Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
Le A 33 599-FC
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Process for preuaring ~lycoconiusates of 20(S)-camptothecin
The present invention relates to a process for preparing glycoconjugates of
20(S)-camptothecin in which a 3-O-methylated f3-L-fucose building block is
attached
to the 20-hydroxyl group of a camptothecin derivative via thiourea-modified
peptide
spacers.
20(S)-Camptothecin is a pentacyclic alkaloid which was isolated in 1966 by
Wall et
al. (J.Am.Chem.Soc. 88, 3888 (1966)). It has a high antitumor activity
potential in
numerous in vitro and in vivo tests. Unfortunately, however, the promising
potential
failed to be realized in the clinic because of toxicity and solubility
problems.
By opening the E ring lactone and formation of the sodium salt, a water-
soluble
compound was obtained which is in a pH-dependent equilibrium with the ring-
closed
form. Here too, clinical studies have been unsuccessful until now.
O p OH
7 17
N~ / ~ \ N ~ ONa
i
\ 1s N 2 s\ ~~"~~ O ~ ~'' N \ ~~~~~~ O
1z 1 ~19 20
H3 a OH HaC OH
Approximately 20 years later, it was found that the biological activity is to
be
attributed to an enzyme inhibition of topoisomerase I. Since then, the
research
activities have been increased again in order to find camptothecin derivatives
which
are more compatible and active in vivo.
To improve the water-solubility, salts of A ring- and B ring-modified
camptothecin
derivatives and of 20-O-acyl derivatives having ionizable groups have been
described
(Vishnuvajjala et al. US 4943579). The latter prodrug concept was later also
applied
to modified camptothecin derivatives (Wani et al. WO 9602546). In vivo,
however,
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the 20-O-acyl prodrugs described have a very short half-life and are very
rapidly
cleaved to give the parent structure.
WO 9631532 describes sugar-modified cytostatics and processes for their
preparation
where improved tumor selectivity is achieved by linking different cytotoxic or
cytostatically active compounds having, for example, regioselectively modified
carbohydrate building blocks via certain spacers.
WO 9851703 describes specific glycoconjugates of 20(S)-camptothecin of the
formula (I) and processes for their preparation:
O
/ / ~ ~N ~ ~O
\N ~ O
H3C O
R, (I)
O
H3C ,,~, O ~,, O ~ SHN O
,
HO ~~~ OH / ~ N R2
H
O ~ CH3
in which
R1 is a sterically demanding non polar side chain of an amino acid and
R' is a basic side chain of an amino acid.
Surprisingly, it has been found that the particular construction of the
camptothecin
derivatives described therein, i.e. the attachment of 3-position-modified f3-L-
fucose
building blocks via a thiourea-modified peptide spacer consisting of a
sterically
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demanding nonpolar and a basic amino acid to the 20-hydroxyl group of 20(S)-
camptothecin leads to very particularly preferred conjugates which, compared
to the
prior art compounds, have particularly high stability, better solubility in
water, better
compatibility, greater therapeutic efficacy against various tumors both in
vitro and
in vivo and considerably higher tumor selectivity, in particular with a view
to bone
man:ow toxicity.
WO 9851703 describes two processes for preparing compounds of the formula (I).
According to the first process, the glycoconjugates of the formula (I)
according to the
invention are prepared by sequential attachment of 20(S)-camptothecin to two
appropriate amino acids via a peptidyl-camptothecin of the formula (II) to
give a
peptidyl-camptothecin of the formula (III), and subsequent attachment of the
isothiocyanate of the formula (IV) (linear synthesis):
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-4-
OH -
O
/ / I ~N I ~O
\ \N \ O
HC
H3C~... O ,..~0 \
HO~~~~ OH / N=C=S
O
~Me (IV)
R~~n (1)
HsC~.., O .,,,0 \
HO ~~~ OH
O~Me
where R' and R2 are as defined above.
However, this process has the disadvantage that the starting material used is
toxic
camptothecin and the further steps of the process give in each case a
camptothecin
_ derivative which is not toxicologically safe.
Accordingly, in WO 9851703, a second process for preparing the compounds of
the
formula (I) has been proposed in which the isothiocyanate of the formula (IV)
is
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-S-
initially attached to an optionally protected terminal basic amino acid to
give a
building block of the formula (V) and this building block is then reacted with
the free
amino group of the amino acid conjugate of the formula (II) formed by 20(S)- -
camptothecin and a nonpolar sterically demanding amino acid (convergent
synthesis):
O
H3C,,~~ O ~~~.0
/ w N o
HO~~~~ OH / N=C=S ~ N ~ O
O~Me OH
(IV)
H N' 'COOH HZN COOH
Y'z
z
R
O
Me,,,, / ~ ~ ~N
O ~~,,0 ~ OOH
/ ~ ~ z \ N \ O
HO ~~~ OH ~ H R
O~Me (V) (II) R~O
/ NHz
H3C'~~,
HO
O
~Me
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In this process, the preparation of the building block (V) does not involve
any
camptothecin derivative intermediates which are toxicologically unsafe, owing
to
which this process is preferred to the first preparation process, for reasons
of safety
and the associated reduction in the safety measures required. Moreover, in the
convergent synthesis route, the longest reaction sequence comprises two steps,
which, compared to the three-step linear synthesis route, means that one
reaction step
is saved, which is associated with economical advantages.
The key step in the second process is the coupling of building blocks (II) and
(V). For
this purpose, the carboxyl group of building block (V) is activated and then
reacted
with the free amino group of building block (II). In WO 9851703, the carboxyl
group
is activated using coupling agents known from peptide chemistry, as described,
for
example, in Jakubke/Jeschkeit: Aminosauren, Peptide, Proteine [Amino acids,
Peptides, Proteins]; Verlag Chemie 1982 or Tetrahedr. Lett. 34, 6705 (1993).
Examples which may be mentioned are N-carboxylic anhydrides, acid chlorides or
mixed anhydrides, adducts with carbodiimides, for example N,N'-diethyl-,
N,N'-diisopropyl-, N,N'-dicyclohexylcarbodiimide, N-(3-dimethylamino-
propyl)-N'-ethyl-carbodiimide hydrochloride, N-cyclohexyl-N'-(2-
morpholinoethyl)-
carbodiimide metho-p-toluenesulfonate, or carbonyl compounds, such as
carbonyldiimidazole, or 1,2-oxazolium compounds, such as 2-ethyl-5-phenyl-1,2-
oxazolium-3-sulfate or 2-tert-butyl-5-methyl-isoxazolium perchlorate, or
acylamino
compounds, such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or
propanephosphonic anhydride, or isobutyl chloroform, or benzotriazolyloxy-tris-
(dimethylamino)-phosphonium-hexafluorophosphate, 1-hydroxybenzotriazole or
N-hydroxysuccinimide esters. Furthermore, it is proposed to employ the amino
acid
component in the form of a L.euchs' anhydride.
However, it was found that, using the coupling agents disclosed in WO 9851703,
coupling of building blocks (II) and (V) could only be effected with moderate
to poor
yields, if at all, in particular with increasing size of radicals R~ and R2.
In particular
in the case where RZ represents the side chain of the amino acid histidine, a
large
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number of side reactions are observed (for example epimerization,
intramolecular
thiolysis of the activated building block (V), other acylations on the
building block
(II), etc.), which either prevent the desired coupling reaction altogether or
result in
very poor yields. If the coupling is carried out, for example, using the
coupling agenf
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI), the
yields
that are obtained are below 10%. Likewise, when the coupling is carried out in
the
presence of benzotriazolyloxy-tris-(dimethylamino)-phosphonium hexafluoro-
phosphate (BOP), there are considerable side reactions of the carboxyl
component
(V), and only very low conversion into the target compound (I).
Accordingly, it was an object of the present invention to improve the
abovementioned convergent process for preparing the compounds of the formula
(I)
and, in particular, to make the process feasible for a greater range of
different radicals
R~ and R2.
Surprisingly, it has been found that, if certain coupling agents are used for
the step of
coupling building blocks (II) and (V), the abovementioned second preparation
process can be carried out with all customary amino acids, giving moderate to
good
yields.
Accordingly, the present invention relates to a process for preparing
compounds of
the formula (I)
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_g_
O
. ~ ~ .
~N ~ O
H3C O _
(I)
O
H3C ,,~, O ~~, O ~ SHN O
,,~~ ~ /
HO OH
O~CH3
in which
R1 represents a sterically demanding nonpolar side chain of an amino acid and
R2 represents a basic side chain of an amino acid.
where the isothiocyanate of the formula (IV)
H3C,,~, O ~~,.0
(IV)
HO OH N=C=S
O~Me
is reacted with an optionally protected terminal basic amino acid
H2N\ 'COON
~R2
in which R2 is as defined above,
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to give an amino acid conjugate of the formula (V)
Men,,, O ~~,, O ~ S OOH
HO ~~ OH ~ NI 'N R2
H H (V)
O
~Me
S in which R2 is as defined above,
which is then reacted with amino acid conjugates of the formula (II),
O
~N ~O
N ~ O
,.
R' O (II)
NH2
in which R1 is as defined above,
the protective group of the side chain is removed and the compounds are, if
appropriate, converted into a suitable salt,
characterized in that
the coupling of the building blocks (II) and (V) is carried out in the
presence of a
coupling agent selected from the group consisting of the compounds below:
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. . - 10 -
+
Me2N NMe Me_N
2
\N PFs 'a _
N'
N + O
+ O~O
Me2N +
NMe2 ~\ ,NJ
C~~-P
\N PF6 PF6 N
+ o_ o
Particular preference is given to using the coupling agent N-[(dimethylamino)-
1H
1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethaneaminium hexa
fluorophosphate N-oxide (HATU):
MaN
Me2
PF6
According to the invention, it is furthermore preferred to use, in the above
process for
preparing compounds of the formula (I), amino acids where R1 represents a
branched
alkyl radical having up to 4 carbon atoms and R' represents a radical of the
formula
-(CH2)~ R3, where
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_ J
R3 denotes -NH2,_ N or NH NH2 and n represents a number from 1
to 4. _
Here, particular preference is given to using amino acids in which R1
represents a
branched alkyl radical of the formulae
CH3 H3
~CH3 , CHs or CH3
and R2 represents a
radical of the formulae
NH
-(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2, ~~ or
-CH2 N
NH
~ .
-(CH2)3 NH' 'NH2
According to the most preferred embodiment of the present invention, in the
above
preparation process the isothiocyanate of the formula (IV) is reacted with
optionally
protected lysine or histdine, preferably unprotected histidine, and the
camptothecin is
reacted with valine. The resulting building blocks (V) and (II) are coupled in
the
presence of N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-
N-
methylmethaneaminium hexafluorophosphate N-oxide (HATU) followed, if
appropriate, by removal of the protective groups, giving, according to the
most
preferred embodiment of the process according to the invention, as compound of
the
formula (I) 20(S)-20-O-{Na-[4-(3-O-methyl-13-L-fucopyranosyl-oxy)-
phenylaminothiocarbonyl]-L-histidyl-L-valyl}-camptothecin or 20(S)-20-O-{Na-[4-
(3-O-methyl-Li-L-fucopyranosyl-oxy)-phenylaminothiocarbonyl]-L-lysyl-L-valyl }-
- camptothecin, particularly preferably 20(S)-20-O-{Na-[4-(3-O-methyl-a-L-
fucopyranosyl-oxy)-phenylamino-thiocarbonyl]-L-histidyl-L-valyl }-
camptothecin.
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Coupling of the first amino acid to camptothecin, with formation of building
block
(II), may result in the formation of mixtures of diastereomers. Pure
diastereomers of
the compounds according to the invention can be prepared by the processes
described
above, for example, by separating the diastereomers in a suitable manner after
the
S first amino acid building block has been attached to the camptothecin and
the
protective groups have been removed. Using a diastereomerically pure
intermediate
(II), it is possible to prepare the diastereomerically pure target compound
(I) by the
route described above.
Mixtures of diastereomers may also be formed when building blocks (II) and (V)
are
coupled. These diastereomers can be separated from one another on the stage of
the
salt-free glycoconjugate of (I), either by column chromatography or by
crystallization
or digestion methods. Preference is given to saturation with methanol or
precipitation
from dichloromethane/methanol using diethyl ether or methyl-t-butyl ether.
The individual steps of the above preparation process according to the
invention can
be carried out under various pressure and temperature conditions, for example
at
from 0.5 to 2 bar and preferably under atmospheric pressure, and/or at from -
30 to
+100°C and preferably from -10 to + 80°C, in suitable solvents,
such as
dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, chloroform,
lower alcohols, acetonitrile, dioxane, water or in mixtures of the solvents
mentioned.
In general, preference is given to reactions in DMF, dichloromethane, THF,
dioxane/water or THF/dichloromethane, at room temperature or with ice-cooling,
and
at atmospheric pressure.
The isothiocyanate of the formula (IV) used as starting material can be
prepared, for
example, according to the process described in WO 98/51703 from commercial
p-nitrophenyl ~i-L-fucopyranoside by selective etherification of the hydroxyl
group in
the 3-position of the saccharide radical using methyl iodide and dibutyltin
oxide,
reduction of the nitro group by catalytic hydrogenation and subsequent
reaction with
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a thiocarbonic acid derivative such as, for example, thiophosgene or
thiocarbonyl-
bisimidazole, in the presence of a base such as ethyldiisopropylamine.
Suitable coupling agents for activating the carboxyl groups are the coupling
agents
known from peptide chemistry, as described, for example, in Jakubke/Jeschkeit:
Aminosauren, Peptide, Proteine [Amino acids, peptides, proteins]; Verlag
Chemie
1982 or Tetrahedr. Lett. 34, 6705 (1993). Preference is given, for example, to
N-
carboxylic anhydrides, acid chlorides or mixed anhydrides.
The carboxyl groups can also be activated by forming adducts with
carbodiimides,
for example N,N'-diethyl-, N,N'-diisopropyl-, N,N'-dicyclohexylcarbodiimide,
N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride, N-cyclohexyl-
N'-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, or carbonyl
compounds, such as carbonyldiimidazole, or 1,2-oxazolium compounds, such as
2-ethyl-5-phenyl-1,2-oxazolium-3-sulfate or 2-tent-butyl-5-methyl-isoxazolium
perchlorate, or acylamino compounds, such as 2-ethoxy-1-ethoxycarbonyl-1,2-
dihydroquinoline, or propanephosphonic anhydride, or isobutyl chloroform, or
benzotriazolyloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate,
1-hydroxybenzotriazole or N-hydroxysuccinimide esters.
Furthermore, the amino acid component can also be employed in the form of a
Leuchs' anhydride. This type of amino acid activation is preferred when 20(S)-
camptothecin is acylated with amino acid components.
As mentioned above, the coupling of the building blocks (II) and (V) requires
a
particular coupling agent selected from the group which consists of the
following
compounds:
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- 14-
+ +
MPN MPN
NMe,
PF6 , - _
+ O~O
MP ' ~(N
,, NJ
- ~l,i~
r6 N O
O
Particularly preferably, the coupling agent used here is N-[(dimethylamino)-1H-
1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethaneaminium
hexafluorophosphate N-oxide (HATU):
Me2N
NMe2
\ PF6
/ NN
N + \O _
These particular coupling agents are commercially available. HATU, for
example,
can be obtained from Perseptive Biosystems GmbH, Wiesbaden, Germany.
Bases suitable for use in the individual steps of the preparation process
according to
the invention are, for example, triethylamine, ethyl-diisopropylamine,
pyridine,
N,N-dimethylaminopyridine or other bases customarily used in such steps.
Suitable protective groups for the third function of the amino acids are the
protective
groups known from peptide chemistry, for example of the urethane, alkyl, acyl,
ester
or amide type.
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In the context of the invention, amino protective groups are the amino
protective
groups customarily~used in peptide chemistry.
These preferably include: benzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,
3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,
4-methoxybenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-
nitrobenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl, tert-
butoxycarbonyl (Boc), allyloxycarbonyl, vinyloxycarbonyl, 3,4,5-
trimethoxybenzyloxycarbonyl, phthaloyl, 2,2,2-trichloroethoxycarbonyl,
2,2,2-trichloro-tert-butoxycarbonyl, menthyloxycarbonyl, 4-
nitrophenoxycarbonyl,
fluorenyl-9-methoxycarbonyl (Fmoc), formyl, acetyl, propionyl, pivaloyl,
2-chloroacetyl, 2-bromoacetyl, 2,2,2-trifluoroacetyl, 2,2,2-trichloroacetyl,
benzoyl,
benzyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, phthalimido,
isovaleroyl
or benzyloxymethylene, 4-nitrobenzyl, 2,4-dinitrobenzyl, 4-nitrophenyl or
2-nitrophenylsulfenyl. Particular preference is given to the Fmoc group and
the Boc ,
group.
Removal of protective groups in the corresponding reaction steps can be
effected, for
example, by action of acid or base, hydrogenolytically or by another reductive
method.
The camptothecin building block used as starting material can be present in
the
20(R)- or the 20(S)-configuration or as a mixture of these two stereoisomeric
forms.
Preference is given to the 20(S)-configuration.
The amino acids used in the process according to the invention can occur in
the L- or
in the D configuration or else as a mixture of D- and L-form.
According to the invention, the term "amino acids" denotes in particular
naturally
occurring a-amino acids but additionally includes their homologs, isomers and
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derivatives. Examples of isomers which may be mentioned are enantiomers.
Derivatives are, for example, amino acids carrying protective groups.
Amino acids having "sterically demanding" side chains are to be understood as
S meaning amino acids whose side chain is branched in the (3- or y-position;
examples
which may be mentioned are valine and isoleucine or leucine.
Typical examples of amino acids having nonpolar side chains are:
alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine,
methionine.
Typical examples of amino acid having basic side chains are:
lysine, arginine, histidine, ornithine, diaminobutyric acid.
The compounds according to the invention are preferably present in the form of
their
salts. In general, salts with organic or inorganic acids may be mentioned
here. These
salts can be prepared by reacting the free compounds of the formula (I) with
organic
or inorganic acids. According to the invention, preferred acids here are
hydrohalic
acids, such as, for example, hydrochloric acid and hydrobromic acid, in
particular
hydrochloric acid, furthermore phosphoric acid, nitric acid, sulfuric acid,
mono- and
bifunctional carboxylic acids and hydroxycarboxylic acids, such as, for
example,
acetic acid, trifluoroacetic acid, malefic acid, malonic aicd, oxalic acid,
gluconic acid,
succinic acid, fumaric acid, tartaric acid, citric acid, salicylic acid,
sorbic acid and
lactic acid, and also sulfonic acids, such as, for example p-toluenesulfonic
acid, 1,5-
naphthalenedisulfonic acid or camphorsulfonic acid.
Below, the present invention is illustrated by nonlimiting examples and
comparative
examples.
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Examples
In the examples below, all amounts are based on percent by weight, unless
indicated
otherwise.
1 Pr~aration of building block (V)
1 a) p-Aminophenyl-3-O-methyl J3-L fucopyranoside:
H3C ''~~~ O ~''' O /
HO ~ OH NH2
OCH3
6 g (21 mmol) of p-nitrophenyl-Li-L-fucopyranoside in 300 ml of absolute
methanol
are treated with 7.84 g (31.5 mmol) of dibutyltin oxide and heated at reflux
for 2 h.
The mixture is then concentrated and the residue is dried and then taken up in
300 ml
of DMF. 15.7 ml of methyl iodide are added, and the mixture is then stirred at
70°C
for 40 h. The solvent is removed under reduced pressure and the residue is
taken up
in 300 ml of dichloromethane. The suspension is filtered and the remaining
solution
is reconcentrated and subjected to flash chromatography
(dichloromethane/methanol
99:1). Concentration gives 3.82 g (83%) of the target product.
3.81 g (12.73 mmol) of the p-nitrophenyl-3-O-methyl-f3-L-fucopyranoside
obtained
in this manner are dissolved in methanol and, after addition of palladium-on-
carbon
(10%), hydrogenated in an atmosphere of hydrogen, at slightly superatmospheric
pressure. The catalyst is filtered off and the product is precipitated with
ether, giving
3 g (88%) of the target product. [TLC: dichloromethane/methanol 9:1 R~ 0.53J.
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1 b) p-Isothiocyanatophenyl-3-O-methyl J3-L-fucopyranoside (IV):
H3C~,~, O , ~~O ~
(~V)
HO~~~, OH N=C=S
O
~Me
A solution of 6.8 g (25.3 mmol) of the p-aminophenyl-3-O-methyl-(3-L-
fucopyranoside obtained according to la) in 600 ml of dioxane/water 1:1 is
stirred,
and 2.72 ml of thiophosgene (1.4 equivalents) are added. After 10 min, 26 ml
of
ethyldiisopropylamine are added and the mixture is stirred at RT for another 5
min
and then concentrated under reduced pressure to a volume of 150 ml. 800 ml of
dichloromethane are added and the phases are separated. The organic phase is
washed twice with water, dried over sodium sulfate and concentrated. The
residue is
stirred into 200 ml of methyl-ten-butyl ether and 200 ml of petroleum ether
and
filtered off with suction. This gives 7.26 g (92%) of the isothiocyanate.
1 c) Na (4-(3-O-methyl f3-L fi~copyranosyl-oxy)-phenylantino-thiocarbonylJ-L-
Itistidine (V):
Me~,,, O '~,, O
(~)
HO ~~~ ~~OH ~1
O~Me
A solution of 10 g (0.0321 mol) of the isothiocyanate obtained in lb) and 4.98
g
(0.0321 mol) of L-histidine are suspended in 400 ml of dioxane/water 1:1 and
treated
with 11 ml of N-ethyldiisopropylamine. The mixture is stirred at room
temperature
for 16 h and then concentrated under reduced pressure and redistilled with
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dichloromethane/methanol 1:1. The crude product is dissolved in 200 ml of
methanol
and added dropwise to 1 1 of methyl-t-butyl ether (MTBE). The residue is
filtered off,
washed with MTBE and dried under reduced pressure. The target product is
obtained -
in a yield of 95% [TLC: acetonitrile/water/glacial acetic acid 5:1:0.2 Rf=
0.14].
2. Preparation of building block (II):
2a) 20(S)-20-O-(N-(ten-butoxycarbonyl)-1.lD-valylJ-camptothecin:
'O
O~NH
O'"t-Bu
With stirring, a suspension of 10 g (28.7 mmol) of 20(S)-camptothecin in 500
ml of
absolute dichloromethane is treated with 14 g (2 equivalents) of N-(tent-
butoxy-
carbonyl)-valine-N-carboxylic anydride and 1 g of 4-(N,N-dimethylamino)-
pyridine.
The mixture is heated at reflux for 4 days and then concentrated under reduced
pressure. The residue and 100 ml of MTBE are stirred for 20 minutes. 200 ml of
petroleum ether are then added, and the mixture is filtered. This gives 14.9 g
(95%)
of the target compound. [TLC: acetonitrile Rf = 0.34].
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2b) 20(S)-20-O-L-Valyl-camptothecin, trifluoracetate (11):
A solution of compound 2a (11.65 g, 21 mmol) in a mixture of 300 ml of
dichloromethane and 70 ml of anhydrous trifluoroacetic acid is stirred at
5°C for 1 h.
Under reduced pressure, the volume of the mixture is reduced, and the product
is
then precipitated using diethyl ether and washed thoroughly with diethyl
ether. The
product is reprecipitated from dichloromethane/methanol using diethyl ether.
If
required, the crude product is taken up once more in 40 ml of methanol, mixed
with
120 ml of methyl-t-butyl ether and cooled to 0°C. The precipitate is
filtered off,
giving 9.4 g (80%) of the desired compound [TLC: acetonitrile/water 20:1 Rf =
0.39].
'O
NH2
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-21-
3. Synthesis of the compound (I)
3a) 20(S)-20-O-(Na (4-(3-O-methyl f3-L-fucopyranosyl-oxy)-phenylamino-
thiocarbonylJ- L-histidyl-L-valylJ-camptothecin
1.04 g (1.96 mmol) of Na-[4-(3-O-methyl-B-L-fucopyranosyl-oxy)-phenylamino-
thiocarbonyl]-L-histidine (V, Example 1) and 1 g (1.78 mmol) of 20(S)-20-O-L-
valyl-camptothecin trifluoroacetate (II, Example 2) are dissolved in 35 ml of
dimethylformamide, and the mixture is cooled to 0°C and then treated
with 1.35 g
(3.56 mmol) of N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-
methylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU) and
616 p,l of N-ethyldiisopropylamine. The mixture is stirred at 0°C
overnight and then
added dropwise to 400 ml of MTBE. The residue is filtered off and then taken
up in
100 ml of methanol and 5 ml of DMF. 3 ml of a 17% strength aqueous ammonia
solution are added and the mixture is stirred for 10 minutes. The mixture is
then
added dropwise to 500 ml of MTBE. The residue is filtered off, washed with
MTBE
and then stirred with 25 ml of water. After 15 minutes, the residue is
recollected and
WO 00/53614 PCT/EP00101480
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_22_
dried overnight. The resulting crude product consists essentially of a mixture
of
diastereomers (L-histidine and D-histidine epimer of the target compound). The
D-
histadine epimer,which is obtained as byproduct, is removed by stirring the
crude
product with 35 ml of methanol for 2 hours. The precipitate is then filtered
off and
subjected twice more to this purification operation. This gives 819 mg (51%)
of the
diastereomerically pure target product [TLC: acetonitrile/waterlglacial acetic
acid
5:1:0.2 Rf= 0.38].
3b) 20(S)-20-O-(Na (4-(3-O-methyl-/.i-L fucopyranosyl-oxy)-phenylamino-
thiocarbonylJ- L-histidyl-L-valylJ-camptothecin, hydrochloride
N
H3
H3C~,,~ O ~~,, O ~ S H N O
.,, ~ / .,,.
HO ~OH ~ '
O
~CH3
HCI i_
806 mg (0.9 mmol) of the compound from Example 3a are suspended in 40 ml of
water and converted into the hydrochloride using 0.86 ml (0.95 equivalents) of
a 1M
hydrochloric acid solution. With stirring, a solution is formed, and this
solution is
then lyophilized. This gives 814 mg (97%) of the target compound [TLC:
acetonitrile/water 10:1, Rf=0.15, [a]ZZp=-37.6° (c=0.21 DMF)].].
