Title of Invention

"A PROCESS FOR THE PREPARATION OF CYCLIC ACETALS OF GLYOCOSIDES OF PODOPHYLLOTOXIN

Abstract

A process for the preparation of cyclic acetals of glycosides of podophyllotoxin of general formula III of the drawing accompanying this specification where in R=H, CH3and R - alkyl, heterocyclo alkyledene which comprises reacting glycosides of podophyllotoxin of general formula II wherein R=H or CH in an organic solvent with a compound like alkylor heterocyclo alkyledene having aldehyde group in presence of a lewis acid catalyst such as herein described at a temperature in the range of 15-3 0oC under inert atmosphere for a period in the range of 4-6 hr, recovering the compound of general formula III by known methods.

Full Text

This invention relates to a process for the Preparation of Cyclic Acetals of glycosides of Podophyllotoxin. Particularly it relates to a process for the synthesis of acetals of glycosides of podophyllotoxin of general formula III of the drawing accompanying this specification where in R=H or CH3 R'=alkylor hetero cyclo alkyledene. More particularly it relates to a preparation of podophyllotoxin -4-O-(4",6"-O-α- fur-furylidene-S -D-glucopyranoside) of the formula(8), 4'-demethylpodophyllotoxin-4-O-(4",6"-O-α-furfurylidene-ß-D-glucopyranoside of the formula(9), podophyllotoxin-4-0-(4",6"-O-isobutyrylidene -ß-D-glucopyranoside) of the formula (10) and 4'-demethylpodophyllotoxin-4-O-(4",6"-O-isobutyrylidene-ß-D-glucopyranoisde) of the formula (11) . The podophyllotoxins are a particular class of natural products which have found extensive usage in the design and synthesis of potential anticancer agents based upon natural product prototype. They have long and fascinating history as medicinals. The aryltetralin lignans have acquired.considerable importance because of their cytotoxic and antitumor activity, De Swet. P.A.G.M.et al . [Adverse effects of herbal Drugs, Vol.2.Springer Verlog Berlin Heildelberg, 1993 ]. Podophyllotoxin and some other structurally closely related lignan & lignan glucosides isolated from roots\rhizomes of P.emodi \ P.hexandrum exert a power- ful and specific inhibition of mitosis. Evaluated with systemic application as tumor damaging agents these natural products failed to act satisfactarily in clini¬ cal trials due to non specific side effects. However the lignan podophyllotoxin present in podophyllum species has been chemically modified to produce the clinically useful drugs namely etoposide [4'-demethyie- pipodophyllotoxin-4 -O- (4",6"-O-ethylidene-i3-D- glucopyranoside)] of the formula 1 and teniposide [ 4'- demethylepipodophyllotoxin -4- 0- (4",6"-O-α- thenylidene- ß-D-glucopyranoside)] of the general formula 1 where in R=H or CH3 and R'=(Formula Removed)or CH3. It has been shown by Stahelin [Planta Medica,22,336(1972), Eur.J.Cancer 6, 303(1970), ibid 9, 215 (1973), ibid 12., 925 (1976)] that etoposide (VM-26) and teniposide (VP-16-273) at low concentrations (e.g. 0.005-0.01 fig/ml) apparently prevent cells from enter¬ing mitosis and arrest cells in the S or G2 phase of cell cycle, Grieder [ Cancer Res.,34, 1788 (1974) ] ; Huang [ Cancer Res.33, 3121(1973) ], Krishnan [ J.Cell.Bio. ,-66, 521 (1975)]; Loike [Biochemistry 15, 5435, 5443 (1976) ]. The class of 4' -demethylated epipodophyllotoxin examplified by clinical prototypes etoposide (VM-26) and teniposide (VP-16-273) have emerged into the forefront of antitumour drug develop¬ment . Scattered early attempts by largely unsuccessful use of podophyllotoxin to treat human neoplasia were summarized by Kelly and Hartwell [ J.Nat 1.Cancer Inst.,14., 967 (1954) ]. The glucosides of podophyllo¬toxin and 4'demethylpodophyllotoxin were found (by workers-at Sandoz Laboratories Basel, Switzerland), to generally inhibit the growth of cell culture as well as experimentally induced tumors in mice: Emmenegeer et al., [ Arzneim, Forsch 11, 327, 459 (1961) ] reported that in vitro the glucosides were found to be 10-10 times less active than their corresponding aglycones, but only 5-20 times less active against Ehrlich's murine ascites tumors. In dose displaying pronounced cytostatic effect, the podophyllotoxin glucosides did not produce the noxious side effects, such as nausea, vomitting, diarrhoea and damage to normal tissue as compared to their corresponding aglycones. However, these glucosides did not act satisfacto¬rily in clinical trials because of non-specific side effects. Kuhn and von Wartburg [ Helv. Chim. Acta.,. 51, 163 (1968) ibid; 51, 1631 (1968)] developed a new procedure for the synthesis of previously unknown glucosides of epipodophyllotoxin type. They also ob¬served that when podophyllotoxin is glucosidated under same conditions inversion of configuration at C-4 of the aglycone moiety occurs leading to epi glucoside. This is presumed to proceed through a common carbonium ion at C-4 by Lewis acid with subsequent substitution by the pyranose group from the less hindered side to give exclusively the epi derivative and reaction was found to be useful for synthesis of glucosides of epipodophyllotoxin as well as 4'-demefehyl-epipodophyl-lotoxin series. The first step of the method reported in litera¬ture (glucosidation with BF3 etherate) gives exclusive¬ly epi isomer thus altering the stereochemistry of the molecule at C-4. The synthesis of cyclic acetals and ketals was achieved; Juslen,C.K. [ J.of Medicinal Chemistry, 14,936 (1971) ] by reaction of the glucoside of epipodophyllotoxin-4-O-ß-D-glucopyranoside or 4'-demethyl-epipodophyllotoxin-4-O- ß'-D-glucopyranoside with the corresponding carbonyl compounds in the presence of Lewis acids such as ZnCl2, BF3 etherate, AlCl3, ion exchange resin. The carbonyl compounds could also be replaced by their acetals or ketals. The con¬densation reaction took place generally with the -OH groups at C-4 and C-6 of the hexapyranose moiety. In the case of aldehydes the formation of two stereoiso¬mers, differing from another in the configuration of newly introduced asymmetric C, is to be expected. However, isomer with an equatorial bond of the aldehyde residue predominates almost exclusively; the isomer with an axial substituent is produced only in minimal amounts and is usually lost during the purification of the main reaction product. The actual configuration of sugar acetals of this type could be recognised espe¬cially with acetals of aromatic aldehydes, by a charac¬teristic chemical shift of the axial proton in the nmr spectrum Bagget,N. [Chemistry & Industry (London), 1832 (1964) ] . In our copending application No.1457/DEL/95 dated 4-8-95 we have described a method for the isolation of glycosides of podophyllotoxin of general formula II where in R=CH3 or H [podophyllotoxin-4-O-ß-D-glucopyra- noside and 4'-demethylpodophyllotoxin- 4-O-ß-D-glucopy- ranoside] from roots/rhizomes of Podophyllum emodi. The mixture was separated on silica gel column by elution with chloroform-methanol 90:10 to yield podophyllotox- in-4-O-ß-D-glucopyranoside(3)followed by elution with chloroform-methanol 85:15 to yield 4'-demethylpodo- phyllotoxin-4-O-ß-D-glucopyranoside(4).In our copending application No.1620/DEL/94 dated 14.12.1994 we have described a method to convert glucosides of general formula II to stereoisomers of etoposide methyl ether of general formula I such as, etoposide and teniposide viz. podophyllotoxin-4-O-(4",6"-O-ethylidene -ß-D- glucopyranoside) where R=R'=CH3; 4'-demethylpodophyllo¬ toxin- 4-O-(4",6"-O-ethylidene-ß-D-gluco-pyranoside), where R = H and R"=CH3 and 4'-demethylpodophyllotoxin-4-O-(4" , 6"-O-α-thenylidene -6-D- gluco-pyranoside), where R=H & R' = In this patent we have described a method to convert glucosides (II) to the acetals of formula III (Formula Removed) and the cytotoxity has been reported for these com¬pounds . The object of the present invention is to provide a process for the preparation of cyclic acetals of glycosides of Podophyliotoxin. Accordingly the present invention provides a process for the preparation of cyclic acetals of glyco¬sides of Podophyliotoxin of general formula III of the drawing accompanying this specification where in R=H, CH3 and R'=alkyl, heterocyclo alkyledene which com¬prises reacting glycosides of podophyliotoxin of gener¬al foirmula II wherein R=H or CH3 in an organic solvent with a compound like alkyl or heterocyclo alkyledene having aldehyde group in presence of a lewis acid catalyst such as herein described at a temperature in the range of 15-30°C under inert atmosphere for a period in the range of 4-6 hr, recovering the compound of general formula III by known methods. In an embodiment of the present invention glyco¬sides of Podophyllotoxin used may be such as podophyl-lotoxin-4-O--D-glucopyranoside, 4'-demethylpodophyllo¬toxin 4-O- -D-glucopyranoside. In another embodiment of the present invention the organic solvent used may be such as nitromethane, acetonitrile, dimethylsulphoxide. In still another embodiment of the present inven¬tion the lewis acid catalyst used may be such as p-toulene sulphonic acid, BF3-ehterate, ZnCl2, AlCl3. In yet still another ambodiment of the present invention the compound having aldehyde group used may be such as furfuraldehyde, isobutyraldehyde. The present invention provides a simple process for the synthesis of cyclic acetals from natu¬rally occuring glucosides of podophyllotoxin and 4'-demethylpodophyllotoxin such as podo-phyllotoxin -4-O-(4",6"-O-α- furfurylidene- -D-glucopyranoside), 4'-demethylpodophyllotoxin -4 -O- (4",6"-O-α-furfurylidene - -D-glucopyranoside, podo-phyllotoxin-4-O-(4",6"-isobutyrylidene- -D-glucopyranoside) and 4'-demethyl-4-O-(4" , 6"-O-isobutyrylidene- -D-glucopyranoisde) . Synthesis of these compounds has been achieved by the condensation reaction of the glucosides of podo¬phyllotoxin and 4'-demethylpodophyllotoxin respec¬tively with the apropriate liquid alicyclic or heterocyclic aldehydes. The above glucosides were suspended in excess of carbonyl compound and stirred after the addition of lewis acid as catalyst at room temperature under nitrogen atmosphere. Usual workup and purification over silica gel and elution with chloroform methanol mixture yielded the pure product. Accordingly the present invention provides a process for the synthesis of podophy11otoxin -4-O-{4",6"-o-furfurylidene-ß-D-glucopyranoside} ,4'-demethyl podophyllotoxin-4-O- {4" , 6" -O-ar-furfurylidene--D-glucopyranoside}, podophyllotoxin-4-O-{4",6"-O-isobutyrylidene -ß-D-glucopyranoside} and 4'-demethylpodo phyllotoxin - 4-O-{4",6"-O-isobutyryli-dene- -D-glucopyranoside}. Synthesis of these com¬pounds has been acheived by the condensation reaction of the glucosides of podophyllotoxin and 4'-demethyl-podophyllotoxin respectively with the appropriate liquid acyclic or heterocyclic aldehydes. The above glucosides were suspended in excess of carbonyl com¬pound and stirred after the addition of lewis acid as catalyst at room temperature under nitrogen atmosphere. Usual workup and purification over silica gel and elution with chloroform methanol mixture yielded the pure product. Accordingly the present invention describes in detail a process for the synthesis of podophyllotoxin -4-O-(4", 6"-O- furfurylidene-JS-D-glucopyranoside) , 4'-demethyl-podophyllotoxin -4-O- (4", 6" -O-α-furfurylidene- -D-glucopyranoside, podophyllotoxin -4-O-(4",6"-O-isobutyrylidene -ß-D-glucopyranoside) and 4'-demethyl -4-O-(4",6"-O-isobutyrylidene -ß-D-glucopyranoisde) respectively. The reaction is conducted by the conden¬sation of glycosides of podophyllotoxin of general formula II with furfuraldehyde or isobutyraldehyde in the presence of p-toulene sulfonic acid or a lewis acid like anhydrous zinc chloride, boron triflouride ether-ate, anhydrous aluminium chloride in an inert atmos¬phere and an organic solvent, passing the reaction over silica gel and an organic solvent, passing the reaction mixture over silica gel and eluting with a mixture of chloroform: methanol. The inert atmosphere may be maintained by using inert gases like nitrogen, argon and the like. The lewis acids which have been used to catalyse the reaction are p-toulene sulphonic acid, anhydrous zinc chloride, boron triflouride etherate, anhydrous aluminium chloride and ion exchange resin (Dowex 50) . The mixture of solvents used for elution may be selected from chloroform:methanol , chloroform:ethyl acetate, chloroform:acetone and the like. In a preferred embodiment of the present invention the novel feature lies in the process for the synthesis of hitherto unknown series of probable anticancer com pounds. Yet another feature of the invention, which has been described in detail in the examples provided below, lies in the approach for the synthesis of final product which is simple and yields are quite high, and therefore economically viable. The examples are given by way of illustration and should not be construed to limit the scope of the invention. Example 1 Podophyllotoxin-4-O- (4", 6"-O-ae-furfurylidene- -D-glu-copyxanoside) Powdered podophyllotoxin-4-O- -D-glucopyranoside (3) 1.0 g suspended in furfuraldehyde (8ml). to this suspension added anhydrous zinc chloride (0.5g) and the mixture was stirred under nitrogen atmosphere for 6 hr. The solution was diluted with chloroform ' (100ml) and washed with water (3x25 ml). The organic layer was dried over anhydrous sodium sulphate followed by dis¬tillation under vacuum to yield semi solid mass. The crude mass was dissolved in chloroform (50 ml) and diluted with dry pet. ether (150 ml). A white solid precipitated which was filtered out. The solid was further purified using column chromatography on silica gel by eluting with chloroform:methanol (97:3). The pure fractions obtained were cooled and crystallised from chloroform/hexane to yield white crytalline solid (650mg)m.p. 173-74; [α] D - 64.6 (c, 0.5, CHCl3); IR (KBr) 3432 (OH); 1768( -lactone); 1590, 1504, 1480(arom. C=C). Example 2 4' -Demethylpodophyllotoxin-4-O- (4" , 6"-O-ar-furfuryli-dene- -D-glucopyranoside) . Powdered 4'-demethylpodophyllotoxin-4-O- -D-glu-copyranoside (3) 0. 5g suspended in furfuraldehyde (8ml). To this suspension added anhydrous zinc chloride (0.5g) and the mixture was stirred under nitrogen atmosphere for 6 hr. The solution was diluted with chloroform (75ml) and washed with water (3x15 ml) . The organic layer was dried over anhydrous sodium sulphate followed by distillation under vacuum to yield semi solid mass. The crude mass was dissolved in chloroform (35 ml) and diluted with dry pet. ether (150 ml) . A white solid precipitated which was filtered out. The solid was further purified using column chromatography on silica' gel by eluting with chloroform:methanol (95:5). The pure fractions obtained were cooled and crystallised from chloroform/hexane to yield white crytalline solid (350mg)m.p. 185-86; [α]D - 70.0 (c, 0.5, CHC13) ; IR (KBr) 3420 (OH); 1764 ( -lactone); 1585, 1500, 1485 Carom. C=C) . Example 3 Podophyllotoxin-4-O-(4', 6'-O-isobutyxylidene- -D-glu-copyranoside) Powdered podophyllotoxin-4-O- -D-glucopyranoside (3) 1.0 g suspended in freshly distilled isobutyralde-hyde (7ml) . To the suspension added freshly fused zinc chloride (0.5g) and the mixture was stirred under nitrogen atmosphere. The reaction was monitored by TLC. After eight hours the reaction was diluted with chloro¬form (100 ml) and washed with water (4x25 ml). The chloroform layer was dried over anhydrous sodium sul¬phate followed by distillation of solvent under reduced pressure. The mass thus obtained was dissolved in chloroform and diluted with pet. ether which yielded white compound which was further purified through column chromatography on silica gel to give white solid (lg) ,m.p.175-76°C; [α]D-59 (C,0.5 CHC13).IR (KBr) 3 44 0 (OH); 17 70 ( -lactone); 1590, 1504, 1499, 1482(arom. C=C). Example 4 4 ' -demethylpodophyllotoxin-4 -O- (4' , 6 " -O-isobutyryli- dene- -D-glucopyranoisde) To a suspension of 4'-demethylpodophyllotoxin-1-O-S-D-gluco-pyranoside (3) 0.7 g in freshly distilled isobutyraldehyde (8ml) added freshly fused zinc chlo¬ride (0.5g) and the mixture was stirred under nitrogen atmosphere. The reaction was monitored by TLC. af-ter 72 hr the reaction was diluted with chloroform (100ml) and washed with water (4x25 ml). The chloroform layer was dried over anhydrous sodium sulphate followed by the distillation of the solvent under reduced pressure. The mass thus obtained was dissolved in chloroform and diluted with hexane. The white compound thus obtained was again purified over silica gel to yield white crystalline solid (11) 300mg, m.p. 168-170°C. [α]D 67.4 (C, 0.5 CHC13) IR (KBr) 3488 (OF!) ; 1768 ( -lactone); 1608, 1600, 1504, 1480 (arom. C=C). Biological Activity The glucoside acetals of podophyllotoxin offer remarkable examples of cytostatic activity. In par¬ticular 4'-demethylpodophyllotoxin-4-O-(4",6"-O-isobut-yrylidene-ß-D-glucopyranoisde) has shown selective cytotoxity for colon cancer sub panel HT2 9. A portion of screening data availble for different glucosides acetals are tabled in Table 1. Advantages of the present invention 1. The present invention gives a new process for the synthesis of hitherto unknown series of probable anti¬ cancer compounds. 2. The approach for the synthesis of, final product is simple and yields are quite high,and therefore economi¬cally viable 3. It is a single step process and eliminates a se¬quence of complicated reactions in which there is lowering of yield at every step. 4. The starting compounds for the synthesis of these anticancer novel acetals have been isolated from marc which is a waste material of podophyllotoxin by extrac¬ tion process. We claim : 1. A process for the preparation of cyclic acetals of glycosides of Podophyllotoxin of general formula III of the drawing accompanying this specification where in R=H, CH3 and R'=alkyl, heterocyclo al-kyledene which comprises reacting glycosides of podophyllotoxin of general formula II wherein R=H or CHo in an organic solvent with a compound like atlkylor heterocyclo alkyledene having aldehyde group in presence of a lewis acid catalyst such as herein described at a temperature in the range of 15-30°C under inert atmosphere for a period in the range of 4-6 hr, recovering the compound of gener¬al formula III by known methods. 2. A process as claimed in claim 1 wherein glycosides of Podophyllotoxin used is such as podophyllotox-in-4-O-ß-D-glucopyranoside, 4'-demethylpodophyllo-toxin 4-0- -D-glucopyranoside. 3. A process as claimed in claim 1-2 wherein the inert atmosphere is maintained by using inert gases like nitrogen & argon. 4. A process as claimed in claims 1-3 wherein the organic solvent employed is such as nitromethane, acetonitrile, dimethyl sulphoxide. 5. A process as claimed in claims 1 to 4 wherein the lewis acid catalyst used are para toulene sulphon-ic acid, zinc chloride, anhydrous aluminium chlo¬ride and other lewis acids. 6. A process as claimed in claims 1-5 whwerein the alkyl or heterocyclo alkyledene containing alde¬hyde group used is furfuraldehyde, isobutyralde-hyde. 7. A process for the preparation of cyclic acetals of podophyllotoxin of general formula III as defined in claim 1 substantially as herein described with reference to the examples and the drawings accom¬panying the specifications.

Documents:

2595-del-1997-abstract.pdf

2595-del-1997-claims.pdf

2595-del-1997-complete specification (granted).pdf

2595-del-1997-correspondence-others.pdf

2595-del-1997-correspondence-po.pdf

2595-del-1997-description (complete).pdf

2595-del-1997-drawings.pdf

2595-del-1997-form-1.pdf

2595-DEL-1997-Form-2.pdf

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