Note: Descriptions are shown in the official language in which they were submitted.
~0465~8
1 This invention relates to a new process for the
chemical synthesis of daunomycin and its beta anomer, of 4'-epi-
daunomycin mixture of alpha- and beta-anomer, and of each single
anomer and to the new products thereby obtained. Daunomycin
and daunomycinone are described and claimed in our British
Patent 1,033,383.
The complete chemical name of ~'-epi-daunomycin alpha
anomer is 7-0-(3'-amino-2'l3',6'trideoxy-alpha-L-arabino-
hexopyranosly)-daunomycinone.
The co~lplete chemical name of 4'-epi-daunomycin beta
anomer is 7-0-(3'-amino-2'l3'l6'trideoxy-beta-L-arabinohexopyra-
noxyl)-daunomycinone.
The process of this invention comprises the condensa-
tion of daunomycinone (I) ~also described and claimed in our
Patent Specification 1l003l383) with a derivative of daunosamine
(3-amino-2l3l6-trideoxy-L-lyxohexosel II) or of 4'-epidauno-
samine (3-amino-2,3l6-trideoxy-L-arabinohexosel III) to obtain
the pharmacologically active glycosides IV (daunomycin), V, VI
and VII. In practice, the hexose II or III has to be protected
and converted into a l-derivativel such as a halidel suitable
for condensation with daunomycinone. After the condensationl ;~
the protecting groups are removed. The formulae of the compounds
referred to herein are as follows:
O OH
CH30 OH OH
3~ I
- 1- `
,
' . '.. . ' ' ~ ' ,.. ' ' .`" "' . :'.: ~ . ~ ....... . . :
i ~ . . ~ ,
i508
Ho~ ,OH
N~12
II III -
O OH
X ~COCH3 ,_~ COCH3
3 H O 3 O OH O
HO~ HO\~
N 2 ~:
O OH O OH
~0 ~ ~ r ~ H
3 H O CH30 O H ~ : .
VI H~l HO~
NH2 . :':
3a ~ ~
~0~6508
1 The 3-amino groups in the hexose II and III have to be
protected with groups whose ramoval can be performed without
further decomposition of the products which contain different
chemically-sensitive groups. The N-trifluoroacetyl group can
satisfactorily be removed by mild alkaline treatment.
The hexoses II and III have to be converted into
derivatives endowed with sufficient stability to be used in the
synthesis. The instability of l-halogeno derivatives of 2-
deoxysugars is well documentated (W.W. Zorbach et al., Advances
in Carbohydrate Chemistry, 1966 21, 273). ~-
The tri-trifluoroacetyl derivatives of the hexoses II
and III have been reacted according to this invention with dry ~
- hydrogen chloride to give the corresponding l-chloro-hexoses. ~;
. .:
These are solid compounds which can be stored for several days
under anhydrous conditions.
As to suitable reaction conditions for the synthesis
. : .
of the glycosidic linkage, the well known Koenigs-Knorr reaction
(J. Conchie, G.A. Lewy and C.A. Marsh, Advances in Carbohydrate -~
Chemistry, 1957, 12, 157) alIows a range of different conditions
which include modifications of the solvent, temperature,
catalyst, and hydrogen chloride or hydrogen bromide acceptor.
The importance of the modifications is such that, usually, an
optimal set of conditions is necessary for a noticeable reaction
rate i
The application of standard conditions to the 1-
halogeno derivatives of 2~deoxy sugars leads to the corresponding `
glycals (~orbach et al., above). The procedure according to
this invention comprises a mild treatment of daunomycinone (I) ~ -
with l-chloxo~N,0 di-trifluoroacetyl derivative of the hexose
II or III in an organic solvent such as chloroform or methylane
~ 3 ~
:- .
10~:i5~1
1 dichloride, in the presence of a catalyst comprising a mercuric
halide (for example mercuric bromide) and a hydrogen chloride
acceptor (for example mercuric oxide).
The final products can be obtained by removal of the
N-trifluoroacetyl with dilute alkali.
The invention is illustrated by the following examples
the temperatures being in degrees centigrade.
EXAMPLE 1
Daunosamine (II) hydrochloride (1 g) is suspended in
anhydrous diethyl ether and treated at 0 with trifluoroacetic
anhydride (8 ml). After standing for two hours at 0 and one
hour at room temperature, the solvent is removed at ~educed
pressure and the residue is crystallized from dichloromethane
to give 1.1 g of tri-trifluoroacetyldaunosan,ine, m.p. 132-134, `
mass spectrum m/e 391 (M-44), 322 (M-113). This (0.5 g) is
treated in anhydrous diethyl ether at 0~ with anhydrous gaseous
hydrogen chloride. After standing at ~5 overnight, the
solvent is removed in vacuo to give l-chloro-N,O-di trifluoro-
acetyldaunosamine as an oily product*:
NMR (CDC13): 1.22~ (d, J-6.5 Hz, 3H, CH3),
2.05-2.70~ (m, 2H, C(2jH2),
4.46~ (dq, J=6.5 Hz and JC lHz, lH, C(5)H),
4.60-5.10~ (m, lH, C(3)EI),
5.37 ~ (c, WH=6.0 Hz, lH, C(4)H), `~
6.29~ (m, WH=6.5 Hz, lH, C(l)H), and ;-
6.37Or (broad s, lH, NH).
Finely powdered daunomycinone (I, 300 mg, 0.75 mM)
is dissolved in anhydrous chloroform (75 ml) and treated with
mercuric oxide (600 mg), mercuric bromide (I50 mg), and
molecular sieve (3A, Merck).
* which is also called: l-chloro-2,3,6-trideoxy-3~trifluoro-
acetamido-4-trifluoroacetoxy-L-]yxohexopyranose
- 4 -
,. .
: .
' ~ ' ' : , ',',, ~ ' , . , :
. . . .. . ... .
~(~4ti~08
1 The suspension is s-tirred for one hour and then 600 mg of
l-chloro-N,O-trifluoroacetyldaunosamine are added.
The mixture is stirred at room temperature for 64 hours,
and then filtered. The solution is evaporated in vacuo. The
residue is taken up in methanol (200 ml) and refluxed for lS
minutes. The residue which remains on removal of the solvent is
chromatographed on a column of silicic acid using a mixture
chloroform:benzene:methanol 100:20:3 (by vol) as eluting agent.
In addition to unreacted daunomycinone, 220 my of N-tri- ;
fluoroacetyl daunomycin m.p. 169-171 (after recrystallization
from tetrahydrofuran and hexane), and 20 mg N-tri-fluoroacetyl
daunomycin (beta isomer), m.p. 138-140 [alpha~23 + ~40 (c 0.1
chloroform), were obtained. N-trifluoroacetyl daunomycin (0.20 g)
is dissolved in 0.1 N aqueous sodium hydroxide (20 ml). The
resulting solution, after 30 minutes standing at room temperature, ;~ -
is treated with 0.1 N aqueous hydrogen chloride to bring the pEI to
8.6, and repeatedly extracted with chloroform.
The extracts are dried over anhydrous sodium sulphate,
concentrated to a small volume, and acidified to pH 4~5 with ~ ;-
0.1 N methanolic hydrogen chloride to allow crystallization of
daunomycin hydrochloride. This is identical in-all respects
with the product ~s obtained by fermentation (see F.Arcamone et al., ~;
Gazzetta Chimica Italiana, 1970, 100, 949). The yield is ;~; ;
practically quantitative.
EXAMPLE 2
2,3,6-Trideoxy-3-trifluoroacetamido-L-arabinohex~se
(1 g) is suspendea in anhydrous diethyl ether (20 ml) and treated
at 0 with trifluoroacetic anhydride. The mixture is further
processed as in Example 1 to give a quantitative yield of
30 2,3,6-trideoxy-N,O-di-trifluoroacetyl-L-arabinohexopyranosyl ;~
chloride. ;~
: . . . .
- .
.. : ~ . , -
~':: : ' ' ' , ' :
-` ~n~sos
1 NMR (CDC13): 1.305 (d, J-6.0 llz 3EI, C113),
2.25-2.80,J (m, 2M, C(2)H2),
4.20-4.65~ (m, lH, C(5)1-1),
4.65-5.15 3 (m, 2H, C(3)H and C(4)H),
6.25J (m, W~1=6.0 Hz, lH, C(l)H), and
6.45~ (broad s, lH, NH).
A solution of daunomycinone (O.5 g) in anhydrous chloro-
form is treated with mercuric oxide (1.0 g), mercuric bromide
(0.25 g), molecular sieve (3~, Merck, g 10), and 2,3,6-trideoxy-N,O-
1~ di-trifluoroacetyl-L-arabinohexopyranosyl chlo'ride (0.5 g). The
mixture is stirred for 24 hours, freed from solids by fiItra-
tion, and evaporated under vacu~n. The residue is taken up in
methanol, refluxed for 15 minutes, evaporated to dryness and '
chromatographed on a silicic acid col~nn using a mixture chloro-
form:benzene:methanol 10:20:3 as eluting agent. The main product ~
which is'obtained is a mixture, in the ratio 70:30 of alpha and ~ '
beta 7-O-(N-trifluoroacetyl-4'-epi-daunosaminyl)-daunomycinone
(yield after crystallization from chloroform, 0.3~g). This material,~
upon treatment with 0.1 N sodium hydroxide as above is converted
20- quantitatively to a mixture'of the corresponding alpha and beta '~
glycosides as free bases. The product is separated into alpha
.
and beta anomers by silica gel chromatography using a chloroform~
methanol:water solvent system 135:20:2 (by vol) as eluting agent. ~ -
There are obtained alpha anomer (4'-epidaunomycinj VI), [alpha]23
320 (c = 0.045, methanol), m.p. 199-201, yield g 0.16; beta-
anomer (VII), m.p. 182-184, yield 0.06 g.
Bioloqical activity of compounds VI (4'-epiaaunom~cin) and
VII (4'-epidaunomvcin, beta-anomer).
_ ... _ . _ .. . ... . .. _ _ .
Compounds VI and VXI display outstanding biological
properties as po~lerful inhibitors of cell mitosis and proliferative
: ~ , , .
- 6 ~
:
.. . .. . .
:~ ~. .. . , ~ .. .
50~3
,. .
1 activity in cultured cells in vitro. They have also shown sub- -
stantial activity on cell trans~ormation induced by oncogenic
viruses. They have an antitumor activity in mice, as shown by an
increase in mean survival time at non-toxlc doses in animals
bearing a number of experimental tumors(Tables 1 to 4) .
rrhe cardiotoxic "in vitro" activity of compound VI is
very low (lower than that of daunomycin) as results from the
following experiment.
The method consists in the cultivation of single cardiac ~ ;
10 cells isolated by tripsinization from heart of new born mice. ;~
After 3~4 days, the cultures~ showing clusters of beating cells r
can be studied both from the point of view of frequency and
rhythm (Necco A., Dasdia T. IRCS r 2 : 1293 r 1974) .
TABLE 1 - Effect of compounds VI and VII on micotic index and
proliferative activity of cultured HeLa cells at
different exposure times.
Results are expressed as percent of untreated controls. ~ ; -
,.,: :: ' ~ . : ''
.. . . .... _ . ., .. , _, . :~
Compound Dose Micotic index _ Colony counts ~ -
~g/ml) 2 h ~ 4 h 8 h 2 h 8 h 2 4 h
- - - ---------~-------.. -_ _ ~ .
0.025 226* 100 117 113 88 48
VI 0.05 189* 103 122 115 56 23 ;
0.1 79 140 0 77 23 3 ~ ~ -
. ........ . ~ .~
0.16 0.0560.027
_. . = .~ . --~ _ _. . ~
0.25 137 103 85 108 86 70
VIl O. S 95 67 8% 101 37 18
1 52 40 0 98 17 6
. .._ . ._ .~. ~
DI50 > 1 0.470.33
--- ..... ~.. ~.. , _;__. .... _ _ '
Dauno- DI50 0. 098 0.036 0.021
30 mycin _ _ _ __ ~,
- :~
*Metaphasic block
.. .. . .
: :
.
~)4~501~3
T~BL~ 2 - Effect of compounds VI and VII on focl formation
and on cell proliferation in cultured mice fibro-
blasts infected with Moloney Sarcoma Virus.
(3 days treatment)
:
Compound Dose Foci formation Cell proliferation B/A
(~Ig/ml) ,
(% of controls) ID50 (% of controls)
(~g/ml) ~g/m
_ ~ . ., . ~ , _
0.0062 51 75
VI o.l25 0 0.006 23 0.013 2.1
= ~ ,. _ , _ . , , . = : ~
0.0062 48 87 ~ 5 ~'
VII 0 025 44 0.01 28 6.4
0.4 0 14
_, .. .~. . . ' ' . _ ~ .
Daunomy-
cin _ 0.006 0~0086 1.4
'
-;
TABLE 3 - Effect of compounds VI and VII on the mean
survival time and on the number of survivors in ~ -
female mice (Swiss CDI) inoculated intraperitoneously
with Sarcoma 180 ascites (1 x 106 cells/mouse).
Compounds were administered on the day following the
transplantation of the tumour.
~, '
- 8 -
. , , -: .. ; ~ ~
: . : .: . . . . . .
4~`50~3
.. . .. . .. .. .. ..
` . . ........ .. . . ........ .
. . .. _~ ,__ ... . __~ ~ ~._
Compound Dose Mean survival time Number of Survivors
(mg/k~)(% of controls) on day 60
. , . _ .. ... .__ _ . . _ ., . . _ .
0.22111.1 1/10 :
VI 1.1120.8 1/10
5.7174.5 0/10 :
. -- . . .. ... __ ... ~ .. . . .
0.26 96.6 0/10 :~.
VII 1.3114.8 1/10 :~
... _. ~ / 118 6 1/10
6/10 animals died as a consequence of drug toxicity ::~
TABLE 4 - Effect of compounds VI and VII on Gross transplantable ;~
. leukemia. Mean survival time of C3H female mice inocu~
.. . .
lated intravenously with a suspens.ion of leukemic lym- '..
phonodes and spleen (2.5 x 106 cells/mouse). Compounds ;~
were administered once daily for five days starting on ~`~
the day following the transplantation of the tumour. .
. .. . . ._ ..... .- - ~
Compound Daily dosage Mean Survival. time : :-
. (m~/kg) (% of controls) : , .. _ . . .. __ _ . _ ._ _ . ... . .. _, .
1.5 115 .
2.25 143
: VI 3 162 ..
. 3.75 122
4.5 120 ~ :
. ' . ~ . .......... , _ ~ ~;
1.5 106
VII 2.25 102
. _ ~ _ . ............. 121 .
_ g - ,~
., ~ ;.
:: - , . ~
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