Note: Descriptions are shown in the official language in which they were submitted.
Wo 93/ 111 50 P~/ US92/0863X
21236~
-1 -
METHOD FOR MAKING STEROIDAL PERACYL GLYCOSIDES
Backqround of the Invention
The present invention relates to processes for the synthesis of steroidal
glycosides, and particularly to the preparation of st~roidal peracyl glycosides ~sed as
intermedi.~es therein.
Tigogenin beta-O-cellobioside is a known compound having utility in the
treatrnent of hypercholesterolemia and atherosclerosis (Malinow, U.S. Patents 4,602,003
and 4,602,005; Malinow e~ ai., Stero;ds, vol. 48, pp. 197-211, 1986). Each patent
discloses a different synthesis of this compound from alpha-D-cellobiose octaacetate;
the first via ~he ~Iycosyl bromide heptaacetate which is couplecl with tigo~enin in the
presence of silver carbonate, and finally hydrolyzed; and the second via direct stannic
chloride catalyzed coupling of the cellobiose octaacetate with tigogenin in methylene
chloride, again foliowed by hydrolysis. In Malinow et al., reaction of collobiose
octaacetate with titanium tetrabromide gave the cellobiosyl brornide heptaacetate, which
was coupled with tigogenin by means of mercuric cyanide, and then hydrolyzed. All
of these methods have sorious drawbacks for producing bulk material to be used as
a pharmaceutica! drug. A desir~ble goa!, met by the present invention, has been to
devise synthetic methods which avoid toxic and/or expensive reagents, and which
cleanly produce the desired tigogenin beta-O~cellobioside, avoiding tedious and
expensive pur^dication steps.
Schmiclt, Angew~ Chem. lnt. Ed. Engl., vol~ 25, pp~ 212-235 (1~86) has reviewed
the synthesis ~and reactions of O-glycosyl trichloroacetimida~es Sormed by the rea~tion
of ~.ugars possessîng a 1-hydroxy group (but wîth other hydroxy groups protected, e~g.,
by benzyl or acet~l) with trichloroacetonitrile in the presen~e o~ a base~ There is
preterential formatîon of the alpha-anomer when sodium hydride is used as base, and
pr~erentialformationofthebeta-anornerwhenthebaseispotassiumcarbonat~. The
alpha anomer of t~rabenzy591ucosyl trichloroacetimi'date when coupled with cholesterol
gave anomeric mi)çtures which~varied with~ catalyst (p-toluenesulfonic acid or boron
trifluoride etherate) and temperature (~0 to ~+20 C~. On the other hand, both the alpha
and beta anomers of tetraacetyigluoosyl analog reporteclly yield exclusively beta-
anomPric products.;
Thus, there has been a eontinuing search in this iield of art for irnproved
methods of stereocontrolled syntheses of steroidal glycosides.
WO 9~/1 1 15(~ PC'r/US92/0~63X
21~6~
-2 -
Sumrnarv ot the Invention
This invention is directed to a process for lhe synthesis of tigogenin 13-O, 11-ketotigogenin ~-O, hecogenin B-O, or diosgenin B-O cellobioside heptaalkanoale that
provides greater f3-anorneric selectivity and increased ylelds. The process is particularly
useful ~or preparing tigogenin B-O-cellobioside heptaalkanoate, which is an interrnediate
~or the known hypocholesterolemic agent tigogenin ~-O-cellobioside. The process
cornprises reacting a-cellobio~yl bromide heptaalkanoate and 13-tigogenin, 11-13-
ketotigogenin, 13-hecogenin or ~-diosgenin in the presence of zinc fluoride or zinc
cyanide under conditions suitabie for 1Orming the tigogenin 13-O-, 11 -ketotigogenin B-O,
hecogenin 13-O-, or diosgenin B-O-cellobioside heptaalkanoate.
Other features and advantages will be apparent from the specification and
claims .
etailed Description of the In~/entlon
Preferably the rnetcl salt used in the stereospecific reaction of a-cellobiosyl
1~ bromide heptaalkanoate and B-tigogenin, 11-keto-B-tigogerlin, 13-hecogenin or fl-
diosgenin is zinc 11uoride or zino cyanide. It is ~sperially preferred ~hat the metal salt
is zinc fluoride. It is preferred that about 0.5 equivalents to about ~ equivalents and
especially preferred that about 1.5 e~uivalents to about 2.25 equivalents metal salt is
used.
It may also be preferred lo conduct the :zinc fluoride or zinc cyanide-activatedcoupling in the presence of additional zinc salts such as zinc halides (e.g., 2inc
bromide, zinc chloride, zinc iodide) or basic salts of zinc (zinc oxide, zinc hydroxide,
zinc hydroxy fluoride, zinc carbonate, etc.) to buffer or to activate the prornoter (i.e.,
zinc tluoride or zinc cyanide metal sait). Trialkyl tertiary amines ~e.g., diisopropylethyl
26 amine, tri~thy!amine, tri~utylamine), te~raalkyiureas ~e.g., tetramethyi urea, tetraethyl
urea) or dia!kylanilines ~e.g., diisopropyl aniline, dibutylanilin~) are alss usefui reaction
buffers. The above a~ditives are generaliy used at 10-50% msle equiYalents of the
promoters.
Aithough any of the alkanoate ~C,~C4) substituted alpha-cellobiosyl bromides
may be used it is preferred th~ acetate (i.e., Cl) is usecl. They may be prepared ~rom
conventional starting materials according to methods described in K. Freudenberg and
.
WO 93/11150 PCr/US~2/0863~
2123~
-3-
W. Nagai, Ann., 494,63 ~1932) (e.g. Example 3). It is preferred that about 0.5 equiva-
lents to about 3 equivalents, and especially preferred that about 1 equivalent to about
2 equivalents alkanoate (C1-C4) substituted alpha-cellobiosyl bromides are used.Any reaction inert solvent may be used. As used above and elsewhere herein,
5 the expression "reaction-inert solvent" refers to a solvent which does not reaet or
decompose with starting materials, reagents, intermediates or products in a rnanner
which adversely affects the yield of the desired product. In general, the solvent can
cornprise a single entity, or contain multiple components. Preferably the solvent is a
non-protic reac~ion inert solvent and it is especially preferred lhat the solvent is
10 acelonitrile because of the excellent stereoselectivity it provides. Other solvents include
rnethylene chloride, ethyl acetate and nitromethane.
It is preferred that ~he reaction is acid catalyzed as 1his can increase the
selectivity of the 13~cellobioside product over the a-cellobioside anomeric produet.
Pre~erably rnineral acids are used. Hydrobromic acid has been shown to be particularly
15 effe~ive in increasing the 13-ce!lobioside product yield. Other pre~erred acids include
hydrochloric, hydrofluoric and sulfuric acid. It is preferred that about 0.05 equivalents
to about 2 equivalents, and especially preferred that about 0.1 equivalents to about 0.5
equivalents acid catalyst is used.
13-Tigogenin's preparation is described by Rubin in U.S. Patents Nos. 2,991,282
20 and 3,303,187, by B. Loken in U.S. Patent No. 3,935,194 and Cag1ioti et aJ.,
Tetrahedron 19, 1127 t~963). Its structure is depicted below.
'
.
H
_H 3 ~.~lC H3
C H 3 - ~ /
~ J
H 0 h
H
.
WO 93/11150 PCr/US92/0f~63X
212'~1i8~ ~-
13-Hecogenin's preparation is described in a paper on Steroidal Sapogenins by
Russell E. Marker et al., in J. Amer. Chem. Soc., 69, 2167-2211 (1947). Its structure is
depictsd below.
- 3 ~ \C H 3
~ ~H3
H H
~'
11-Keto-B-tigogenin switches the carbonyl group from th~ 12 position to the 11
position of the structure ~epicted above. 11~Keto-B-tigogerlin is prepared ~rom
hecogenin by lhe following procedure. According to the proc0dure of Conforth, et al.,
). Chem. Soc., 19~4, 907), hecogenin is acetylated, bromina~ed, treated with sodium
hydroxi~e and reduced with zinc to give the 12-hydroxy-11-keto analog. Then 12-
hyclroxy~ keto ana!og is acetylated and reduced with calcium and amrnonia ts give
1 1-ketotigogenin.
Diosgenin's:preparation is described in "Diosgenin and C)ther Steroidal Dr~g
Precursors" by Asolkar, L.V.,~ Chadha, Y.R., and Rawat, P.S" Council of Sci~ntific and
Industrlàl Research, New D~lhi, India, 1~83 pages, 1979 and also in T. Kawasaki et al.,
C~hem, Pharm. Bull., Japan~10 698 t1962). Its ~tructure is depicted below.
.
:
~ .
Wo 93t 1 1 15(1 PCl / US92/1)~63X
~123(i8~
~ H 3 J. ~ C H 3
HO R
Pr~ferably about 1 equivalent to about 2 equivalents of the steroid is used. It
is esp~clally pref~rred that about 1 equivalent to about 1 5 equivalerlts of the steroid is
. used.
Any envirorlment or conditions (e.g., lernperature, time, solvent) suitable for (i.e,
capable of) forming the desir~d tigogenin, 11-ketotigogenin, hecogenin- or diosgenin-
10 beta-O-cellobloside heptaalkanoate may ~e used However, it is preferred that the
reaction occurs at a temperature of about 20C to about 100C and preferably ~omabout ~OQC t~ about 65C. Below:about 2ûC the reaction can b~ slow and above
about 100~ undesir~d side rea~tions (e.g. anorneri~ation~ can occur This reaction
conveniently ca~ried out at ambient pressure how ver, pressures from about 0.5 to
15 about 3 atmospheres may be~ used.
Pre~erably ths steroid, metal salt and solvent are heated to retlux and sufficient
solvent is a~eotropically:distilled~:to remove substantially all the water. Then the
ce!l~biosyl bromide heptaacetate is~ added to the above mixture and heated ~or about
~:0.5 to::about 6.0 hours,~ typioally under nitrogen The desired cornpounds are then
20 isola~ed by conventional methsds
:: For example, the ~Iycosides may be precipitated from the erud2 ~iltered reaction
mlxture (e.g. acetonitrile ~product solution) by the addition of about 25% to 75% water
: ~ :
:
WO 93/11150 Pcr/l-ls92/ox63x
2~.2~j8 9i -6-
and the remainder alcohol (e g. methanol). Precipitation of the product ~rom aqueous
rnethanol/acetonitrile requires less processing than an extractive isolation, and provides
a product of greater purity.
The steroidal peracyl glycosides rnay be deacetylated by conventional methods
5 such as treatment with triethylarnine in methanol, basic anion exchange resins or
5 sodiurn methoxide in rrethanol or methanol/THF solvents (e.g. Example 2 below). For
exarnple~ the deacetylated pro~uct may be prepared by refluxing in methanol/THF using
a non-cataiytic amount of sodium methoxide followed by conventional work-up. Theexcess methoxide is used to decompose the fluoro sugar, if any ~3-cellobiosyl fluoride
heptaacetate is present, otherwise the deacetylation would be catalylic in sodium
10 methoxide. The tigogenyl-~-O-cellobioside or analogs are then isolated by conventional
methods such as ~iltration.
Although the above process is designed to synthesize steroidal glycosides of
the 13 con~iguration, the more therrnodynamically stable a-anorners are accessible by
acid-catalyzed isomeriza~ion of the 13-glycosides. For example1 tigogenyl a-O-
1~ cellobioside heptaalkanoate can be prepared from tigogenyl. 13-0-cellobiosidehept~alkanoate by heating the 13-glycoside in a methylene chloride solution containing
hydrogen bromide.
The influence of r~action stoichiometry, lemperature, solvents, molecular sieves,
and vestigial hydrogen bromide on the stereoselectivity and yield o~ tigogenyl 13-O;cello-
20 bioside heptaac~etate (using the process o~ Example 1) are summarized in Table 1.
.
j ,
.. . WO93/11150 P~1`/~ ;92/0~63X
~123~
Table 1
Zinc Fluoride or Cyanide Activated
Glycosi~ic Couplings with Tigogenin
Equivalents Solvenl Sieves T~ remp. ~G~coside
Activator Tiqoqenin ~leld
2.00 ZnF2~4.00~ 1.0 CH3CN No 2.5 hrs/65C 79%
1.50 ZnF2(3.00) 1.0 CH3CN No 2.5 hrs/65C 68%
0.50 ZnF~(1.00) 1.0 CH3CN No 3.0 hrs/65C 32%
O.S0 ZnF~(1.00) 1.0 CH3CN No 1.5 hrs/65C 30%
Hbr (0.38)
2.00 ZnF2(4.00) 1.0 CH2CI2 No 3.0 hrs/4~C 10%
25%~anome~
2.00 ZnF2(4.00) 1.0 Toluene 4A 20 hrs/65C 41%
2.00 ZnF2(4.00) 1.0 CH2CI2/CH3(:;N No 1.5 hrs/65C 64%
(2/1 3)
1.00 . ZnF2(2.00) 2.0 CH~CN No 1.0 hrs/65C 30%
Hbr(0.75)
0.50 ZnF2(0.~0j 1.0 CH3CN No 22 hrs/50C 38%
2.00 ZnF2(4.00) 1.0 CH3CN No 1.75 hrs/80C 76%
0.~0 ZnF2~0.50) 1.0 CH3CN No 1.75 hrs/B0C 53%
1.25 ZriF2(Z.2s) 1.0 CH~CN No 1.75 hrs/80C ~1%
2.00 Zn~CN)2(4.00) 1.0 CH3CN No 2.0 hrs/65C ~3%
2.10 ~n(C~N)2(5-60) ~ 1-0 ~H3CN No 3.0 hr~/65C 55%
1.50 2n(~N)2(4,00) 1-0 CH3CN No 2.5 hrs/6~C 4~%
The zinG fluoride-acthtatecl gly~oside c~upling was repeated with hecogenin and
diosgenin in analogous processes to the B-tigogenin glycosidic coupling o~ Example
1 below. The results with these other sterols are summarized in Table 2.
~`
WO 93/1 1 150 PC'r/US92/OX63X
~ ~ 236 '~ ~ -8-
Tabl2 2
Zinc Fluoride-Mediated Glycosidic Couplings
with Hecogenin or Diosgenin
Euuivalents Solvent Sieves TimerremP. Yleld
Glv~ ~ Activator Sterol Gh,~e
Cholesterol
2.00 2.25 (1.0) CH3CN No 2.25 hrs/65C 63%
Hecogenin
2.00 2,25 (1.0) CH3CN No 3.0 hrs/65C 72%
Diosgenin
2.00 2.25 (1.0) CH3CN No 2.5 hrs/65C 65%
This invention makes a significant advance in the ~ield of steroidal glycosides
by providing efficient methods of preparing steroidal peracyl glycosides. The
deacetylated end products are useful as antihyper~holesterolemic agents.
6 It should be understood that the invention is not limited to the particular
embodiments shown and described herein, but that various changes and rnodifications
may be made without departing from the spirit and scope of this novel concept asde~ined by the following claims.
Example_1
Tiqo~enyl 13-O-Cellobios~acetate
To a dry ~lask equipped with a mechanical stirrer, thermometer, and distillation head
were added 13-tigogbnin (4.1 6g; 0.01 mole), anhydrous zinc fluoride (4.1 3g; 0.04 mole),
and 160 ml of dry acetonitriie. The slurry was hea~ed to reflux (8~C) and 9û ml of
distillates was removed overhead while 60 ml of fresh, dry acetonitrile was added to the
slurry. ~he mixture was cooled to 2~C and then a sarnp!e was removed ~or a KarlFisher determin~t3On (K.F.= 0.0~% H20).
.
a-Cellobiosyl bromide heptaacetate (14.00g; 0.02 mole) was added to the ~lask, and
th~n the slirred slurry was heated to 65 C under a nitrogen atmosphere. The mixture
vvas mairltained at 65~C for 2.5 hours when lthin-layer chrom~tography1 (tlc) showed
that the reaction was complete. The reaction was oooled to ambient temperature and
130 mi of methyl ne chloride was add~d. The lhin slurry was filtered through Celite and
the filtrate (300 ml) was washed with a sa~urated sodium bicarbonate solution (70 ml)
Wo 93/1 l 150 PCT/US92/OX63X
2123~34
g
followed by an aqueous wash (70 ml). After lhe organic layer was dried over
anhydrous sodium sulfate (20 grams~ and filtered; the solution was then concentrated
to 50 ml via a distillation at atmospheric pressure. Two hundred milliliters of 2B-ethanol
was added to the warm concentrat~ and the turbid solution was concentrated to
5 approxirnately 50 ml. The thin slurry was cooled to 25C and then it was granulated
for 90 minutes at room t~mperature. The crude product was filtered, the cake waswashed with 25 rnl of 2B-~thanol, and then ~ried at 40C in V~cuo for 17 hours to give
10.8 grams o~ white crystalline solids (m.p. = 22~-231 C).
The solids were dissolved in 25 ml of methylene chloride and then 75 rnl ot 2B-
ethanol was added. The thin slurry was heated to reflux (760 mm) and 35.ml of
distillate was removed overhead. The resulting slurry was cooled to room temperature
and then was granulated ~or 90 minutes. The B-glycoside was filtered, and then dried
at 40C in vacuo ~or 18 hours to give 9.65 grams of a while crystalline solid (m.p. =
229-234C). Thin-layer chromatography' and high pressure liquid chromatography2
~hplc) show that the product contains 77% (w/w) tigogenyl 13-O-cellobioside
heptaacet~te and 15% (w/w) a-cellobiosyl fluoride heptaacetate. The a-celiobiosyl
fluoride heptaacetate is most easily removed trom the product during the deacetylation
stepi
Example 2
Tiaogenvl 13-0-cellobioside
Crude tigogenyl R-O-cellobioside heptaacetate (50.0g; ~.048 moles~ was dissolvedin 250 ml of tetrahydro~uran and 250 ml of methanol while maintained under a nitrogen
atmosphere. The hazy solution was ~iltered through a b~d of Ce,ite and then a solution
of ~odium rnethoxide (0.46g; 0.008 moles3 in methanol ~10 ml) was added to the filtrate.
. 2~ The solution was hea~ed to reflux (60C) and mairltained at reflux for 1.25 hours
generating a thick white slurry. A reaction aliquot was removed and analyzed by thin-
I ay~r chrornatography which indicated that the reaction was complete. The slurry was
con~entrated by removing 200 ml of distillate and then 200 ml of water was added to
the refluxing slurry. Another 20û ml of distillate was removed, and additional water (200
~0 ml) was added. The slurry was cooled to ambient ternperature and filtered. The
product cake was washed with water (50 ml) and then pulled dry on the fiiter. The
wat~r-wet~cake was heated to reflux (65C:: in 600 mls of THF and 92 mls of waler).
DARC0 G-60 (1.53 grams~ was added to the solutionl stirred for 15 minutes, and then
WO 93/11150 Pcr/~ls92/o~63x
2 1 2 3 t; ~ 4 -10-
the mixture was iiltered through Celite The solution was concen~rated by removing 460
ml of distillates and 460 ml of methanol was then added. The methanol addition and
concentration sequence was repeated twice again removing an addition 800 mls o~
distillate and 800 mls of ~resh methanol was added. The resultiny slurry was cooled
to 20C and then granulated ~or one hour. The product was filtered, rinse with f!esh
methanol (50 ml), and then the wet cake was reslurried in 300 mls of fresh methanol
(24C). The product was filtered and then dried at 40C in vaCuo overnight. Tigogenyl
13-O-cellobioside (24.4g; 0.036 moles) was isolated in 74% overall yield. Spectral and
physical properties were identical to an authentic sample.
Example 3
a-D-Cellobiosvl 8romide Heptaacetate
a-D-Cellobiosyl bromide heptaacetate was prepared ~orm a-D-cellobiose
octaacatate and hydrogen bromide in glacial acetic acid using a modified procedure
of Freudenberg a~d Nagari1.
A 20% (w/w) h~drogen bromide solution (178.7g; 0.44 mole of HBr) in glacial acetic
acid was prepared by bubbling gaseous hydrogen bromide into glacial acetic acid until
a derlsity of 1.212 was obtained. In a separate dry reactor maintained under a nitrogen
atmosphere, o-D-cellobiose octaacetate (50.0g; 0.074 moles) was dissolved in 408 ml
of rnethylene chioride. The HBr/HOAC solution was added to the disaccharide sol~ltion
to give a yellow solution. Afterthe solution was stirred for ~ hours at ~ 17-255, a
small aliquot o~ solution was removed for a reaction completion assay. Once thin-layer
chromatography2 indicated th~t the reaction was complete, the solution was cooled
to 10C and 0.5 liters o~water was added. The mixture was stirred ~or 10 rninutes, the
stirring was stopped, z~nd th~ iayers~ were allowed ts separate. The rnethylene chloride
layer was decanted and then washed with 7.50/D W/W sodium bicarbonate solution (0.5
liters~ ~ollowed by water io.5 liters). Finally, the methylene chloride sol~tion was dried
over 8 grams of anhydrous magnesium sulfate and then ~iltered. The MgSO4~hydrate
~: :
:: : . _
K. Freudenberg and W. Nag~arij Ann., 494, 63 (1932).
2Merck Pre-Coated TLC Silica Gel 60F-264 Plates using a toluene/acetic acid (4:1 ) eluant.
Plates were sprayed with 10% (w/w) H2SO4 in water and heated ~or charring, after the plates
were cleveloped.
.
.~ Wo 93/11150 PC~/lJS92/0863~
2123~
cake was washed with fifty milliliters of fresh methylene chloride, and the filtrate and
wash were combined. The methylene chloride solution was concentrated to approxi-mately 0.15 liters by an atmospheric distillation and then cooled to ambient
temperature. Diisopropyl ether (0.6 liters) was slowly added over 15 minutes with
stirring to generate a thick slurry. The product was granulated for 1 hour at 25C,
filtered, and then dried in v~cuo at 40C for 4.5 hours. a-Cellobiosyl bromide
heptaacetate (47.6g; 92% yield) was obtained as a white crystalline solid (m.p. = 192-
194C) whose 'H NMR spectrum (Cl:)C13) was consistent with its structure.
Example 4
1 O 11 -Ketotiqoqenvl-~-O-(:~ellobioside Heptaacetate
To an appropriately equipped one liter, 3-necked round bottom flask were added
acetonitrile (305 mls), 11-ketotigogenin (5.00g; 0.011 moles), and rhombohedral,crystalline zinc fluoride (1,65g; 0.016 rnoles). The slurry was heated to reflux (80C)
and then 100 ml of distillate was removed overhead. The slurry was coolecl to room
temperature, and then 15.39 grams (0.022 moles) of a-ce!lobiosyl bromide heptaacetate
was added. The reaction mixture was reheated to ~0-65~ and then maintain~d at 60-
, ~ ,
65C: for 2 hours. A reaction sample was removed ~or a reaction completion assay.
Thin-layer chromatography assay (EtOAc/hexanes 1.5:1 ) showed the complete
disappearanc0 ot the glycosyl bromide starting material so the reaction was cooled to
25C and 1~2 ml of ~methylene chloride was added. After stirring for 10 minutes~ the
mixture was tiltered through~ Celite and the filter cake was washed with 25 ml of CH2CI2.
Th~ oombine~ r~action ~lltrate and wash were washed with water ~81 mls), saturated
sodium bi~arbonate solution~(75 mls),~and water (137 ml). The organic layer was ~inally
dried over 11 grams of anhydrous magnesiurn sulfate. The MgSO4 was tiltered and
26 ~ washed with 16 mls~of ~fresh ÇH2CI2. The filtra~e ~nd wash were combined and then
con~entrated at r~duced pressure to one fourth its original volume (300 mls~. 2B-
khanoi (250 ml) was added and the resulting solution was concentra~ed to one-half
volume (170~mls). The slurry was cooled to 20-25C and then granulated ~or 1 hour.
`The whit~ waxy solids wer~ ~lltered, washed with ~resh 2B-ethanol (50 mls), and then
dried in vacuo at 40~:: overnight. 11-ketotigogenyl-B-O-cellobioside heptaacetate ~9.7
grams~ m.p. = 205-219C) was iso!ated in 84% overall yield. Chromatographic and
spectral ~haracterization were identical to an authentic sample ot 11 -ketotigogenyl-~-O-
cellobioside heptaacetate. Crude 11-ketotigogenyl-B-O-cellobioside could also be
:
:
WO 93/11150 P~/l)S92/08638
21 ~ 3 t1~ ~
isolated in 64% yield by the ~ollowing aqueous isolation sequence: The crude reaction
mixture was dilutecl with additional fresh acetonitrile (50 ml) and then ~iltered through
Celite. Methanol (290 mls) was added to the filtrate and the resulting solution was
heated at reflux (65C) lor one howr. One hundred millili~ers of deionized water was
5 slowly added to the refluxing solution to give a ha~y mixture. Alter 20 minutes at rellux
(72C), the mixture was slowly cooled to room temperature and then granulated at 23-
25C for 1 hour. The crude product was ~iltered and washed with water. The filter
cake was suspended in 2B-ethanol (75 ml) and the rnixture was heated to re~lux. The
mixture was then cooled to ambient temperature, liltered, and the solids were dried in
10 vacuo at 40C overnight. 1 1 -Ketotigogenyl-e-O cellobioside heptaacetate was isolated
in 64% overall yield.
Examp!e 5
1 1-Ketotiqoqer~YI~
11-Ketotigogenyl-B-O-cellobiosid~ heptaacetate (9.7g; 9.2 mmoles) was suspended
15 in 50 mls o~ methanol and SO rnls of tetrahydrofuran. The system was purged with
nitrogen and then a solution o~ sodium rnethoxide (O.lOg; 1.9 mmoles~ in rnethanol (1
ml) was added. The solution was heatcd to reflux (61 C) and then maintained at reflu
tor 1 hour. Thin-layer chromatography (CH2CI2/methanol 4:1) of the resulting slurry
showed that the reaction was complete. The tetrahydroluran was removed ~rom the
20 reaction by an atmospheric distillation and evsntually displacement with methanol (220
mls). A total of 180 ml o~ distillate was collected. The slurry was cooled to 20-25C
and then granulated overnight. The product was ffltered, wash~d with methanol (2 x
20 mls) and then the wet cake was reslurried (2C hours) in 100 ml o~ deionized water.
Alter fllltratio~ and drying in vacuo at 25~ overnight, 4.7 grams of 11^ketoti~ogenyl~
25 O-cellobioside was isolated in 65.5% overall yield. The product was homogenous by
tlc and analytical characterizations was consistent wnh the product's structure.1~ ExamPlQ6
Ln Situ Hydrobromic Acld Generation
e1-Cellobiosyl bromid~ heptaacetate (5.00g; 7.15 mmole) and 50 ml ol acetonitrile
30 were added to a 75 ml round bottom flask which was equipped with a mechanicalstirrer, thermometer, and vacuum distill~tion head. The system was purged wilh
nitrogen and then the pressure was reduced to approximately 425 mm Hg. The
solution was heated to 56-58C:; and approxim~tely 13 ml o~ distillate was removed
WO 93~ 0 PCI /US92/û863X
~123~
-13-
overhead. The solution was cooled to room temperature and the vacuum was slowly
released. Methylene chloride (37 rnl) was added to the glycosyl bromide solution and
then the solution was extracted with water (2 x 25 ml). The aqueous phases were
combined and then titrated to a phenolphthalein endpoint using 0.100~ N NaOH
sohJtion.
Tho millequivalents of HBr acid contained in the a-cellobiosyl bromide heptaacetate
solution before and after the ~eotropic ~trip are reported below. The azeotropic strip
increas~ the tetr~table acid approximately 8-10 fold depending upon the water content.
TITRATED ACIDS
Initial a-Cellobiosyl Bromide 2.1 milliequivalents
Heptaacetate Solution
, , _ _ . . .~
Azeotroped a-Cello~iosyl ~romide 16.9 milliequivalents
Heptaa etate Solution
_ -_
Wh~n th~ azeotropic distillation was used in Example 1 to dry the glycosyl brornide
solution and to increase its acid content, the reaction time was decreased to 1.0 hour
at 65C. In addition, high yields and high 13-anomeric selectivity were maintained.
Fxample 7
Aqueous Isolation of Tiqoqenyl ,,
C~
A zinc ~luoride-mediated glycosidic coupling of B-tigogenin (0.915 mole~ with a-cellobiosyl bromid~ heptaacetate (1.830 mole3 in ac~tonitrile was conducted according
to the procedure of Example 1. Once the reaction was complete, the crude reac~ion
mixture was worked-up by an aqueous method to give high-quality tigoyenyl 13-O-
cellobiosicle heptaacetate by the following sequence.
The crude reaction mixture was filtered through Celite to afford approxirnately 20
liters of a golden colored ~iltrate. The filtrate was heated (55-60~C~ and concentrated
at reduced pressure to about 10 liters. The concentrated solution was cooled to 50C
and 6.0 Iiters of methanol was added. Subsequently, 7.5 liters of deionized water was
slowly added over 30 minutes. A solid precipitated from solution once about 2 liters
of water was charged. The mixture was heated to reflux ~73C) and then maintained
WO g3/ 111 50 PCr/ U S9 2/O~s~ ? ~
2 1 2 3 ~ 4
at reflux for 2 hours. The slurry was cooled to 25~C and granulated overnight. The
crude product was filtered, washed with methanol (2 x 1.5 liters), and then dried in
v~cuo at 40C. The crude solid (1.01 kg) was 9~.5% pure by a hplc assay. In
addition, the crude product contained only 1.3% of tigogenyl a-O-cellobioside
heptaacetate and no B-cellobiosyl fluoride heptaacetate.
Crude tigogenyl 13-O-cellobioside heptaacetate (1.0 kg) was slurried in 10.2 liters
of 2B ethanol ùnder nitrogen and lhen the mixture was heated to reflux (78C). Af~er
the slurry was at re~lux tor 1.5 hours, the mixture was cool~d to 25C and then
granulated for 12 hours. The product was filtered, washed with tresh ethanol (2 x 300
ml), and finally dried in vacuo at 4~ C overnight. Tigogenyl (3-O-cellobioside
heptaacetate (0.89 kg) was isolated in 74% overall yield frorn tigogenin. The white
orystalline product was 98.90io pure by high pressure liquid chromatography and
contained only 0.5% (w/w) o~ the isomeric a-anomer~
.
~ ~ : Y
,
