopropylidene-~-D-glucofuranose at the 6-position, followed ; by cyclization of 3 and 5-positions with dimethoxypropane and finally hydrolysis of 6-pivaloyl ester. mh-s compound was treated with dry powdered sodium hyd_oxide and nonylbromide by exactly the same procedure as des_r bed ln Exampie "
step 1.
SUBSrlTl)TE SHEET
~: :
~ .
,~ , .
W092/04359 2 0 9 ~ ~ 87 PCT/~S91/06~8 Ste~ 2: ~ydrolysis according ~ the generai ~roceaure described in Example 6 gave the partiaily blockeà
compound 1,2-O-Isopropylidene-6-O-n-nonvl-~-D-glucofuranose, (IIIb).
Exam~le 15 Preparation of ',2:3,5-Di-O-isopropylidene-6-deoxy-6-amino-~-D-glucofuranose and i,2-O-isopropvlidene-~-deoxy-6-amino-~-D-glucofuranose.
SteD 1: Preparation of 1,2:3,5-Di-O-isopropylidene-6-deoxy-6-amino-~-3-glucoruranose.
A mixture or 1,2:3,5-Di-O-isoDro~vliàene-~-tos-v_-~-L-glucofuranose (prepared as descrlbed in ~'.S. Paten- No.
4,996,195) (lOg) and sodium azide (2.5 equivalents) ~n drv dlmethylformamide (100 ml) was hea~ed at 80-qOC fo- 2 hours.
The progress of the reaction was monitored ~y TLC and G~.
After the completion of the reaction, tne D~ was removed under diminished pressure. The res~due was dissolvec in ether (150 ml), washed with water (~ x 50 mi), sodium bica-bonate sol~ ion (1 ~ 50 ml)~ rhe ^-gar. _ 1~YQ~ e_ (~gSO4) and ~he solvent removed. The _-oduc_ 1,2:3,;-di-O-isopropylidene-~-deoxy-6-azido-~-~-glucof~znose so -crmed was found to be > 98% pure by GC, T_~ anc N.MR. 1,2:3,5-di-v-isopropylidene-6-deoxy-6-azido-~-D-glucofuranose was then reduced catalytical}y 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-O-isopropylidene-6-deoxy-5-amino-~-D-glucofuranose was 96%.
Steo 2: Hydrolysis according to the general procedure described in Example 6 gave the partiallv blocke~
- compound 1,2-O-isopropylidene-6-deoxy-5-amlno-~-G-glucofuranose.
Exam~le 15 The general procedure for the preparation oî 6-deoxv-6-amino or 6-amino substituted glucofuranose was t~ reac_ 1,2:',5-di-O-isopropylidene-6-tosyl-~-a-glucofuranose with an appropriately substituted amine (2.2 equivalents), fo~
. . .
-~ SUB~ITUTE SHEEr ., , .
, W092/043~9 2 ~ 9 1 5 ~ ~ PCT/~S91/0~8 example 3-phenyl-1-propylamine (II~j), at 80-90~ -or ~-3 hours. The progress of the reaction was fcllowed Dy T~C and GC. After the completion of the reaction, the product was dissolved in ethylacetate (100 ml),_washed with a saturztec solution of sodium bicarbonate (2x20 ml), brine (lx20 ml), dried (MgS04) and solvent removed. The i,2:3, 5-Di-O-isopropylidene-6-deoxy-6-amino-~'-(pher.yl?ropyl)-~-D-glucofuranose was then purifieà by flash chromatography usinc the appropriate solvent system.
Steo 2: Hydrolysis according to the gener21 procedure described in Example 6 gave the par~ a . ~_ocXed compound I,2-0-IsopropvlLdene-6-deoxy-6-2r.iro-1 -(phenylpropylj-~-D-gl~cofuranoce.
ExamDle 17 Pharmacological Activl~y of Compounds _- ~cnmula Compounds of formula III were tested f~r immunomodulatory and anti-proliferative effects. ResuI s c-these studies are desc-ibed in ..e d2r~ ~abIes r^_oh Compounds (~IIe), (-I_i), ( ~.-. -r.~ ) were -cun_ to inhibi~ the mouse spleen ceIl bias_ogeni^ -espor.se _.-.-dose aependent manner, wlth s~rcns 2ntip~0 _-erati~e e__-c-(greater than -50~ of control) wi~h ail compounds tes~ed (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 (IIIp) sianificantly decreased the proliferative response of splenic T lymphocytes at concentrations ranging from 10 to 7~0 ua/~l. Additional immunomodulatory effec~s of the compounds o_ the p-esent invention were observed in ~R cultures (Table XVII) where compounds (IIId), (IIIe) and (IILi) were demonst-aled to exert strong non dose dependent inhibition of ~ iymphccyte proliferation. Compound (IIIc) was ~ound to exhibit st-ong inhibitory effects cn ~he MLR a- cor.cenlrations -angLr.c frc~
SUBSTITUTE SHEET
WO 92/~359 2 0 9 1 ~ 8 7 PCT/~S91/064~
10 to 300 uM whereas compounds (IIIe) and ~IIId ! were 2c~ e at the highest dose levels only.
Results of the cesting of compounds or the present invention for modulatory effects on mouse peritoneal mac_ophage production are presented ir. ~able XVII T, -L-i activity was demonstrated to be signi~icantly lowereà in cultures containing 2.;, 10, and lOC u~.~m' _- compouni (IIIi). Compound (IIIe) was signif~cantiv inhibitory to I~-' activity at 2.5, 10, 25 and 100 ug~ml. Both compounas appeared to exer~ inhibitory effects cn ~-1 activi y i-. no.
dose dependent manner.
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W092/04359 2 Q ~ 1 5 8 7 pr~T/~s9l/06~s~
( - 67 -The compounds of the present invention also lnclude the following monosaccharides:
- (3S) 1,2-O-isopropylidene-,-D-ribo-hexos-3-ulose-1,4:3,6-difuranose; and c~J (IV) - Methyl 3-O-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside.
C~3 O
N ~~ O- c ~3 (v) C ~/, Exam~le 18 Preparation of 1,2-O-Isopropylidene-,-~-r~bo-hexos-3-uiose-1,4:3,6-difuranose, (IV).
Two grams of 1,2:5,6-di-O-isopropylidene-,~-u-r~bo-hexofuranos-3-ulose were dissolved in tetrahydrofuran (2mLj and cooled to 0 - 5C. Aqueous perchloric acid (2mL, 3Q%) 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 neutralized with a saturated solution of potassium carbona~e and extracted wLth ethyl acetate. Removal of the solvent gave a crude product which was puriied 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 1 (broad OH), No > C = O (stretching). H
NMR (CDC13): ~ 5.97 (d, lH), 4.51 - 4.41 (m, 4H), 4-26 (m, lH), 3.78 (m, lH), 3.07 (d, lH), 1.58 (d, 3H), 1.40 (s, 3H). C NMR (CDC13, P~T): ~ 113.99, 110.93 (C-3 and - SUBSTITU~E SHEET
W092/04359 2 0 9 ~ ~ 8 7 PCT/~Sgl /06~5~
C-9), 106.98, 84.04, 82.78, ,1.07 ~C-1, C-^, C-4, C-5), ?3.5 (C-6), 27.22, 27.18 (C-8, C-9). CIMS: 21q ~ r 1) -Examole 13 -PharmacologLcal Activity of 1,2-O-Isopropylidene~ ribo-hexos-3-ulose-1,4:3,6-difuranose, (IV).
Compund IV was tea~ed in the Bud-8 skin celi fib o~las_-assays as described in Example I2. As can been seen --om Tables XIX, XX and XXI below, the compounds of the inven~i~n produced significant, non dose dependent decreases in the ~ud-8 skin cell fibroblast proliferation in .hose cul~ures that contained 1, 10, 25 and 100 ug/~.l, _espec-ivel~ ner.
compared to the control cultures tha~ did not contain ~ne compounds of the invention. Cyto~oxicily was not obse-ved ar these concentrations of the test com~ound. Cytotoxic effe~s were o~served in the highest concentratLons tested (300 ugimi and 750 ugfml). These anti-proliferative effects co~rola~es with decreases in Bud-8 skin cell f_b-~las. ?rodu_~isn cf PGE2 and LTB4 (~ables XX and XXI) whero signif can~l~
decrease o- levels of bc~. pro-ir. lamm2 __-. me~ a-c-~seen a~ several non-toxic doses o_ com?3un~ ~..
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W092/04359 2 0 3 1 5 8 7 PCT/~S91/064~8 _ 73 -Exam~le 20 Preparation of Methyl 3-0-3 -(N',N -dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside (V).
Ste~ 1: The prepara~ion of 1,2-O-isopropylidene-3-0-3 -(N',N'-dimethylaminopropyl)-6-O-p-toluenesulfonyl-^a-D-glucofuranose, (1).
Ten grams of 1,2-O-isopropylidene-3-C-3 -(N~,N~-dimethylaminopropyl)-^a-D-glucofuranose were dissolved in 30mL of anhydrous pyridine and cooled to 5C in an ice-water bath. p-Toluenesulfonyl 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 altowed to attain ambient temperature 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 carbona~e solution. The combined organic phase was washed with wa~er and brine, dried ove- anhydrous magnesium sulfate and evaporated to give 1~._ g (95%) of the tosylate as a glassy material, that soli- fied on trituration with ether. CIMS: ~60 (M - 1) Ste~ 2: The preparation of 1,2-O-isopropylidene-5-deoxy-3-0-3'-(N',N'-dimethylaminopropyl)-^a-D-glucofuranose, (2).
Ten grams of 1,2-O-Isopropylidene-3-O-3'-(N',N'-dimethylaminopropyl)-6-O-p-toluenesulfonyl-^a-D-glucofuranose (1) were dissolved in 60m^P 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 THF. After the addition was complete, the mixture was refluxed for 2 hours. It was then cooled in an ice bath and the excess lithium alu~inum hydride was quenched by the addition of 2m^P of water, followed by 5m^P of 15~
- sodium hydroxide solution. The mixture was fi'tered through Celile and the filtrate was evaporated to give 5.91 g (94%) of the 1,2-O-isopropylidene-6-deoxy-3-0-3'-(N',N' SUBSrlTUTE SHE~ET
. ~ .
., ` :: . . . `.
, ` ' W092t043~9 PCT/~S9l/06458 2091~7 ~-dimethylaminopropyl)-^a-D-glucofuranose (2) product which was used without purification for the-ne~t s~ep. CIMS: 290 (M + 1) NMR (CDC13): ^w 5.92 (d, lH, H~ .55 (d, lH~ H2), 3.95 (m, 2H, H3 and H4), 3.83 (m, lH, H5), 3.48 lm, 2H, OCH2), 2.28 (s, 3H, CH3), 1.48 and 1.32 (s~ 3H each, C(CH3)2) -Ste~ 3: The prepara~ion of 6-Deoxy-3-0-3'-(N',N'-dimethvl-am~nopropyl)-D-glucopyranose, (3).
Three grams of 1,2-0-isopropylidene-3-0-3'-(N',N'-dimethylaminopropyl)-6-deoxy-^a-D-glucofuranose (2) was dissolved in lOmL of tetrahydrofuran and 5m^~ o 3 ~
hydrochloric acid was added. The mixture was stir-ed for ^
hours at 50C, cooled and neutralized with 20% scc um 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. mhe 6-Deoxy-3-0-3~-(N',N'-dimethylaminopropyl)-D-glucopyranose (3) was used directly for the next step.
Ste~ 4: The preparation of methyl 3-0-3'-(N',N'-dimethylaminopropyl)-6-deoxy-3-gluco?yr2noside, (V) -The 6-Deoxy-3-0-3'-(N',N'-dimethylaminopropyl)-D-glucopyranose (3) (1.8 g) was dissolved in lOmL 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 neutralized with saturated potassium carbonate and extracted several times with dichloromethane. 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 ethyi acetate) to give 1.64 g (86.5%) of the methyl 3-0~3'-~N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside compound. The lH N~R showed the presence of a mixture of ^a and ^b-isomers in the ratio of 6:4. CIMS: 264 (M + 1) -SUE~SrlTlJTE SHEE~T
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W092/04359 2 0 3 1 j ~ 7 PCT/~'S91/0S~8 Exam~le 21 PharmacologiCal Activity of ~ethyl 3-0-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside, (v).
Methyl 3-o-3~-~N~N~-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside of this invention has_~emonstrated inhibitory effects on the proliferation of GS-10~-V-20 human skin cell fibroblasts.
A compound that inhibits fibroblast proliferztion has the potential to be utilized as a dermatological drug used to treat chronic dermatorse, such as psoriasis and autolm~une disorders which result in join~ inflamma~ion, such as rheumatoid arthritis. Alsc, an an~i-prol-ferative effecr may well be observed with other tissues, such as those tha~ line the blood vessels, or joints, the uncontrolled prolifera~ on of which produce disease, thereby broadening the scope of potential applications.
Svecific Method: Fibroblast Assav The effect of methyl 3-0-3'-(N',~'-dimethylamir.o-r.-propyl)-6-deoxy-D-glucopyr2noside on the proi _erative capacity or human GS-109-V-20 skin fibroblasts was measured with the use of 3H-thymidine incc-poration assay. Cultu-e~
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. Thess cells were then plated in triplicate at a density of lX103 cells/O.lml/microtiter well. To these cells was added O.lm^P incubation medium containing the methyl 3-0-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside compound.
After 3 days of culture, l~uCi 3H-thymidine was added in 50ul volume to each culture well of the micro~iter plates.
Eighteen hours later, each of the GS-109-V-20 cultures was examined morphologically fcr evidence of drug-induced toxicity such as cell rounding or granularity. The ~r.-thymidine-pulsed cells were then precipitated and the amoun~
SUBSrlTt)TE SHEFI
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W092/04359 pCT/~'S91/06458 209~587 _ 76 -of 3H-thymidine incorporation was counted on a liquid scintillation counter.
Methyl 3-O-3'-(N',N~-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside was suspended directly into the medium by extensive sonication, without being filter-sterilized. A
range of doses of this compound was usled to measure effects of this compound upon GS-109-V-20 cell proliferation. The following doses were used:
Group 1: 0 ug~ml Group 7: 10 ug/ml Group 2: 0.001 ug/ml Group 8: 25 ug/ml Group 3: 0.01 ug/ml Group 9: 100 ug/ml Group 4: 0.1 ug/ml Control G-oups:
Group 5: 1 ug~ml Control: 2X10 5 M Indomethacin Group 6: 2.5 ug/ml Control: 2X10 6 M NDGA
SDecific Method: Incubation Medium The incubation medium used for culturing the GS-109-'Y'-20 cells was RPMI-1640 medium containing 10% fetal bovine serum, 100 ug~ml streptomycin, 100 U/ml peniclllin, 0.2 ~ Hepes buffe- solution, 5x10-5 M 2-mercaptoethano' and ~ m~
glutamine.
Specific M.ethod: Human 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 polyps of the colon (American Type Culture Collection, Catalogue of Cell Lines and Hyb-idomas, -6th Ed., 150, 1988). These cells were selected for use because they are considered to exis~ in an initiateà state, as opposed to being normal or transformed, and have a more extensive population doubling time and sur~ival pe-iod in culture than do normal fibroblasts. In this regard, they would more closely reflect ~he biological characteristics of psoriatic or rheumatoid synovial fibroblasts which also proliferate more extensively than do normal ~i~roblasts, but not as extensively as immortalized transformed tumo_ cells.
SllBSrlTUTE SHEET
W092/04359 ~ 8 ~ P~T/US91/0~58 .
The number of GS-lO9-V-20 cells was expanded for use in the described assays by culture in 25 cm2 flasks at 37C in an atmosphere of 5% CO2 in air. At approximately 4-5 day intervals, or when confluence was reach~d, the cells were passaged. This was accomplished by detaching the cells ~y trypsinization, washing and reseeding the cells at a lower density into fresh tissue culture flasks.
Statistical Anal~sis_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 prolifer2tive abil ties of the skin cells cultured in the presence of methyl 3-C-3'-(N',N'-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 'rable XXII
below.
A significant inhibitory effect was observed in a non-dose-dependent manner for cultures receiving the methyl 3-~-3'-(N',N'-dimethyl2mino-n-propyl)-6-deoxy-D-glucopyr2noslde.
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 exer~s these effects throuh novel mechanisms.
SU8SrlTUTE SHEEr ,, . :
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~W092/04359 2 0 9 1 ~ 8 7 PCT/~:S91/0645~
The compounds of the present invention as shown by formulae I, II, III, IV and V are useful for treating mammals with inflammatory and/or autoimmune disord~rs such as psoriasis, atopic dermatitis, rheuma~oid~ arthritis, ostearthritis, 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 ~o their valuable pharmacological properties, the compounds of the present invention or their physiologically acceptable salts are particularly suitabie 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 hhich comprise an effective amount of at least one compound of the present invention with or without a pharmaceutically accepta~le carrier. If appropriate, compounds contair.ins an amino functionality may be in the form of an acid-add ~ion salt. Preferred acid addition salts are hydrochloric acid salts.
The present invention also encompasses a method of treating animal-~ 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 or an acid-addition salt thereof, with or without a pharmaceutically acceptable carrier. The compositions according to the invention can be adminis~ered orally, topically, rectally, internally, or, if desiredt parenterally. Oral administration is preferred.
Suitable solid or liquid galenical formulations, fo-example are granules, powders, coated tablets, microcapsules, suppositories, -~yrups, elixirs, suspensions, emulsions, drops or injectable solutions. Preparations having a protracted SUElSrITUTE SHEEl .
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W0~2/04359 2 0 9 1 5 8 7 PCT/US91/0645~
release of the active compound may also be used. These formulations can also contain additiv~s such as excipients, disintegrants, binders, coating agents, swelling agents, glidants, or lubricants, flavors, sweeteners or solubilizers.
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 ana monohydric 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 cf 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 ln vi~-o testing, the effective amount to achieve a 50~ inhibition of the cultured cells range from about 1 - 200 ug/ml of cul ~re medium, preferably 10 - 100 ug/ml.
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W092t04359 ~ PCT/~S91/0~58 Mouqe peritoneal macrophage I~-1 production was also found to be inhibited by compounds of the present invention as described in Table XI, Compound (IIg) exerted dose dependent inhibition of macrophage IL-l production with significant decreases in L-l activity observed at 10, 25 and 100 ug/ml of compound. Since IL-l is a potent stimulator c-B and T lymphoc~tes, bo~h of ~-nich are acti~e in inflammat^~v and autoimmune diseases, these data are indicative of the extensive immunomodulatory effects cf he compounds of the present invention. Because uncontrolled fibroblast proliferation and biosynthetic activity are hallmarks c-inflammatory diseases in which joint damags is obser~ed !suc-.
as rheumatoid arthritis)~ compounds of ~he present inver. ior.
were teste~ f^~ activity fn fi~roblast cul~ es. Studies were performed to de~ermine the anti-p~c iCerat~ve effec~s Oc the compounds, and these effec~s were cG__elated with 's~tels of the pro-infla~matory mediators PGr~ and ~TB~ in the fibroblast cultures.
Compounds of the present invent~on were aiso testec 'cr potential effects on fibroblast prcliferative and biosvn~hetic ac'ivity. Com?our.a !--5~ ~as observed ~- ex--=
non dose deDendent anti-proliferat ve efrec~s on Bud-8 skin cell fibroblasts with significant an~i-proliferative ac__~
seen at 100, 25 and 10 ug/ml o compounc (Table XLI).
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 decreaseà in Bud 8 skin cell cultures at concen~rations of 1-100 ug/ml OI comvound (TIg) (Table XIII). Cell toxicity was again observed in cultures which included 300 and 750 ug/ml o_ the compound. Non dose dependent inhibitory effects on fibroblas~ PGE2 produc~ion are exhibited i-. Table XIV where concentrations o. ', '~ ana 25 ug/ml OI compound are seen to correla~e ~i-h significar._ decreases in PGE2 levels in Bud 8 skin cell f brcblas~s SUE~ UTE SREEI`
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W092/04359 2 0 9 ~ ~ 8 7 PCT/~:S91tO6458 ~
cultures. Compound induced cell ~oxicity was observed on y at the highest dose level tested. .
Since uncontrolled fibroblast proliferation and activation are dominant features in ir.~lammatory diseases such as rheumatoid arthritis and psoriasis, ~hese data strongly suggest the potentizl for _he use of the compounàs of the present invention as therapeutic agents for ~he treatment of these diseases.
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W092/04359 2 0 9 1 ~ 8 7 PCT/US91/0~S8 i _ 5; _ The fully blocked 6-substituted derivatlves of glucofuranose and allofuranose are shown by the followina general formula III: ~s~
~0 ~o 2 ~'~ ~ ( III) wherein X is O, R is C8-C20 alkyl~ or CnH2nY' wherein n= 1~2~3~ o and Y is selected from phenyl, cyano, pyrrolyl, methy'pyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyi, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidiny_, imidazolidinyl, piperidinyi, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (C5 Cl0 N(CH3)2;
or X is NH
and R8 is H, or CnH2nY, wherein n= 1,2,3 or 4 and Y is selec~ed from OH, cyano, pyr-olyl, pyrrolidinyl, methylpyr-olidinyl, pipecol_~
imidazolyl, pyrazolyl, pyrazcl ny:, pyrazolidinyl, oxazolyl, oxazolidinyl isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, 2 (C~2)3N~CH3)2 or (C5-C10 alkoxy) or phenyl;
or X is S
and R8 is C5-Cl0 alkyl, or CnH2nY wherein n= 1,2,3 or 4 and Y
is selected from OH, phenyl, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazol-.~!, oxazo!idinyl, isooxazolyl, isooxazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N~CH3)2 or ~ Fs cl o alkoxy);
SUBSTITUTE SHEEI' . , ~. .
~u~la~ ~
W092/043s9 PCT/~'S91/0~5~
- 56 - ~-and R9 and RlO are hydrogen or form an isopropylidene group.
Selective removal of the protecting group at the 3,5-positlon is achieved by the general procedure described in Example 6 to yield partially blocked compounds of formula III where R-and RlO are hydrogen. Preferred compounds of formula III
are:
l,2-O-Isopropylidene-6-O-h2ptyl-~-D-glucofuranose (~IIa!;
l,2-O-Isopropylidene-6-O-nonyl-~-3-glucofuranose (IIIb);
l,2-O-Isopropylidene-6-O-dodecyl-~-D-glucofuranose (IIIc);
l,2-O-Isopropylidene-6-O-pentadecyl-~-D-glucofuranose (IIId);
l,2-O-Isopropylidene-6-0-3'-(pher.yl?-opyl)-~-D-glucofuranose (IIIe);
l,2-O-Isopropylidene-6-0-3'-(N',N'-dime~r.ylamino-n-propy1!-~-D-glucofuranose (IIIf);
l,2:3,5-Di-0-isopropylidene-6-0-met.~oxyoctyl-~-C-glucofuranose (IIIg);
1,2-0-Isopropylidene-6-0-propionitrlle-~-D-g!uco~uranose (IIIh);
l,2:3,5-Di-0-Isopropylidene-5-deoxy-o-amino-[2'-~minoeth~
2"-(N'-methylpyrrolidyl)-~-3-s1_cof~_-2ncse (II-~);
l,2:3,5-Di-0-Isopropylidene-6-àeoxy-5-zmino-3'-(phenylpropyl)-~-D-glucofuranose ( TT-, );
l,2:3,5-Di-Q-Isopropylidene-6-deoxy-6-N-(N'-propylpipecolinyl)-a-D-glucofuranose (IIIk);
l,2:3,5-Di-0-Isopropylidene-6-deoxy-o-amino-ethoxyerhanol-~-D-glucofuranose (IIIl);
l,2-O-Isopropylidene-6-deoxy-6-amino-3~-(propan-l~-ol~ D-glucofuranose (IIIm);
l,2:3,5-Di-0-Isopropylidene-6-deoxy-6-thio-n-heptyl-~-D-glucofuranose (IIIn);
l,2-O-Isopropylidene-6-deoxy-6-thio-n-heptyl-~-3-glucofuranose (IIIo);
l,2:3,5-Di-0-Isopropylidene-6-deoxy-6-thio-2r-(ethyl-N~-pyrrolidyl)-~-D-glucofuranose (IIIp);
l,2-O-Isopropylidene-6-deoxy-6-thio-2~-(ethyipyrrolidyl)-~-D-glucofuranose ~IIIq);
:
SUBSTIT~)TE SHEET
. .
W092/04359 2 0 91 ~ 8 7 PCT/~'S91/O~SB
- S7 - _ l~2:3~5-Di-o-Isopropylidene-6-deoxy-6-thio-3~-(N/~N
dimethyl-amino-isobutyl)-~-D-glucofuranose (IIIr);
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-~hio-}'-(propan-1'-ol)-~-D-glucofuranose (IIIs); and 1,2-O-Isopropylidene-6-deoxy-6-thio-3'-(phenylpropyl)-~-D-glucofuranose (IIIt).
Synthetic procedures ror represenbative compounds according to this embodiment are shown in the Fxamples wnl-h follow. The activity of selected compounds is shown in Example 17.
Exam~le 13 Preparation of _,2:3,5-di-O-isop-opyiidene-o-deoxy-O-_hio-n-heptyl-~-D-glucofuranose and 1,2-O-Isopro?yl dene-5-deoxv-5 thio-n-heptyl-~-D-glucofuranose, (IIIo).
Ste~ 1: The preparation of 1,2:3,5-di-O-isopropylidene-o-deoxy-5-thio-~-D-glucofuranose has been dsscr~bed in U.S. Paten~ No. ~,996,195, the disclosure o which is incorporated by reference.
Ste~ 2: mhe preparation of :,2:3,5-ai-O-isop~o?yli~en.e-~-deoxv-6-thio-n-heptyl-~,D-glucofuranose:
mixture o~ 1,2:3,5-di-O-isopropylidene-5-dec~.~-6-_n o-~-D-glucofuranose (2.76g; 0.C1 mol) and dry, powdered soc~um hydroxide (1.20g) were mixed togethe- and heated at 90-95C, in an oil bath, under diminished pressure (o~lmm Hg).
When the formation of water bubbles in the flask ceased (40 minutes)l the vacuum line was disconnected and l-bromohep~ane (2.15g, 0.012 mol) was added. The mixture was hea~ed at the same temperature for 45 minutes (the progress of the reac~ion as followed by TLC) and then the flask was cooled to ambient temperature. Dichloromethane (75mL) was added anà stl--ed well. The resultant mixture was filtered and the residue was washed with 75mL more dlchloromethane in small po-. ons. ~he solvent was removed and the residue was purlfied by 'lash chromatography using ether:hexane = 30:70. mhe yield o' ~he pure compound was 3.33g (89.2~). NMR (CD~13): ~ 6.0' (d/
SUBSTlTlJTE SHEET
-~U~15~ ~
W092/043ss PCT/~:S9l/06458 lH, H1), 4.59 (c, lH, H~), 1.34 (m, 12H) 0.89 (~r 3H, CH2CH3)- CIMS: 375 (~
Ste~ 3: One gram of 1,2:3,5-di-0-isoprooyiidene-6-deoxv-o-thio-n heptyl-~-D-glucofuranose was dissolved in tetrahydrofuran (lm~) and hydrochloric acld (3~, lmL) was dropwise-added, over a period of 10 minutes, at O-10C. The reaction mixlure was stirred at the same temperaturA for 1.5 hours, ar.à
then neutralized with saturated potassium carbona~e solution to pH = 9Ø The mixt--re was extracted with ethylacetate (lOOmL) a~.d the solven~ removed using a rotary evaporator. ~2p~-a-ion c- the solvent gave the crude produc- ~h~ch W2S ?u_ - eA
by flash chromatography uslng E 20:hexane = 50:~0.
The yield of the pure compound wa_ 0.77g (86.2~`.
NMR (CDC13): ~ 5-96 (d, lH, H~ -54 (d~ H~ ~2) 0-87 (t, 3H, CH2CH3)- CIMS: :'35 (~ I 1), 277 (~ -C4Hg).
Examole l~
~reparation of ,2:3,5-Di-O-~sop-_?ylidene-~ n-nor.~
glucofuranose ar.d 1,2-0-lsooropv' dene-o-û-n-nenyl-~-D-giucofuranose, (IIIb).
Ste~ 1: 1,2:3,5-Di-O-Isopropylidene-6-0-n-nonyl-~-D-glucofuranose (R8 = C9~18) The synthesis of 1,2:3,5-Di-O-isopropylidene-~-D-glucofuranose (as described in U.S. Patent No. 4,996,195) was achieved by reacting pivaloyl chloride with 1,2-0-isopropylidene-~-D-glucofuranose at the 6-position, followed ; by cyclization of 3 and 5-positions with dimethoxypropane and finally hydrolysis of 6-pivaloyl ester. mh-s compound was treated with dry powdered sodium hyd_oxide and nonylbromide by exactly the same procedure as des_r bed ln Exampie "
step 1.
SUBSrlTl)TE SHEET
~: :
~ .
,~ , .
W092/04359 2 0 9 ~ ~ 87 PCT/~S91/06~8 Ste~ 2: ~ydrolysis according ~ the generai ~roceaure described in Example 6 gave the partiaily blockeà
compound 1,2-O-Isopropylidene-6-O-n-nonvl-~-D-glucofuranose, (IIIb).
Exam~le 15 Preparation of ',2:3,5-Di-O-isopropylidene-6-deoxy-6-amino-~-D-glucofuranose and i,2-O-isopropvlidene-~-deoxy-6-amino-~-D-glucofuranose.
SteD 1: Preparation of 1,2:3,5-Di-O-isopropylidene-6-deoxy-6-amino-~-3-glucoruranose.
A mixture or 1,2:3,5-Di-O-isoDro~vliàene-~-tos-v_-~-L-glucofuranose (prepared as descrlbed in ~'.S. Paten- No.
4,996,195) (lOg) and sodium azide (2.5 equivalents) ~n drv dlmethylformamide (100 ml) was hea~ed at 80-qOC fo- 2 hours.
The progress of the reaction was monitored ~y TLC and G~.
After the completion of the reaction, tne D~ was removed under diminished pressure. The res~due was dissolvec in ether (150 ml), washed with water (~ x 50 mi), sodium bica-bonate sol~ ion (1 ~ 50 ml)~ rhe ^-gar. _ 1~YQ~ e_ (~gSO4) and ~he solvent removed. The _-oduc_ 1,2:3,;-di-O-isopropylidene-~-deoxy-6-azido-~-~-glucof~znose so -crmed was found to be > 98% pure by GC, T_~ anc N.MR. 1,2:3,5-di-v-isopropylidene-6-deoxy-6-azido-~-D-glucofuranose was then reduced catalytical}y 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-O-isopropylidene-6-deoxy-5-amino-~-D-glucofuranose was 96%.
Steo 2: Hydrolysis according to the general procedure described in Example 6 gave the partiallv blocke~
- compound 1,2-O-isopropylidene-6-deoxy-5-amlno-~-G-glucofuranose.
Exam~le 15 The general procedure for the preparation oî 6-deoxv-6-amino or 6-amino substituted glucofuranose was t~ reac_ 1,2:',5-di-O-isopropylidene-6-tosyl-~-a-glucofuranose with an appropriately substituted amine (2.2 equivalents), fo~
. . .
-~ SUB~ITUTE SHEEr ., , .
, W092/043~9 2 ~ 9 1 5 ~ ~ PCT/~S91/0~8 example 3-phenyl-1-propylamine (II~j), at 80-90~ -or ~-3 hours. The progress of the reaction was fcllowed Dy T~C and GC. After the completion of the reaction, the product was dissolved in ethylacetate (100 ml),_washed with a saturztec solution of sodium bicarbonate (2x20 ml), brine (lx20 ml), dried (MgS04) and solvent removed. The i,2:3, 5-Di-O-isopropylidene-6-deoxy-6-amino-~'-(pher.yl?ropyl)-~-D-glucofuranose was then purifieà by flash chromatography usinc the appropriate solvent system.
Steo 2: Hydrolysis according to the gener21 procedure described in Example 6 gave the par~ a . ~_ocXed compound I,2-0-IsopropvlLdene-6-deoxy-6-2r.iro-1 -(phenylpropylj-~-D-gl~cofuranoce.
ExamDle 17 Pharmacological Activl~y of Compounds _- ~cnmula Compounds of formula III were tested f~r immunomodulatory and anti-proliferative effects. ResuI s c-these studies are desc-ibed in ..e d2r~ ~abIes r^_oh Compounds (~IIe), (-I_i), ( ~.-. -r.~ ) were -cun_ to inhibi~ the mouse spleen ceIl bias_ogeni^ -espor.se _.-.-dose aependent manner, wlth s~rcns 2ntip~0 _-erati~e e__-c-(greater than -50~ of control) wi~h ail compounds tes~ed (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 (IIIp) sianificantly decreased the proliferative response of splenic T lymphocytes at concentrations ranging from 10 to 7~0 ua/~l. Additional immunomodulatory effec~s of the compounds o_ the p-esent invention were observed in ~R cultures (Table XVII) where compounds (IIId), (IIIe) and (IILi) were demonst-aled to exert strong non dose dependent inhibition of ~ iymphccyte proliferation. Compound (IIIc) was ~ound to exhibit st-ong inhibitory effects cn ~he MLR a- cor.cenlrations -angLr.c frc~
SUBSTITUTE SHEET
WO 92/~359 2 0 9 1 ~ 8 7 PCT/~S91/064~
10 to 300 uM whereas compounds (IIIe) and ~IIId ! were 2c~ e at the highest dose levels only.
Results of the cesting of compounds or the present invention for modulatory effects on mouse peritoneal mac_ophage production are presented ir. ~able XVII T, -L-i activity was demonstrated to be signi~icantly lowereà in cultures containing 2.;, 10, and lOC u~.~m' _- compouni (IIIi). Compound (IIIe) was signif~cantiv inhibitory to I~-' activity at 2.5, 10, 25 and 100 ug~ml. Both compounas appeared to exer~ inhibitory effects cn ~-1 activi y i-. no.
dose dependent manner.
SUBS~ITUTE SHEEl-,, , ~ ~ '. ` ' , ' ~U~l~o ~
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WO 92/04359 PCT/I_:S91/06458 2~91~87 - 6~
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^~ o s ~ ~ v ~ _ 0~ v c SUBSrlTUTE SHEEJ
W092/04359 2 Q ~ 1 5 8 7 pr~T/~s9l/06~s~
( - 67 -The compounds of the present invention also lnclude the following monosaccharides:
- (3S) 1,2-O-isopropylidene-,-D-ribo-hexos-3-ulose-1,4:3,6-difuranose; and c~J (IV) - Methyl 3-O-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside.
C~3 O
N ~~ O- c ~3 (v) C ~/, Exam~le 18 Preparation of 1,2-O-Isopropylidene-,-~-r~bo-hexos-3-uiose-1,4:3,6-difuranose, (IV).
Two grams of 1,2:5,6-di-O-isopropylidene-,~-u-r~bo-hexofuranos-3-ulose were dissolved in tetrahydrofuran (2mLj and cooled to 0 - 5C. Aqueous perchloric acid (2mL, 3Q%) 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 neutralized with a saturated solution of potassium carbona~e and extracted wLth ethyl acetate. Removal of the solvent gave a crude product which was puriied 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 1 (broad OH), No > C = O (stretching). H
NMR (CDC13): ~ 5.97 (d, lH), 4.51 - 4.41 (m, 4H), 4-26 (m, lH), 3.78 (m, lH), 3.07 (d, lH), 1.58 (d, 3H), 1.40 (s, 3H). C NMR (CDC13, P~T): ~ 113.99, 110.93 (C-3 and - SUBSTITU~E SHEET
W092/04359 2 0 9 ~ ~ 8 7 PCT/~Sgl /06~5~
C-9), 106.98, 84.04, 82.78, ,1.07 ~C-1, C-^, C-4, C-5), ?3.5 (C-6), 27.22, 27.18 (C-8, C-9). CIMS: 21q ~ r 1) -Examole 13 -PharmacologLcal Activity of 1,2-O-Isopropylidene~ ribo-hexos-3-ulose-1,4:3,6-difuranose, (IV).
Compund IV was tea~ed in the Bud-8 skin celi fib o~las_-assays as described in Example I2. As can been seen --om Tables XIX, XX and XXI below, the compounds of the inven~i~n produced significant, non dose dependent decreases in the ~ud-8 skin cell fibroblast proliferation in .hose cul~ures that contained 1, 10, 25 and 100 ug/~.l, _espec-ivel~ ner.
compared to the control cultures tha~ did not contain ~ne compounds of the invention. Cyto~oxicily was not obse-ved ar these concentrations of the test com~ound. Cytotoxic effe~s were o~served in the highest concentratLons tested (300 ugimi and 750 ugfml). These anti-proliferative effects co~rola~es with decreases in Bud-8 skin cell f_b-~las. ?rodu_~isn cf PGE2 and LTB4 (~ables XX and XXI) whero signif can~l~
decrease o- levels of bc~. pro-ir. lamm2 __-. me~ a-c-~seen a~ several non-toxic doses o_ com?3un~ ~..
S~BSTITUT~ SH~ET
-, ~0 92/04359 2 0 ~ 1 5 ~ 7 Pcr/~591/o64~
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W092/04359 2 0 3 1 5 8 7 PCT/~S91/064~8 _ 73 -Exam~le 20 Preparation of Methyl 3-0-3 -(N',N -dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside (V).
Ste~ 1: The prepara~ion of 1,2-O-isopropylidene-3-0-3 -(N',N'-dimethylaminopropyl)-6-O-p-toluenesulfonyl-^a-D-glucofuranose, (1).
Ten grams of 1,2-O-isopropylidene-3-C-3 -(N~,N~-dimethylaminopropyl)-^a-D-glucofuranose were dissolved in 30mL of anhydrous pyridine and cooled to 5C in an ice-water bath. p-Toluenesulfonyl 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 altowed to attain ambient temperature 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 carbona~e solution. The combined organic phase was washed with wa~er and brine, dried ove- anhydrous magnesium sulfate and evaporated to give 1~._ g (95%) of the tosylate as a glassy material, that soli- fied on trituration with ether. CIMS: ~60 (M - 1) Ste~ 2: The preparation of 1,2-O-isopropylidene-5-deoxy-3-0-3'-(N',N'-dimethylaminopropyl)-^a-D-glucofuranose, (2).
Ten grams of 1,2-O-Isopropylidene-3-O-3'-(N',N'-dimethylaminopropyl)-6-O-p-toluenesulfonyl-^a-D-glucofuranose (1) were dissolved in 60m^P 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 THF. After the addition was complete, the mixture was refluxed for 2 hours. It was then cooled in an ice bath and the excess lithium alu~inum hydride was quenched by the addition of 2m^P of water, followed by 5m^P of 15~
- sodium hydroxide solution. The mixture was fi'tered through Celile and the filtrate was evaporated to give 5.91 g (94%) of the 1,2-O-isopropylidene-6-deoxy-3-0-3'-(N',N' SUBSrlTUTE SHE~ET
. ~ .
., ` :: . . . `.
, ` ' W092t043~9 PCT/~S9l/06458 2091~7 ~-dimethylaminopropyl)-^a-D-glucofuranose (2) product which was used without purification for the-ne~t s~ep. CIMS: 290 (M + 1) NMR (CDC13): ^w 5.92 (d, lH, H~ .55 (d, lH~ H2), 3.95 (m, 2H, H3 and H4), 3.83 (m, lH, H5), 3.48 lm, 2H, OCH2), 2.28 (s, 3H, CH3), 1.48 and 1.32 (s~ 3H each, C(CH3)2) -Ste~ 3: The prepara~ion of 6-Deoxy-3-0-3'-(N',N'-dimethvl-am~nopropyl)-D-glucopyranose, (3).
Three grams of 1,2-0-isopropylidene-3-0-3'-(N',N'-dimethylaminopropyl)-6-deoxy-^a-D-glucofuranose (2) was dissolved in lOmL of tetrahydrofuran and 5m^~ o 3 ~
hydrochloric acid was added. The mixture was stir-ed for ^
hours at 50C, cooled and neutralized with 20% scc um 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. mhe 6-Deoxy-3-0-3~-(N',N'-dimethylaminopropyl)-D-glucopyranose (3) was used directly for the next step.
Ste~ 4: The preparation of methyl 3-0-3'-(N',N'-dimethylaminopropyl)-6-deoxy-3-gluco?yr2noside, (V) -The 6-Deoxy-3-0-3'-(N',N'-dimethylaminopropyl)-D-glucopyranose (3) (1.8 g) was dissolved in lOmL 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 neutralized with saturated potassium carbonate and extracted several times with dichloromethane. 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 ethyi acetate) to give 1.64 g (86.5%) of the methyl 3-0~3'-~N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside compound. The lH N~R showed the presence of a mixture of ^a and ^b-isomers in the ratio of 6:4. CIMS: 264 (M + 1) -SUE~SrlTlJTE SHEE~T
, : , :
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W092/04359 2 0 3 1 j ~ 7 PCT/~'S91/0S~8 Exam~le 21 PharmacologiCal Activity of ~ethyl 3-0-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside, (v).
Methyl 3-o-3~-~N~N~-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside of this invention has_~emonstrated inhibitory effects on the proliferation of GS-10~-V-20 human skin cell fibroblasts.
A compound that inhibits fibroblast proliferztion has the potential to be utilized as a dermatological drug used to treat chronic dermatorse, such as psoriasis and autolm~une disorders which result in join~ inflamma~ion, such as rheumatoid arthritis. Alsc, an an~i-prol-ferative effecr may well be observed with other tissues, such as those tha~ line the blood vessels, or joints, the uncontrolled prolifera~ on of which produce disease, thereby broadening the scope of potential applications.
Svecific Method: Fibroblast Assav The effect of methyl 3-0-3'-(N',~'-dimethylamir.o-r.-propyl)-6-deoxy-D-glucopyr2noside on the proi _erative capacity or human GS-109-V-20 skin fibroblasts was measured with the use of 3H-thymidine incc-poration assay. Cultu-e~
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. Thess cells were then plated in triplicate at a density of lX103 cells/O.lml/microtiter well. To these cells was added O.lm^P incubation medium containing the methyl 3-0-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside compound.
After 3 days of culture, l~uCi 3H-thymidine was added in 50ul volume to each culture well of the micro~iter plates.
Eighteen hours later, each of the GS-109-V-20 cultures was examined morphologically fcr evidence of drug-induced toxicity such as cell rounding or granularity. The ~r.-thymidine-pulsed cells were then precipitated and the amoun~
SUBSrlTt)TE SHEFI
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W092/04359 pCT/~'S91/06458 209~587 _ 76 -of 3H-thymidine incorporation was counted on a liquid scintillation counter.
Methyl 3-O-3'-(N',N~-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside was suspended directly into the medium by extensive sonication, without being filter-sterilized. A
range of doses of this compound was usled to measure effects of this compound upon GS-109-V-20 cell proliferation. The following doses were used:
Group 1: 0 ug~ml Group 7: 10 ug/ml Group 2: 0.001 ug/ml Group 8: 25 ug/ml Group 3: 0.01 ug/ml Group 9: 100 ug/ml Group 4: 0.1 ug/ml Control G-oups:
Group 5: 1 ug~ml Control: 2X10 5 M Indomethacin Group 6: 2.5 ug/ml Control: 2X10 6 M NDGA
SDecific Method: Incubation Medium The incubation medium used for culturing the GS-109-'Y'-20 cells was RPMI-1640 medium containing 10% fetal bovine serum, 100 ug~ml streptomycin, 100 U/ml peniclllin, 0.2 ~ Hepes buffe- solution, 5x10-5 M 2-mercaptoethano' and ~ m~
glutamine.
Specific M.ethod: Human 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 polyps of the colon (American Type Culture Collection, Catalogue of Cell Lines and Hyb-idomas, -6th Ed., 150, 1988). These cells were selected for use because they are considered to exis~ in an initiateà state, as opposed to being normal or transformed, and have a more extensive population doubling time and sur~ival pe-iod in culture than do normal fibroblasts. In this regard, they would more closely reflect ~he biological characteristics of psoriatic or rheumatoid synovial fibroblasts which also proliferate more extensively than do normal ~i~roblasts, but not as extensively as immortalized transformed tumo_ cells.
SllBSrlTUTE SHEET
W092/04359 ~ 8 ~ P~T/US91/0~58 .
The number of GS-lO9-V-20 cells was expanded for use in the described assays by culture in 25 cm2 flasks at 37C in an atmosphere of 5% CO2 in air. At approximately 4-5 day intervals, or when confluence was reach~d, the cells were passaged. This was accomplished by detaching the cells ~y trypsinization, washing and reseeding the cells at a lower density into fresh tissue culture flasks.
Statistical Anal~sis_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 prolifer2tive abil ties of the skin cells cultured in the presence of methyl 3-C-3'-(N',N'-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 'rable XXII
below.
A significant inhibitory effect was observed in a non-dose-dependent manner for cultures receiving the methyl 3-~-3'-(N',N'-dimethyl2mino-n-propyl)-6-deoxy-D-glucopyr2noslde.
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 exer~s these effects throuh novel mechanisms.
SU8SrlTUTE SHEEr ,, . :
WO92/04359 ~ PCl/US91/064~8~
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SUE~SrlTUTE SHEEr . .
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~W092/04359 2 0 9 1 ~ 8 7 PCT/~:S91/0645~
The compounds of the present invention as shown by formulae I, II, III, IV and V are useful for treating mammals with inflammatory and/or autoimmune disord~rs such as psoriasis, atopic dermatitis, rheuma~oid~ arthritis, ostearthritis, 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 ~o their valuable pharmacological properties, the compounds of the present invention or their physiologically acceptable salts are particularly suitabie 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 hhich comprise an effective amount of at least one compound of the present invention with or without a pharmaceutically accepta~le carrier. If appropriate, compounds contair.ins an amino functionality may be in the form of an acid-add ~ion salt. Preferred acid addition salts are hydrochloric acid salts.
The present invention also encompasses a method of treating animal-~ 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 or an acid-addition salt thereof, with or without a pharmaceutically acceptable carrier. The compositions according to the invention can be adminis~ered orally, topically, rectally, internally, or, if desiredt parenterally. Oral administration is preferred.
Suitable solid or liquid galenical formulations, fo-example are granules, powders, coated tablets, microcapsules, suppositories, -~yrups, elixirs, suspensions, emulsions, drops or injectable solutions. Preparations having a protracted SUElSrITUTE SHEEl .
. .
W0~2/04359 2 0 9 1 5 8 7 PCT/US91/0645~
release of the active compound may also be used. These formulations can also contain additiv~s such as excipients, disintegrants, binders, coating agents, swelling agents, glidants, or lubricants, flavors, sweeteners or solubilizers.
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 ana monohydric 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 cf 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 ln vi~-o testing, the effective amount to achieve a 50~ inhibition of the cultured cells range from about 1 - 200 ug/ml of cul ~re medium, preferably 10 - 100 ug/ml.
SU8STITUTE SHE~E~
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Claims (30)
1. A fructofuranose compound of formula (I):
(I) wherein A is H, methyl or ethyl;
R1 and R2 are H, methyl, ethyl C5-C10 alkenyl or together form an isopropylidene ring;
R3 is H, C5-C10 alkyl, C5-C10 alkenyl, C5-C10 alkynyl, 2-octyne, benzyl, or C5-C10 ester; and R is H, C5-C10 alkyl, C5-C10 alkenyl, C5-C10 alkynyl, 2-octyne, benzyl, or C5-C10 ester.
(I) wherein A is H, methyl or ethyl;
R1 and R2 are H, methyl, ethyl C5-C10 alkenyl or together form an isopropylidene ring;
R3 is H, C5-C10 alkyl, C5-C10 alkenyl, C5-C10 alkynyl, 2-octyne, benzyl, or C5-C10 ester; and R is H, C5-C10 alkyl, C5-C10 alkenyl, C5-C10 alkynyl, 2-octyne, benzyl, or C5-C10 ester.
2. A fructofuranose of formula (I) as claimed in claim 1, wherein A is methyl;
R1 and R2 are each ethyl, cis-2-octane, or together form an isopropylidene ring;
R3 is H, C7H15, cis-2-octene, trans-2-octene, 2-octyne, or octanoyl; and R4 is H, C7H15, cis-2-octene, trans-2-octene, 2-octyne, or octanoyl.
R1 and R2 are each ethyl, cis-2-octane, or together form an isopropylidene ring;
R3 is H, C7H15, cis-2-octene, trans-2-octene, 2-octyne, or octanoyl; and R4 is H, C7H15, cis-2-octene, trans-2-octene, 2-octyne, or octanoyl.
3. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.
4. 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 1.
5. A compound according to claim 1 selected from methyl 1,3-O-isopropylidene-6-O-heptyl-^a-D-fructofuranoside;
methyl 1,3-di-O-ethyl-6-O-heptyl-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4-O-heptyl-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4,6-di-O-(2-octynyl)-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4,6-di-O-(trans,2-octenyl)-^a-D-fructofuranoside;
methyl 1,3-o-isopropylidene-4,6-di-O-(cis,2-octenyl)-^a-D-fructofuranoside;
methyl 1,6-di-O-(cis,2-octenyl)-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4-O-octanoyl-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-6-O-octanoyl-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4,6-di-O-octanoyl-^a-D-fructofuranoside; and 2,3-O-isopropylidene-4-O-heptyl-^b,D-fructofuranose.
methyl 1,3-di-O-ethyl-6-O-heptyl-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4-O-heptyl-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4,6-di-O-(2-octynyl)-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4,6-di-O-(trans,2-octenyl)-^a-D-fructofuranoside;
methyl 1,3-o-isopropylidene-4,6-di-O-(cis,2-octenyl)-^a-D-fructofuranoside;
methyl 1,6-di-O-(cis,2-octenyl)-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4-O-octanoyl-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-6-O-octanoyl-^a-D-fructofuranoside;
methyl 1,3-O-isopropylidene-4,6-di-O-octanoyl-^a-D-fructofuranoside; and 2,3-O-isopropylidene-4-O-heptyl-^b,D-fructofuranose.
6. A pharmaceutical composition comprising a compound according to claim 5 and a pharmaceutically acceptable carrier.
7. 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 5.
8. A compound of formula II
(II) wherein:
x1 is O
and R is C12-C20 alkyl, or CnH2nY, wherein n= 1,2,3 or 4 and Y is selected from cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (C5-C10 alkoxy), CH2CH(CH3)CH2N(CH3)2, CH2CH2N(C5-C10 alkyl)2, or (C3-C7 alkenyl), or X1 is NH
and R5 is C2-C10 alkyl, or CnH2nY wherein n= 1,2,3 or 4 and Y is selected from hydroxy, cyano, pyrrolyl, pyrrolidinyl methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, (CH2)3N(CH3)2, (C5- C10 alkoxy)/ or phenyl;
and R6 and R7 are hydrogen or form an isopropylidene ring.
(II) wherein:
x1 is O
and R is C12-C20 alkyl, or CnH2nY, wherein n= 1,2,3 or 4 and Y is selected from cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (C5-C10 alkoxy), CH2CH(CH3)CH2N(CH3)2, CH2CH2N(C5-C10 alkyl)2, or (C3-C7 alkenyl), or X1 is NH
and R5 is C2-C10 alkyl, or CnH2nY wherein n= 1,2,3 or 4 and Y is selected from hydroxy, cyano, pyrrolyl, pyrrolidinyl methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, (CH2)3N(CH3)2, (C5- C10 alkoxy)/ or phenyl;
and R6 and R7 are hydrogen or form an isopropylidene ring.
9. A compound of claim 8 wherein R6 and R7 together form an isopropylidene ring.
10. A compound of claim 9 wherein saLd compound is a glucofuranose.
11. A compound of claim 9 wherein said compound is an allofuranose.
12. A compound of claim 8 wherein R6 and R7 each hydrogen.
13. A compound of claim 12 wherein said compound is a glucofuranose.
14. A compound of claim 12 wherein said compound is an allofuranose.
15. A compound according to claim 9 selected from 1,2-O-Isopropylidene-3-O-n-dodecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-n-pentadecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-n-octodecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-3'-(phenylpropyl)-^a-D-glucofuranose;
1,2:5,6-Di-O-isopropylidene-3-O-3'-(morpholinylpropyl)-^a-D-ylucofuranose;
1,2:5,6-Di-O-isopropylidene-3-O-3'-n-propoxy-n-heptyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-3'-n-propoxy-n-heptyl-^a-D-glucofuranose;
1,2:5,6-Di-O-isopropylidene-3-O-2'-(ethylpyrrolidyl)-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-2'-(ethylpyrrolidyl)-^a-D-glucofuranose;
1,2-O-isopropylidene-3-O-3'-(propan-1'-ol)-^a-D-glucofuranose;
1,2:5,6-Di-O-isopropylidene-3-deoxy-3-amino-(3'-hydroxy-n-propyl)-^a-D-glucofuranose;
1,2-O-isopropylidene-3-O-3'-(N',N'-dimethylamine-n-propyl)-^a-D-allofuranose;
1,2:5,6-Di-O-isopropylidene-3-O-3'-(phenylpropyl)-^a-D-allofuranose;
1,2-O-Isopropylidene-3-deoxy-3-N-3'(N',N'-dimethylamino-n-propyl-^a-D-allofuranose;
1,2-O-Isopropylidene-3-deoxy-3-N-3'-(phenylpropyl)-^a-D-allofuranose;
1,2-O-Isopropylidene-3-deoxy-3-amino-heptyl-^a-D-allofuranose;
1,2-O-isopropylidene-3-deoxy-3-amino-2'-(propan-1'-ol)-^a-D-glucofuranose;
1,2-O-isopropylidene-3-deoxy-3-amino-n-heptyl-^a-D-glucofuranose; and 1,2-O-Isopropylidene-3-deoxy-3-amino-3'-(phenylpropyl)-^a-D-glucofuranose.
1,2-O-Isopropylidene-3-O-n-pentadecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-n-octodecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-3'-(phenylpropyl)-^a-D-glucofuranose;
1,2:5,6-Di-O-isopropylidene-3-O-3'-(morpholinylpropyl)-^a-D-ylucofuranose;
1,2:5,6-Di-O-isopropylidene-3-O-3'-n-propoxy-n-heptyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-3'-n-propoxy-n-heptyl-^a-D-glucofuranose;
1,2:5,6-Di-O-isopropylidene-3-O-2'-(ethylpyrrolidyl)-^a-D-glucofuranose;
1,2-O-Isopropylidene-3-O-2'-(ethylpyrrolidyl)-^a-D-glucofuranose;
1,2-O-isopropylidene-3-O-3'-(propan-1'-ol)-^a-D-glucofuranose;
1,2:5,6-Di-O-isopropylidene-3-deoxy-3-amino-(3'-hydroxy-n-propyl)-^a-D-glucofuranose;
1,2-O-isopropylidene-3-O-3'-(N',N'-dimethylamine-n-propyl)-^a-D-allofuranose;
1,2:5,6-Di-O-isopropylidene-3-O-3'-(phenylpropyl)-^a-D-allofuranose;
1,2-O-Isopropylidene-3-deoxy-3-N-3'(N',N'-dimethylamino-n-propyl-^a-D-allofuranose;
1,2-O-Isopropylidene-3-deoxy-3-N-3'-(phenylpropyl)-^a-D-allofuranose;
1,2-O-Isopropylidene-3-deoxy-3-amino-heptyl-^a-D-allofuranose;
1,2-O-isopropylidene-3-deoxy-3-amino-2'-(propan-1'-ol)-^a-D-glucofuranose;
1,2-O-isopropylidene-3-deoxy-3-amino-n-heptyl-^a-D-glucofuranose; and 1,2-O-Isopropylidene-3-deoxy-3-amino-3'-(phenylpropyl)-^a-D-glucofuranose.
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 effective amount of the compound according to claim 9.
18. A pharmaceutical composition comprising a compound according to claim 15 and a pharmaceutically acceptable carrier.
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:
(III) wherein x2 is O, R8 is C8-C20 alkyl, or CnH2nY, wherein n= 1,2,3, or 4 and Y is selected from phenyl, cyano, pyrrolyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, p?azolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (C5-C10 alkoxy), NH or N(CH3)2;
or x2 is NH
and R8 is H, or CnH2nY, wherein n= 1,2,3 or 4 and Y is selected from OH, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2 or (C5-C10 alkoxy) or phenyl;
or x2 is S
and R8 is C5-C10 alkyl, or CnH2nY wherein n= 1,2,3 or 4 and Y
is selected from OH, phenyl, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isooxazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2 or (C5-C10 alkoxy);
and R9 and R10 are hydrogen or form an isopropylidene group.
(III) wherein x2 is O, R8 is C8-C20 alkyl, or CnH2nY, wherein n= 1,2,3, or 4 and Y is selected from phenyl, cyano, pyrrolyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, p?azolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2, (C5-C10 alkoxy), NH or N(CH3)2;
or x2 is NH
and R8 is H, or CnH2nY, wherein n= 1,2,3 or 4 and Y is selected from OH, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isoozazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2 or (C5-C10 alkoxy) or phenyl;
or x2 is S
and R8 is C5-C10 alkyl, or CnH2nY wherein n= 1,2,3 or 4 and Y
is selected from OH, phenyl, cyano, pyrrolyl, pyrrolidinyl, methylpyrrolidinyl, pipecolinyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, oxazolyl, oxazolidinyl, isooxazolyl, isooxazolidinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, O(CH2)3N(CH3)2 or (C5-C10 alkoxy);
and R9 and R10 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 where:in said compound is an allofuranose.
23. A compound according to claim 13 selected from 1,2-O-Isopropylidene-6-O-heptyl-^a-D-glucofuranose;
,2-O-Isopropylidene-6-O-nonyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-dodecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-pentadecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-3'-(phenylpropyl)-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-3'-(N',N'-dimethylamino-n-propyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-isopropylidene-6-O-methoxyoctyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-propionitrile-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-amino-[2'-aminoethyl-2"-(N'-methylpyrrolidyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-amino-3'-(phenylpropyl)-^a-D-glucofuranase;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-N-(N'-propylpipecolinyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-amino-ethoxyethanol-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-deoxy-6-amino-3'-(propan-1'-ol)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene 6-deoxy-6-thio-n-heptyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-deoxy-6-thio-n-heptyl-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-thio-2'-(ethylpyrrolidyl)-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-deoxy-6-thio-2'-(ethylpyrrolidyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-thio-3'-(N',N'-dimethyl-amino-isobutyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-thio-3'-(propan-1'-ol)-^a-D-glucofuranose; and 1,2-O-Isopropylidene-6-deoxy-6-thio-3'-(phenylpropyl)-^a-D-glucofuranose.
,2-O-Isopropylidene-6-O-nonyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-dodecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-pentadecyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-3'-(phenylpropyl)-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-3'-(N',N'-dimethylamino-n-propyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-isopropylidene-6-O-methoxyoctyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-O-propionitrile-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-amino-[2'-aminoethyl-2"-(N'-methylpyrrolidyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-amino-3'-(phenylpropyl)-^a-D-glucofuranase;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-N-(N'-propylpipecolinyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-amino-ethoxyethanol-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-deoxy-6-amino-3'-(propan-1'-ol)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene 6-deoxy-6-thio-n-heptyl-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-deoxy-6-thio-n-heptyl-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-thio-2'-(ethylpyrrolidyl)-^a-D-glucofuranose;
1,2-O-Isopropylidene-6-deoxy-6-thio-2'-(ethylpyrrolidyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-thio-3'-(N',N'-dimethyl-amino-isobutyl)-^a-D-glucofuranose;
1,2:3,5-Di-O-Isopropylidene-6-deoxy-6-thio-3'-(propan-1'-ol)-^a-D-glucofuranose; and 1,2-O-Isopropylidene-6-deoxy-6-thio-3'-(phenylpropyl)-^a-D-glucofuranose.
24. A pharmaceutical composition comprising a compound according to claim 20 and a pharmaceutically acceptable carrier.
25. A method of treating an animal or human suffering from an inflammatory and/or autoimmune disorder comprising administer?g thereto an effective amount of the compound according to claim 20.
26. A pharmaceutical composition comprising a compound according to claim 23 and a pharmaceutically acceptable carrier.
27. 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 23.
28. A compound selected from (3S) 1,2-O-isopropylidene-^a-D-ribo-hexos-3-ulose-1,4:3,6-difuranose; and Methyl 3-O-3'-(N',N'-dimethylamino-n-propyl)-6-deoxy-D-glucopyranoside.
29. A pharmaceutical composition comprising a compound according to claim 28 snd a phsrmaceutically 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.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US58154290A | 1990-09-12 | 1990-09-12 | |
US581,542 | 1990-09-12 | ||
US75781791A | 1991-09-11 | 1991-09-11 | |
US757,817 | 1991-09-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2091587A1 true CA2091587A1 (en) | 1992-03-13 |
Family
ID=27078356
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002091587A Abandoned CA2091587A1 (en) | 1990-09-12 | 1991-09-12 | Monosaccharides having anti-proliferation and anti-inflammatory activity, compositions and uses thereof |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0548226A1 (en) |
JP (1) | JPH06503813A (en) |
AU (1) | AU8622991A (en) |
CA (1) | CA2091587A1 (en) |
IE (1) | IE913228A1 (en) |
IL (1) | IL99454A0 (en) |
WO (1) | WO1992004359A2 (en) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5360792A (en) * | 1991-12-20 | 1994-11-01 | Greenwich Pharmaceuticals Incorporated | Anti-proliferative and anti-inflammatory compounds: 5- or 6-deoxy hexose monosaccharides having a saturated nitrogen-containing heterocycle at the 5- or 6-position bound through the nitrogen atom |
FR2694753B1 (en) * | 1992-07-09 | 1994-12-23 | Picardie Jules Verne Universit | Processes for the specific synthesis of new aminothioethers by substitution of hydroxylated sites on mono or polyhydroxylated molecules, products obtained by these processes and their applications. |
US5432163A (en) * | 1992-11-13 | 1995-07-11 | Greenwich Pharmaceuticals Incorporated | Anti-proliferative and anti-inflammatory compounds: derivatives of pentose monosaccharides |
CA2164827A1 (en) * | 1993-06-11 | 1994-12-22 | David S. Thomson | Immunomodulatory, anti-inflammatory, and anti-proliferative compounds: 5,6-dideoxy, 5-amino derivatives of idose and 6-deoxy, 6-amino derivatives of glucose |
US20090048186A1 (en) * | 2005-04-19 | 2009-02-19 | Vishwajanani Jitendra Sattigeri | Monosaccharide Derivatives as Anti-Inflammatory and/or Anti-Cancer Agents |
EP1842855A3 (en) * | 2006-03-29 | 2007-12-05 | Ranbaxy Laboratories Limited | Monosaccharide derivatives as anti-inflammatory and/or anti-cancer agents |
EP1864992A2 (en) | 2006-05-30 | 2007-12-12 | Ranbaxy Laboratories, Ltd. | Monosaccharide derivatives |
EP1953170A1 (en) * | 2006-10-03 | 2008-08-06 | Ranbaxy Laboratories Limited | Monosaccharide derivatives as anti-inflammatory agents |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB1589916A (en) * | 1976-11-16 | 1981-05-20 | Prodotti Antibiotici Spa | Glucofuranose derivatives |
JPS55154991A (en) * | 1979-05-23 | 1980-12-02 | Hisamitsu Pharmaceut Co Inc | Beta-d-fructopyranoside derivative |
US4735934A (en) * | 1981-02-17 | 1988-04-05 | Greenwich Pharmaceuticals Incorporated | Method of therapeutically treating a warm blooded animal afflicted with an autoimmune disease |
US4996195A (en) * | 1989-01-09 | 1991-02-26 | Greenwich Pharmaceuticals Inc. | Derivatives of α,D-glucofuranose or α,D-allofuranose and intermediates for preparing these derivatives |
US5010058A (en) * | 1989-06-22 | 1991-04-23 | 501 Greenwich Pharmaceuticals Incorporated | 3,5,6-substituted derivatives of 1,2-O-isopropylidene-α,D-glucofuranose and intermediates for preparing these derivatives |
-
1991
- 1991-09-12 WO PCT/US1991/006458 patent/WO1992004359A2/en not_active Application Discontinuation
- 1991-09-12 AU AU86229/91A patent/AU8622991A/en not_active Abandoned
- 1991-09-12 CA CA002091587A patent/CA2091587A1/en not_active Abandoned
- 1991-09-12 IE IE322891A patent/IE913228A1/en unknown
- 1991-09-12 EP EP91916966A patent/EP0548226A1/en not_active Withdrawn
- 1991-09-12 IL IL99454A patent/IL99454A0/en unknown
- 1991-09-12 JP JP3515897A patent/JPH06503813A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO1992004359A3 (en) | 1992-12-10 |
EP0548226A1 (en) | 1993-06-30 |
AU8622991A (en) | 1992-03-30 |
IE913228A1 (en) | 1992-02-25 |
WO1992004359A2 (en) | 1992-03-19 |
IL99454A0 (en) | 1992-08-18 |
JPH06503813A (en) | 1994-04-28 |
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