Note: Descriptions are shown in the official language in which they were submitted.
~2 ~ 7 ~
BEHRIN~WERKE AKTIENGESELLSCHAFT HOE 89/B 014 - Ma 763
Dr. Ha/Sd
A ~rocess for the Preparation-of etoposides
~he invention relates to a process for the preparation of
4-0-~4,6-0-alkylidene-beta-D-glucopyranosyl)-4'-0-de-
methyl-4-epi-podophyllotoxins, which are called etopo-
sides, especially of etoposide (VP-16~ which, by reason
of its cytostatic a~tivity, i~ ~ui~able for the treatment
of cancexs. The invention particularly relates to a
process for the preparation of 4,6-0-alkylidene-2,3-di-
O-acyl-beta-D-glucopyranoses which are used a8 glycosyl
donors for the synthesis of 4-0-beta-D-glucopyranosyl-
epi-podophyllotoxins, and to a process for the deacyl-
ation of the glycoside intermediates.
Etoposide (VP-16) and the closely structurally related
teniposide (VM-26) have been introduced as pharmaceuti-
cals. They are particularly valuable for the treatment of
small-cell lung cancer and testicular cancer.
The preparation, action and clinical use of etoposides
has been described by T.W. Doyle in "Etoposide ~VP-16) -
current status and new developments" (Editor. B.F. Issell,
F.M. Muggia and S.K. Carter; Academic Press, 1984, pp.
15-32).
Etoposides are usually prepared starting from a function-
alized glucopyranose building block and a 4'-0-protected
epi-podophyllotoxin dPrivative.
The synthesis of etoposides starting from the glycosyl
donors 2,3-di-0-acetyl-4,6-0-ethylidene-D-glucose or 2,3-
di-O-halogenoacetyl-4,6-0-ethylidene-D-gluco~e has been
described in Swi8s Patent No. 514,578 (GB 823,06B) or
EP 0,111,058. The preparakion described therein of these
donors, which are obtained from 1-0-benzyloxycarbonyl-
beta-glucose precursors, is costly and, because of the
use of benzyl chloroformate as reagent, ha~ardous ko
2 ~ 7 ~
~ 2 -
health. In addition, these precursors in the beta fonm
must be isolated from an alpha,beta mixture because only
the beta form provides the beta-hydroxyglucopyranose
donors required for the glycoside synthesis.
EP 0,226,202 A2 describes the preparation of etoposides
using 1-O-trialkyltin-glucose donors.
Because of the chemical in~tability of the podophyl-
lotoxin aglycone, by~products are formed during the
conventional deblocking of the etoposide precursoxs.
Deace~ylation as described in the abovementioned patent
applications is effected by methanolysis catalyzed by
~inc(II) salts. The by-products occurring in this case
are the corresponding methyl esters of the hydroxy acid
formed by opening of the lactone ring, as well as a picro
lS compound, which comprise up to about 30 % of the xeaction
product.
Because of the disadvantages which have been described,
there is a need for a straightforward and low-cost
process for the preparation of etoposides.
It has been found, surprisingly, that hydrogenolysis of
benzyl 2,3-di-O-acyl-4,6-O-ethylidene-beta-D-glucopyrano-
side provides the glycosyl donor with retention of the
beta con$iguration. Since the alpha-hydroxy form of the
glucose unit does not occur in this reaction, the ~ubse-
quent glycosylation of the epi-podophyllotoxin deriva-
tives takes place selectively to give the desired beta-
glycosides. It has furthermore been found that ~he
deacylation of the etoposide precursors using a basic ion
exchanger takes place without formation of the by-products
described above.
~he invention has the ob~ect of developing a process which
provide~ the glycosylation component 4,6-O-alkylidene-
2,3-di-O-acyl-beta-D-glycopyranose in improved yields and
in pure beta-hydroxy form, and signifies a simplification
2 ~
-- 3 --
compared with the known processes, the use thereof for
the glycosylation of (epi)-podophyllokoxin derivatives
and the working out of an improved protective-group
chemistry on acylated 4-0-glucosyl-epi-podophyllotoxins
which permits preparation of etoposides without the
formation of by-products.
This object i5 achieved by the process for the prepara-
tion of a beta-glucopyranose derivative of the formula I
o \ \~n
,0 ~ ~ oR3
Rl R2 0,
in which
Rl and R2 are H or an acyl protective group and
- R3 is H, benzyl or the radical of the formula II
<0~,
~ Orle
with R4 being H or a benzyloxycarbonyl or chloroacetyl
protective group and A being Cl-C4-alkyl, which compri~es
eliminating the benzyl group in a benzyl glucopyranoside
of the formula I in which
Rl and R2 are an acyl protective group,
R3 iS a benzyl protective group and
A is C1-C4-alXyl,
2 ~ 7 ~
-- 4 --
by hydrogenolysis in the presence of a hydrogenation
catalyst and of an or~anic sol~ent, there being formation
of a glucopyrano~e derivative of the formula I which is
in the beta-hydro~y form and in which Rl, RZ and A retain
their meaning, and R3 is a hydrogen atom, subsequently
reacting this ~lycosidation component with an epi-podo-
phyllotoxin derivative of the formula III
OH
<0~
~ O
MeO ~~ Olte
o-R4
in which
R4 is benzyloxycarbonyl or chloroacetyl protective group,
in the presence of a promoter ~uch as, preferably,
BF3.ether or of a tri-(Cl-C4)-alkylsilyl trifluoromethane-
sulfonate and of an organic ~olvent, where appropriate in
the presence of a desiccant at -30C to 0C, there being
formation of a beta-glycoside of the formula I in which
R1, R2, R4 and A retain their meaning/ and R3 is a radical
of the formula II, and eliminating in the product which
is formed the benzyloxycarbonyl protective group by
hydrogenolysis and the acyl protective group using basic
ion exchangers in the presence of a polar ~olvent, there
being formation of a product of the formula I in which
and * are hydrogen, R3 is a radical of the formula II
with R4 equal to hydrogen, and A i~ Cl-C4-alkyl.
Preferably prepared by the process according to the
invention ar~ compounds of the formula I in which R1 and
R2 are H, the acetyl or chloroacetyl protective group, R3
is H, benzyl or a radical of the formula II with R4 being
2 ~
-- 5 --
H or a benzyloxycarbonyl or chloroacetyl protective
group, and A is methyl.
The detailed procedure for this is as follows;
The ~tarting compound benzyl 2,3-di-O-acyl-4,6-O-alkyli-
dene-beta-D-glucopyranoside can be prepared by the
proce~ses customary in carbohydrate chemis~ry. ~tarting
~rom 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl
fluoride, which is easily obtainable industrially, and
benzyl alcohol, the ~eta-benzylglucoside is selectively
prepared in the pxesence of a Lewis acid such as
BF3.ether. The product is deacetylated with sodium methy-
late and sub~equently con~erted with a Cl-C4-aldehyde
under acid catalysis into a 4,6-0-alkylidene compound
which is finally reacted at ~-2 and 0-3 u~ing an acid
anhydride or halide in the presence of a base such as
pyridine or triethylamine at -40C to O~C to give a
benzyl 2,3-di-O-acyl-4,6-O-alkylidene-beta-D-glucopyrano-
side.
The glucopyranose glycosylation component which is in the
2V beta-hydroxy form can be prepared in the following
manner: benzyl 2,3-di-O-acyl-4,6-O-alkylidene-beta-D-
glucopyranoside is hydrogenated in the presence of
palladium/carbon or palladium/barium sulfate and of a
solvent such as methanol, ethanol, acetone or ethyl
acetate or mixtures thereof, preferably acetone/ethanol
mixture~, preferably under atmospheric pre~sure, there
being quantitative formation of the qlucopyranose com-
ponent in the beta-hydroxy form.
The reaction of the beta ~ydroxy donors with a 4'-O-
protected 4'-O-demethyl-4-epi-podophyllotoxin deri~ati~e
of the formula III i carried out using a promoter such
as BF3.ether or of a tri-(Cl-C4)-alkylsilyl trifluoro-
methanesulfonate in an anhydrous organic solvent such as
dichloromethane, ethyl acetate, ether, acetone or aceto-
nitrile, where appropriate in the presence of a desiccant
2 ~ 7 ~
such as a molecular sieve at -30~C to 0C. It is particu-
larly advantageous to employ the promoter trimethylsilyl
trifluoromethanesulonate.
~he benzyloxycarbonyl protective group is eliminated in
5 a customary manner by hydxogenolysis in the pre~ence of
palladium/carbon or palladium/barium sulfate. The acyl
protective groups in the c~rbohydrate moiety and, where
appropriate, on the aglycone of the etoposide derivatives
of the formula I are eliminated usin~ a basic ion
lD exchanger, preferably Dowex lx8 in an organic solvent
such as methanol, ethanol, propanol, ethyl acetate,
dichloromethane, chloroform or mixtuxes thereof. Deacyl-
ation of chloroacetyl derivatives of the formula I u~ing
the ion exchanger Dowex lx8 is particularly preferred and
takes place quantitatively in methanol/chloroform as
solvent to give etoposides without the formation of
by-products.
Acyl protective groups are, in particular, acetyl or
mono-, di- or trihalogenoacetyl protective groups with
fluorine, chlorine or bromine as halogen, preferably the
chloroacetyl protective group.
The examples which follow are intended to illustrate the
invention without, however, confining it to the compounds
mentioned.
Example 1
Preparation of benzyl beta-D-glucoside
` - Benzyl beta-D-glucopyranoside (Compound 1)
5 g (14.2 mmol) of 2,3,4,6-tetra O-acetyl-alpha-D-gluco-
pyranosyl fluoride were dissolved in 50 ml of dry aceto-
nitrile, and 2.2 ml (1.5 eq) of benzyl alcohol wereadded. The reaction mixture was cooled to 0C and, while
stirring, 2.1 ml of BF3.ether were added dropwise. After
2 0 ~
-- 7 --
2.5 h (TLC- hexane/ethyl acetate), 2.5 ml of ~riethyl-
amin~ were added to the reaction mixture, which was then
concentrated in vacuo. ~he residue was dissolved in chloro-
form, and the solution was washed with phosphate buffer,
pH 7.5, and then with ~aturated brine, dried over sodium
sulfate and evaporated in vacuo. The product ~6.3 g) was
dissolved in 50 ml of dry methanol, and the ~olution was
adjusted to pH 11 with sodium methylate. After 4 h, it
was neutralized with Dowex ~x8 and evaporated in vacuo.
The resulting product, which is identical (lH NMR, rotation~
to the product described in the literature, was employed
without further purification steps in the next stage.
Exam~le 2
Preparation of the 2,3-di-O-acyl-4,6 ethylidene-gluco-
sides
Benzyl 4,6-O-ethylidene-beta-D-glucopyranoside
(Compound 2)
58.8 g (157.0 mmol) of compound 1 were suspended in
600 ml of dioxane. 17.7 ml of acetaldehyde were added,
and then the mixture was cooled to 10C and, while stir-
ring, 2 ml of concentrated H2SO4 were added dropwise.
After 1.5 h, a further 9 ml of acetaldehyde and 0.5 ml of
concentrated HaSO4 were added. The reaction mixture was
then stirred at room temperature for 12 h. The re~ulting
red-brown ~olution was adjusted to pH 7 with methanolic
sodium methylate solution ~ evaporated in vacuo and then
distilled with toluene. ~he residue was taken up in ethyl
acetate, and the solution was thoroughly washed with ice-
water. The organic phase was dried over sodium sulfate
and evaporated. The product was dissolved in chloroform/
me~hanol 7:1 and filtered through 150 g of silica gel,
and the residue after evaporation of the solvent was
crystallized from ether~
Yield: 52.5 g (81 %), ~23 = -79.1 (c = 1 in chloroform)
Melting point = 150C
2 ~ 7 ~
-- 8 --
Ben~yl 2,3-di-0-acetyl-4,6-0-ethylidene-be~a-D-gluco-
pyranoside ICompound 3)
3.51 g (11.8 mmol) of compound 2 were dissolved in 30 ml
of dichloromethane and 30 ml of pyridine and, at 0C,
5.3 ml of acetic anhydride were added. The reaction
mixture was stirred for 12 h and then worked up as
customary in carbohydrate ch~mistry.
Yield: 5.28 g (100 %), ~]D3 Y -87 (c = 1 in rhloroform)
Melting point = 155C
Benzyl 2,3-di-0-chloroacetyl-4,6-0-ethylidene-beta-D-
glucopyranose (Compound 4)
25 g ~54.3 mmol) of compound 2 were dissolved in 350 ml dry
dichloromethane, and 56 ml of triethylamine were added.
The mixture was cooled to -20C and 20 ml of chloroacetyl
chloride dissol~ed in 250 ml of dichloromethane were
added in portions. After 4 h, a further 11 ml of tri-
ethylamine and 3.5 ml of chloroacetyl chloride were added
to the reaction mixture. After a further 4 h, the mixture
was filtered, and the organic phase was thoroughly washed
~0 with ice-cold phosphate buffer, pH 7Ø The organic phase
was dried over magnesium sulfate and evaporated in vacuo.
The residue was dissolved in chloroform/ethyl acetate and
filtered through 100 g of silica gel. The product from
evaporation crystallized from chloroform and petroleum
ether. ~ield: 31.0 g (82 ~), [~ ID = -54 . 9 ( C = 1 in
dichloromethane)
E~amPle 3
Preparation of beta-hydroxy-glycosylation components
2,3-Di-0-acetyl-4,6-~-ethylidene beta-D-glucopyranose
(Compound 5)
1 g (2.6 ~mol) of compound 3 were dissolved in 40 ml of
ethyl acetate, and 1 g of palladium/carbon was added. The
20~5~ ~
g
reaction mixture was hydrogenated under atmospheric
preæsure at room t~mperature for 1 h. The cataly~t was
filtered off and the solution was evaporated in vacuo at
35C, and the residue was then dis~illed with toluene,
The resulting produc$ was employed without further
purification steps in the next reaction stage. Yield:
0.76 g (99 %), [~] 23 = - 16.4 (c = 1 in ethyl acetate)
2,3-Di-O-chloroacetyl-4,6-O-ethylidene-beta-D-gluco-
pyranose (Compound 6)
25 g (55.6 mmol) of compound 4 were dissolved in 600 ml
of acetone and 60 ml o~ ethanol. 15 g of palladium/carbon
were added and then the mixture was hydrogenated for
l.S h. ~he catalyst was then filtered off and the organic
phase was evaporated at 35C. The residue was distilled
1~ with toluene and employed without further purification
steps in the next reaction stage.
Yield: 19.8 g (98 %), [~]23 = -19.8 ~c = 1 in acetone~
Example 4
Glycosylation of podophyllotoxin derivatives
4'-O-Benzyloxycarbonyl-4 O-(2,3-di-O-chloroacetyl-4,6-O-
ethylidene-beta-D-glucopyranosyl)-4'-O-demethyl-4-epi-
podophyllotoxin (Compollnd 7)
Process A:
15.2 g (42.3 mmol) of compound 6, 22.6 g ~4~.3 mmol) of
4'-O benzyloxycarbonyl-4'~0-demethyl-4-epi-podophyl~
lotoxin and 20 g of 4 A molecular sieves were suspended
in 1000 ml of dichloxomethane and, at -20C under protec-
tive gas, 45.5 ml of BF3~ether were added. The mixture was
fitirred at -20~C for 17 h, then 46 ml of ~riethylamin~
were added and the mixture was fil~ered. The ~iltrate wa~
evaporated in vacuo, and the residue was then distilled
with toluene. The resulting crude product was dissolved
20~7~
- 10 -
in chloroform and then thoroughly washed with phosphate
buffer, pH 7.5. The organic phase wa~ dried over ~odium
8ulfate and evaported in vacuo. The residue was dissolved
in a dichloromethane/petroleum ether/acetone 8:2~1
~olvent mixture and filtered through lO0 y of ~ilica gel.
The product from evaporation of the solvent was
crystallized from chloroform/petroleum ether. Yield:
31.8 g (56 %)~ 23 = _37.9D (C = 1 in chloroform)
Process B:
15.2 g (42.3 mmol) of compound 6, 22.0 g (42.3 mmol) of
4'-0-benzyloxycarbonyl-4'-0-demethyl-4-epi-podophyl-
lotoxin and 20 g of 4 A molecular sieves were suspended
in 550 ml of dichloxomethane~acetone 10:1 and, at -30C
under protective gas, 9.4 g of trimethylsilyl trifluoro-
methanesulfonate were added. The reaction mixture wa~
stirred at -25C for 6 h, and then 50 ml of triethylamine
wers a~ded and the mixture was filtered. The subseguent
working up was carried out as described in process A.
Yield: 34 g (92 ~)
4'-0 Benzyloxycarbonyl~4-0-t2,3-di-0-acetyl 4,6-0-ethyl-
ide~e-beta-D-glucopyranosyl3-4'-0-demethyl-~-epi-podo-
phyllotoxin (Compound 8)
1.22 g ~4.23 mmol) of compound 5, 2.20 g ~4.23 mmol) of
4'-0-benzyloxycarbonyl-4'-0-demethyl-4-epi-podophyl-
lotoxin and 2.5 g of 4 ~ molecular ~ieve~ wer~ ~uspende~
in 60 ml of dichloromethane/acetone 10:1 and, at -30C
under protective gas, 0.94 g of trimethyl~ilyl trifluoro-
methanesulfonate was added. The reaction mixture was
stirred at -25C for 8 h and then 5 ml of tri~thyl~mine
were added, and the working up was as for the preparation
of compound 7~
YIeld: 2.8 g tB2 ~)
4'-0-Chloroacetyl-4-0-(2,3-di-0-chloroacetyl-4~6~0-
ethylidene-beta-D-glucopyranosyl)-4'-0-demethyl-4-epi-
2 ~ 7 ~
11
podophyllotoxin (Compound 9)
11 g (30.6 mmol) of compound 6, 14.6 g (30.6 mmol) of 4'-
0-chlsroacetyl-4'-0-dem~thyl-4-epi-podophyllotoxin and
20 g of molecular sieves were stirred in 330 ml of
dichloromethane/acetone 10:1 and, at -30C, 6.~ g of
trimethylsilyl trifluoromethanesulfonate were added.
After 7 h the reac~ion mixturs was worked up as described
for the preparation of compound 7.
Yield: 21.3 g (86 ~)
Example 5
Deacylation of the etoposide precursors
4' 0-Benzyloxycarbonyl-4'-0-demethyl-4-epi-4-0-(4,6-0-
ethylidene-beta-D-glucopyranosyl)-podophyllotoxin
(Compound 10)
27.5 g (31 mmol) of compound 7 were dissolved in 780 ml
of dichloromethane and 1225 ml of methanol. 50 g of
Dowex lx8 ion exchanger were added and then the reaction
mixture was stirred at room temperature for 1 h. The
suspension was filtered, and the resin was washed with
methanol. ~he filtrates were evaporated, the residue was
dis~olved in chloroform, and the solution was thoroughly
wa~hed with phosphate buffer, pH 7.5, and then with
water. The organic phase was dried over sodium ~ulfate
and evaporated in vacuo. The residue crystallized from
ether/petroleum ether.
Yield: 21.5 g (96 %~
4'-0-Demethyl-4-epi-4-0-(4,6-0-ethylidene-beta-D gluco-
- pyranosyl3-podophyllotoxin (Compound 11)
~0 g (24.6 mmol~ of compound 9 were dissolved in S00 ml
of dichloromethane ~nd 900 ml of methanol, and 50 g of
Dowex lx8 wsre added. The reactisn mixture was stirred at
room temperature for 2 h and then filtered, washing the
resin with methanol. The combined filtrates were e~aporated
2 ~
12
in vacuo. The residue was taken up in dichloromethane,
and the solution was then washed with phospha~e buffer,
pH 7.5, and with water. The organic phase was dried over
sodium 8ul fate and evaporated in vacuo. The product
crystallizes from dichloromethane and hexane.
Yield: 13.6 g (94 ~)
Example 6
Elimination of the benzyloxycarbonyl group by
hydrogenolysis
4'-0-Demethyl-4-epi-4-0-(4,6-0-ethylidene-beta D-gluco
pyranosyl)-podophyllotoxin (Compound 11)
9.7 g (13.4 mmol~ o~ compound 10 were di6solved in 260 ml
of methanol and hydrogenated with 5 g of palladium/car-
bon~ After 40 min, the catalyst was filtered off, and the
solution was evaporated in vacuo. The residue was dis-
solved in dichloromethane and thoroughly washed with
phosphate buffer, pH 7.5, and then with water. The organi~
phase was dri~d over sodium sulfate and evaporated in
vacuo. The product crystallizes from dichloromethane and
hexane.
Yield: 7.25 g (92 %)
