CA2366632A1 - Method for producing glyco-conjugates of 20(s)-camptothecin - Google Patents
Method for producing glyco-conjugates of 20(s)-camptothecin Download PDFInfo
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- CA2366632A1 CA2366632A1 CA002366632A CA2366632A CA2366632A1 CA 2366632 A1 CA2366632 A1 CA 2366632A1 CA 002366632 A CA002366632 A CA 002366632A CA 2366632 A CA2366632 A CA 2366632A CA 2366632 A1 CA2366632 A1 CA 2366632A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B53/00—Asymmetric syntheses
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The invention relates to a method for producing glyco-conjugates of 20(S)- camptothecin in which a 3-O-methylated .beta.-L-fucose building block is linked to the 20 hydroxyl group of a camptothecin derivative via thiourea- modified peptide spacers, whereby the decisive step exists in the linking of the building block (II) with the building block (V) in the presence of a particular coupling reagent such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5 - b]pyridin-1-yl-methylene]-N-methylmethane-aminium hexafluorophosphate-N-oxid e (HATU).
Description
Le A 33 599-FC
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
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, ' WO 00/53614 PCT/EP00/01480 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
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 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):
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 -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 _ -6-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 . -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) _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, 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) 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:
' WO 00/53614 PCT/EP00/01480 . . - 10 -+
Me2N NMe Me_N
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
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, ' WO 00/53614 PCT/EP00/01480 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
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 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):
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 -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 _ -6-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 . -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) _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, 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) 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:
' WO 00/53614 PCT/EP00/01480 . . - 10 -+
Me2N NMe Me_N
\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)-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethaneaminium hexa fluorophosphate N-oxide (HATU):
MaN
Me2 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 _ 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 , CHs or CH3 and R2 represents a radical of the formulae NH
-(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2, ~~ or 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.
_12_ 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 . -13-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:
+ +
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.
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 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.
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 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.
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 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].
WO 00/53614 PCTlEP00/01480 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
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 _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
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)].].
+ O~O
Me2N +
NMe2 ~\ ,NJ
C~~-P
\N PF6 PF6 N
+ o_ o Particular preference is given to using the coupling agent N-[(dimethylamino)-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethaneaminium hexa fluorophosphate N-oxide (HATU):
MaN
Me2 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 _ 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 , CHs or CH3 and R2 represents a radical of the formulae NH
-(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2, ~~ or 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.
_12_ 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 . -13-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:
+ +
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.
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 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.
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 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.
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 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].
WO 00/53614 PCTlEP00/01480 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
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 _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
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)].].
Claims (7)
1.~A process for preparing compounds of the formula (I), 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) is reacted with an optionally protected terminal basic amino acid in which R2 is as defined above, to give an amino acid conjugate of the formula (V), in which R2 is as defined above, which is then reacted with amino acid conjugates of the formula (II), 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:
where the isothiocyanate of the formula (IV) is reacted with an optionally protected terminal basic amino acid in which R2 is as defined above, to give an amino acid conjugate of the formula (V), in which R2 is as defined above, which is then reacted with amino acid conjugates of the formula (II), 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:
2. A process as claimed in either of claims l, characterized in that the coupling of the building blocks (II) and (V) is carried out in the presence of N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU)
3. A process as claimed in either of claims 1 or 2, characterized in that amino acids are used in which R1 represents a branched alkyl radical having up to 4 carbon atoms and R2 represents a radical of the formula -(CH2)n-R3, where R3 denotes -NH2, and n represents a number from 1 to 4.
4. A process as claimed in any of claims 1 to 3, characterized in that amino acids are used in which R1 represents a branched alkyl radical of the formulae and R2 represents a radical of the formulae -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2, or
5. A process as claimed in any of claims 1 to 4, characterized in that the isothiocyanate of the formula (IV) is reacted with optionally protected lysine or histdine.
6. A process as claimed in any of claims 1 to 5, characterized in that the isothiocyanate of the formula (IV) is reacted with unprotected histdine.
7. A process as claimed in any of claims 1 to 6, characterized in that the camptothecin is reacted with optionally protected valine.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19909979A DE19909979A1 (en) | 1999-03-06 | 1999-03-06 | Process for the preparation of glycoconjugates of 20 (S) -camptothecin |
| DE19909979.0 | 1999-03-06 | ||
| PCT/EP2000/001480 WO2000053614A1 (en) | 1999-03-06 | 2000-02-23 | Method for producing glyco-conjugates of 20(s)-camptothecin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2366632A1 true CA2366632A1 (en) | 2000-09-14 |
Family
ID=7899996
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002366632A Abandoned CA2366632A1 (en) | 1999-03-06 | 2000-02-23 | Method for producing glyco-conjugates of 20(s)-camptothecin |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1173454A1 (en) |
| JP (1) | JP2002539132A (en) |
| AU (1) | AU3159500A (en) |
| CA (1) | CA2366632A1 (en) |
| DE (1) | DE19909979A1 (en) |
| WO (1) | WO2000053614A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2200617B1 (en) | 2001-01-19 | 2005-05-01 | Almirall Prodesfarma, S.A. | DERIVATIVES OF UREA AS ANTAGONISTS OF INTEGRINAS ALPHA 4. |
| EP2968381A4 (en) * | 2013-03-15 | 2016-11-30 | Sidney Hecht | Sugar-linker-drug conjugates |
| US10046068B2 (en) | 2013-03-15 | 2018-08-14 | Arizona Board Of Regents On Behalf Of Arizona State University | Saccharide conjugates |
| JP2021095424A (en) * | 2018-03-28 | 2021-06-24 | 持田製薬株式会社 | Anti-cancer agent-bonded alginic acid derivative |
| WO2020156189A1 (en) * | 2019-01-30 | 2020-08-06 | 四川科伦博泰生物医药股份有限公司 | Camptothecin derivative and water-soluble prodrug thereof, pharmaceutical composition containing same, preparation method, and use |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ID23424A (en) * | 1997-05-14 | 2000-04-20 | Bayer Ag | GLIKOKONJUGUS OF 20 (S) -CAMPTOTESIN |
-
1999
- 1999-03-06 DE DE19909979A patent/DE19909979A1/en not_active Withdrawn
-
2000
- 2000-02-23 EP EP00909241A patent/EP1173454A1/en not_active Withdrawn
- 2000-02-23 AU AU31595/00A patent/AU3159500A/en not_active Abandoned
- 2000-02-23 JP JP2000604049A patent/JP2002539132A/en active Pending
- 2000-02-23 WO PCT/EP2000/001480 patent/WO2000053614A1/en not_active Application Discontinuation
- 2000-02-23 CA CA002366632A patent/CA2366632A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO2000053614A8 (en) | 2002-01-24 |
| AU3159500A (en) | 2000-09-28 |
| EP1173454A1 (en) | 2002-01-23 |
| JP2002539132A (en) | 2002-11-19 |
| DE19909979A1 (en) | 2000-09-07 |
| WO2000053614A1 (en) | 2000-09-14 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |