CA2112482A1 - Polymer-bound paclitaxel derivatives - Google Patents
Polymer-bound paclitaxel derivativesInfo
- Publication number
- CA2112482A1 CA2112482A1 CA002112482A CA2112482A CA2112482A1 CA 2112482 A1 CA2112482 A1 CA 2112482A1 CA 002112482 A CA002112482 A CA 002112482A CA 2112482 A CA2112482 A CA 2112482A CA 2112482 A1 CA2112482 A1 CA 2112482A1
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- Prior art keywords
- phe
- ala
- gly
- leu
- beta
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
- C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Epoxy Compounds (AREA)
- Medicinal Preparation (AREA)
- Peptides Or Proteins (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Analysing Materials By The Use Of Radiation (AREA)
Abstract
A polymer conjugate consisting essentially of: from 90 to 99.9 mol % of units represented by formula (a); from 0.1 to 5 mol % of units represented by formula (b), wherein one of R1 and R2 is a copolymer residue of formula (c), and the other one is hydrogen atom; from 0 to 9.9 mol % of units represented by formula (d), wherein R is a phenyl or t-butoxy group, R3 is H or an acetyl group, A and A1 which may be the same or different, represent a chemical single bond, an amino acid residue or peptide spacer selected from .beta. Ala, Gly, Phe-Gly, Phe-Phe-, Leu-Gly, Val-Ala, Phe-Ala, Leu-Phe, Leu-Ala, Phe-Leu-Gly, Phe-Phe-Leu, Leu-Leu-Gly, Phe-Tyr-Ala, Phe-Gly-Phe, Phe-Phe-Gly, Phe-Leu-Gly-Phe, Gly-Phe-Leu-Gly-Phe, Gly-.beta.Ala, Phe-Gly-.beta.Ala, Phe-Phe-.beta.Ala, Leu-Gly-.beta.Ala, Val-Ala-.beta.Ala, Phe-Ala-.beta.Ala, Leu-Phe-.beta.Ala, Leu-Gly-.beta.Ala, Phe-Leu-Gly-.beta.Ala, Phe-Phe-Leu .beta.Ala, Leu-Leu-Gly-.beta.Ala, Phe-Tyr-Ala-.beta.Ala, Phe-Gly-Phe-.beta., Phe-Phe-Gly.beta.Ala, Phe-Leu-Gly-Phe-.beta.Ala or Gly-Phe-Leu-Gly-Phe-.beta.Ala. The compounds are endowed with antitumor activity and show improved water solubility and decreased toxicity in comparison with paclitaxel or its known analogs. A method for their preparation and pharmaceutical compositions containing them are also described.
Description
WO 94/Ofll56 PCI`/EPg3/01433 2112 ~l~2 POLYMER-BOUND P~CLITAXEL DERIVATIVES
The present invention is directed to polymer-bound paclitaxel and polymer-bound paclitaxel derivatives endowed with antitumor activity, to a method for their preparation and to pharmaceutical compositions containing them.
Paclitaxel (also named Taxol in several publications) is a member of the ~axane family of diterpenes, isolated and characterized from an extract of bark of Taxus brevifolia L.; other analogues of paclitaxel are also known and were prepared by semisynthesis starting from 10-deacetyl baccatin III, extracted from the needles o~
Taxus baccata L., see Wain et al. in JACS, 93, 2325 " .. ,..,,;- .
(1971) and Lovelle et al., Proc. Am. Assoc. Cancer Res., 31, p. 417, (1990). These compounds have been shown to possess potent antitumor activity, but they are very slightly soluble in water and some toxic side-effects are associated also with their administration by injection or intravenous infusion using as Garrier cremophor EL ~TM).
The present invention provides a polymer conjugate of the formula I consisting essentially of:
from 90 to 99.9 mol % of units of the formula:
~H2 from 0.1 to 5 mol % units represented by the formula WO 94/00156 P~EP93/01433 2'~ ~4~
R O
~C~
~ ~ :
H Rl-O ~ ~C~CH~ ~ -~6 O
wherein one of R~ and R2 is a copolymer residue of the formula l~2 CH3--C-CC)NH-CH2-CO-A~
and the other one is hydrogen atom; ~ --from O to g;9 mol % of units represented by the formula ~;
l~2 `;- -CH3--C-CONH-CH2-CO-A,-NH-CH2-CHO~}-CH3 ~ .:
'~"", ~ '~
', '~ ' ",` '.:
~. ,` `, .
wherein R is a phenyl or t-butoxy group, R3 is ~ or an :~
acetyl group, A and Al which may be the same or ;;
differ~, represent a chemical slngle ~ond, an amino acid residue or peptide spacer selected from ~ Ala, i-Gly, Phe-Gly, Phe-Phe , Leu Gly, Val-Ala, Phe-Ala, Leu~
Phe, Leu-Ala, Phe-Leu-Gly, Phe-Phe-Leu, Leu-Leu-Gly, Phe~
Tyr-Ala, Phe Gly-Phe, Phe-Phe-Gly, Phe-Leu-Gly-Phe, Gly~
Phe-Leu-Gly-Phe,Gly-~Ala, Phe-Gly-~Ala, Phe-ihe-~Ala, `~
W094/OD156 PCT/EP~3/01433 2 I ~ 2 Leu-Gly-~Ala, Val-Ala-~Ala, Phe-Ala-~Ala, Leu-Phe-~Ala, Leu-Gly-~Ala, Phe-Leu-Gly-~Ala, Phe-Phe-Leu ~Ala, Leu-Leu-Gly-~Ala, Phe ~yr-Ala-~Ala, Phe-Gly-Phe-~, Phe-Phe-Gly-~Ala, Phe-Leu-Gly-Phe-~Ala or Gly Phe-Leu-Gly-Phe-~Ala.
More particularly the preSent invention prcvides pol~mer conjugates of paclitaxel and derivatives of paclitaxel with improved wa~er solubility and decreased toxicity.
Preferably, the mol ~ ~f units containing the paclitaxel and paclitaxel derivatives is from 0.5 to 2, more preferably, the content of paclitaxel in the polymer was from 2 to lO ~ (w/w), most preferred compounds are those characterized by a content of from 4 to 7 % ~w/w). The wavy line denotes that the ~xygen linked at position 7 of lS thP paclitaxel structure may be in both configurations, i.e. ~ (natural) or Preferably R represents a phenyl group, R3 is an acetyl group and A is a Phe-Leu-Gly or Phe-Leu-Gly-~Ala residue.
All the amino acid residues have the natural L-20~ configuration. According to a preferred embodiment, the polymer conjugate is a copolymer of l-methacryloylamino-2-hydroxypropane, ~methacryloyl~lycyl-phenylalanyl~
leucylglycyl~3~amino-2 hydroxypropane an~ 2'~
(methacryloylglycylphenylalanylleucylglycyl-~alanyl) paclitaxel.
The % w/w content was determined after enzymatic hydrolysis by HPLC method analogous to that described in Cancer Treatment Rep. 71 (l), 19~7, p. 53~59.
WO~4/001~6 PC~/EP93/01433 æ
Enzymatic hydrolysis To 1 ml of murine or human plasmas various concen~rations of the polymer-bound paclitaxel were added and at appropriate time (24, 48, 72, g6 h) 100 ~1 were collected and stored at -70~C until further processing.
Extraction procedure Samples were extracted by adding 75 ~1 of Tetra Butyl Ammonium Phospha~e (TBAP3 O. 5 M, 1~50 ~1 CH3C~ and 150 ~1 of NaCl SM and vigorously shacked for 20' a~ 4~C.
After that time samples were spun at 15000 x ~ for 10' and the supernatants were collected and evaporated using a high va~uum centrifuge . Samples were recovered by aclding 500 ~1 o~ MeOH:H20 (75:25 V/~ and injected into HPLC for de~erminlng ~he total paclitaxel percentage content. ~ ~
HPLC sy tem ~ `
CGlumn : Nova Pak C~8 (Waters) 3.~ x 300 mm :~
Flow Rate : 1.5 ml/m -:
Detector : W 231 nm ~ :~
2~ Injection : 20 ~
Mobile Phase : 57.5 % HtO brouqht to pH 2 42.5 % CH3CN ;~
The pr.Psent invention also provides a process for ~`~
preparing a polymer conjugate which process comprises reacting a compound of the formula II
, ` ~, ; ~.
WO~4/001S6 PCr/~P93/01433 2 ~ 2 ~ ~ ~
R~ ~
2 OH , oeou~
~360 ;~
: .. . .:
wherein one of A2 and A3 is a chemical bond and the o her one is A, and A, R and R3 are as defined above, with an activated polymer consisting essentially of from 90 to 99.9 mol % of units representecl by formula 1~H2 -CH3 - C-CO-NH-CH2-CHOH-CH3 ~:
'~
and from 10 to 0.1 mol % of units represented by the formula wherein A, is as de~ined above and the eatin~ the resultant polymer conjugate with 2-hydroxypropylamine.
Preferably the reaction between the compounds of ~he formula II and the activated polymer is carried out in an anhydrous polar organic solvent such as dimethylsulfoxide or dimethylformamide optionally in presence of an organic or inoryanic base such as an alkaline carbonate, dimethylaminopyridine or W~9~/00156 , PCT/~P93/0~433 `~i3~J
triethylamine. The reaction can typically be effected for ~`
from 1 to 24 hours.
The reaction is typically carried out at a temperature of from 15 to 40C, preferably at room temperature. The 5 alkaline carbonate is, for example, an alkali metal carbonate or an alkaline earth metal carbonate. :~
Some of the starting compounds of the formula II are .~
known compounds, i.e. paclitaxel or paclitaxel analogues - ;
or may be prepared starting from known compounds. ~
For example 7-epi derivatives may be prepared by .~.
refluxing in toluene paclitaxel or its analogs in the presence of a base (Na2CO3 or diazabicycloundec~ne~ . -Other compounds of the formula II are new, in particular those in which A3 is ~Ala residue and those in which either A2 or A3 represents di,, tri, or te~ra peptide .
spacer as defined above fQr A and are within the scope of , ,.
the invention. ~:
The compounds of formula II wherein A2 is not a chemical - sinyle bond may be prepared ~y reacting a paclitaxel or a 2~ paclitaxel analog with protected amino acid or peptide in the presence of a condensating reagent, and with or without the additional presence of a catalyst, preferably at room .temperature, followed by the removal of the protecting group with known methods.
The condensation may be also carried out using activated esters such as paranitrophenyl ester of peptide or amino acid. Suitable condensing reagents include carbodiimides - such as dicyclohexyl carbodiimide (DCC).
WO94~00l~fi PCT~EP93/0i433 2~
,~
Suitable catalysts include 4-dimethylamino-pyridine (DM~P~, pyridine or triethylamine.
Various known amino protecting groups can be utilized and commercially available protected amino acid or peptides can be utilized as the starting materials. Amino acids or peptides protected with t-BOC, trityl, FMOC or carbobenzyloxy ~CBZ) can be utilized. Amino Acid or peptides procted wlth t-BOC, trityl or FMOC groups are preferred.
The compounds of the formula II wherein A3 is not a chemical single bond may be prepared by protecting or blocking the 2'-hydroxy group and then esterifying the 7-position hydroxyl and then removing the 2'-protecting or blocking group.
More preferably, the compounds of the formula II wherein A3 is Gly or ~Ala residue are prepared by reacting paclitaxel with 2-3 equivalents of N-prote ted amino acid to produce 2',7-disubstituted paclitaxel, the 2'-position amino acid is cleaved and then the 7-position amino acid 2Q is deprotected.
Reaction of paclitaxel and the protected amino a~id is conducted in the presence of a condensing reagent and a catalyst, like those above defined.
,-- , Cleavage o the 2'-amino acid is conducted by adjusking the pH of the 2'-7-(amino acid) paclitaxel solution to pH
7-7.4 for example by mixture of the 2',7-di(amino acid) paclitaxel in a phosphate buffer at pH 7-7.4 or with a slight excess of NaHCO3. ,.
~.;
W~94/0~1~6 PCI`/EP93/0143 ~ 8 -Deprotection of the amino acid is conducted under a known amino acid deprotection method, such as mild acid treatment with, for example, acetic acid, or by reduction.
Thus, for example, paclitaxel is allowed to react with 2-3 mol. equivalent of N-protected amino acid (t.boc, CBZ
or FMOC pro~ected) in methylene chloride in the presence of DCC and a catalytic amount of 4-dimethylaminopyridine.
In this manner, the protected amino acid is introduced at 2' and 7-position. The 2',7-bis amino acid derivative of paclitaxel is allowed to stand in presence of NaHCO3 in H2O/MeOH for 2-5 hours, whereby selertive deprotection at the 2'-position occurs to yield the 7-substi.tuted derivative of paclitaxel. The protecting groups are removed by appropriate deprotecting agent (e.g., acid, mild base or hydrogenolysis).
The activated polymer is a synthetic, water soluble polymer prepared by the copolymerization of N'-(2~
hydroxypropyl) methacrylamide with p-nitrophenylesters 20~ of N-methacryloyl oligopeptides, as described in US-A~
4062~31 and US-A-4097470.
The polymer conjugates of the formula I and the new paclitaxel derivatives of the formula II exhibit good water solubility, biocompatibility and release the paclitaxel or paclitaxel derivative in the plasma or after internalization into ce~ls by cleaving of the oligopep~ide spacers~
WO ~4/()0156 PCI`/EP93/01433 2 1 ~ 2 ~
g :.-.. ;
Biols:~qical ac~ivity Microtubule a~3sembly and disa~sembly a.~say Calf brain tubulin was prepared by two ~ycles of assembly-disassembly ~Shelanski M.L. Gaskin F. and Cantor C.R., Proc. Natl, Acad.Sci. U.S.A. 70, 765-768, 1973) and stored in liquid nitrogen in MAB (0.1 M MES, 2.5 mM EGTA, O.S mM MgSO4, O.1 mM EDTA, 0.1 mM DTT pH 6.4).
All the experiments were carrie~ ou~ on protein stored for less than 4 weeks.
~efore each experiment, the tuhulin was kept 30 min at 4C.
Ass mbly was moni~ored ~y the method of Gaskin et al s-(Gaskin F., Cantor C.R. Shelanski M.L. J. Molec . Biol 89, 737-758, 1974). The cuvette (1 cm path) containing tubulin ~1 mg/ml) and 1 mM GTP was shifted to 37C a~d continous turbidity measurements were made at 340 nm on Perkin-Elmer 557 Double Wavelenght `Double Beam ~:
Spectrophotometer equipped with an automatic recorder and a termostatically regulated sample chamber.
20~ After 30 minutes 4 mM CaCl2 was added and depolymerisation was measured for 10 minutes as decreased turbidity. At regular intervals of 15 minutes scalar doses of. the tested compounds were added and variations in the turbidity were monitored. Data are espressed as percentage of ripolymerisation induced by the tested compounds. The results are shown in Table I.
W~94/~0156 ~ PCT/EP93/0l433 -- 10 -- ,~
In vitro druq sensitivity assay Exponentially growing B16-F10 murine melanoma cells were seeded (2 x 104/ml) in RPMI 1640 medium were supplemented -with 10% heat-inactivated fetal calf serum and 2 mM
glutamine in 24 well-plates (Costar). Scalar concentrations of tested compounds were added immediately . .
after seeding. The inhibition of cell growth was ;~
evalua~ed by counting cells with a coulter~counter after 72 hrs incubation. For each kested compound:conc~ntration :~-triplicate cultures were used. The antiproliferative activity of the tested compounds was calculated from .
dose-response curves and espressed as IC50 (dose causing 50% inhibition cell growth in treated cultures relative to untreated controls).~The resu~lt are shown in Table I.
~ :~ T~ble I
.
Compound prepared in ~ubulin As~embly :%~ ICso 6 : ~ 0.5~M ~ ;s~
Example l ; ~ o : 15~ 19 ~ :~
. ~ . _ : ~ : :., :: Example 9 : ~ 79 145~ 47 .- ~ - - _ Example 3 46 : ~86 ~ 23~ ;
. . . _. __ _ : _ ~ ., ,- ~ .
Example 4 ~ ~10 ~ ~ .~. .
: ~ ~
Example 6 : ~ 0 ~ ~lO~ 51~ : : ,~ .
~ . ,. . . - -- ~ _ :
Reference compound : : ~ ~ ~ ;
Paclltaxel 41 96 39 v~l~
: ; : ^',:,~''''`",-, '`, ~;
WO94/00156 PCT~EP93/01433 2 1 1 2 ~
The copolymer-paclitaxel prepared in Example 6 was tested in vivo agains~ Bl6 FlO murine melanoma in compariYon with paclitaxel.
Mice C57Bl6 female mice were obtained from Charles River ~ , Italy. -Animals were 8 to lO weeks old at the beginning of the experiment.
, Drugs Because of its limited a~ueous solubility, paclitaxel: was dissolved in a vehicle consisting of polyoxyethylated -~
castor oil (Cremophor EL) 50% and ethanol ~50~ and then:
diluted with glucose 5% solution at the desidered concentrations. The isolution was slightly hazy and I5~ precipitates formation~was observed after hort time.~
The compound o~ example 6 was easily dissolved in gluGo~e 5% and the resultlng solutlon was clear:~for:~long time (more than 2 hours). Fi~al concentrations~were referred to the ~paclitaxel conteDt of the compound (4~ of total).
2~ Tumor : The~murine melanoma Bl~6FlO was used. A suspension of :lO~5~
tumor cells in 0.2 ml was injected subcutaneously in the ~ ~-fla~k of.mice.
Tumor size was measured with caliper and tumor weight was calculated with~the formula~
~: a~+b 2 ~
'., .:
~;~.";.., ' ~'. ',:
:~'''''' -WOg4/n0l56 PCT~EP93/01433 ?, 4~q~
- 12 - ~ .
Druqs administration Paclitaxel was given intraperitoneally because of its ;~
poor solubility and vehicle toxicity. ~ :
The compound of example 6 was injected intravenously.
Both compounds were administered at day 1, 5, 9 after tumor implantation.
The data reported in Table 2 show that the compound of the present invention is more active than paclitaxel~
The dose of the polymer-conjugate is referred to the paclitaxel content.
Table 2 _ _- ~
Compound Do~e Tumor Tox -~:
~mg/~g) Inhibition ~%) ,..... _ ~ .. ".. ". :'"": .
Control _ ~ . _ _ Pacli~axel 14.~ 53 0/10 . ,_. _ ~ .. .. _ 22 3~ 0/10 ........ .. _ .. . . _ . _. .. . .. , . ." , . ~.,,., ~. ,,.:
33 ~ 92 l/7 .
. .. _ . .
15' Compound of ex. 6 14.6 77 0/9 ., _ . _ -. -- 22 ~ 0/10 ~ ~
~, .. . ~. ~,.
TOX: number of mice which died for toxicity. ~ ;`
TOX determination was made when mice died before the .
control or when significant body weight loss and/or spleen and or/or liver size reduction were observed. ~;
From the above data, it can be seen that the polymer . ~
' .":~',' W094/OOlS6 21.1 2 ~ ~ 2 PCT/EP93/01433 conjugates of the present invention exhibit excellent antitumor activity. These compounds, therefore, are useful a~titumor agents due to the lower toxicity and :~
increased water solubility as compared to paclitaxel or its derivative. Examples of tumors that can be treated are for istance, sarcomas, carcinoma, lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor, leukemlas and adenocarcinoma. ~::
The improved solubility a~ decreased toxicity of the`
polymer-conjugates of the present invention means that~
they are suitable for intravenous injectisn or infusion.
The dosage dep~nds upon the age, weight and condition of :~
the patient. The dosage may be from l mg/kg body weight to I g/kg body weight, preferably from 4 to 800 mg/kg ~:~
body wei~ht~ Typical formulations contain a quantity of polymer-bound paclitaxel/polymerbound ~ paclitaxel ~ -derivative equivalent~ to 0.5,~l.5t lO, 20, 25, or~50 mg~~:
: of the active pa;clitaxel/pacIitaxel derivative.
The polymer conjugates ~ may~;~ be formulated ~ as 20- pharmaceutical ~compositions with a pharmaceutically acceptable carrier or diluent. Any approprlate carrier:~or -.. :
diluent may be used. The solutlons for intraYenous injection or infusion may contain as carrier:or di~luent, for example, sterile water or preferably they may be ln :
the form of ster~ile, ^ aqueous: or isotonlc saline~
solutions.
The following:examples illustrate the invention.
.."'`;'~, WO94/~0156 PCT/EP93/01433 q~
9~ - 14 -Example 1 Copolymer of l-methacryloylamino-2-hydroxypropane~
1 (methacryloyl-glycyl-phenylalanyl-leucyl-glycyl)amino- -2-hydroxypropane and 2'(methacryloyl-glycyl-phenyl -alanyl-leucyl-glycyl~paclitaxel. -To a solution of 1.4 g of copolymer of 1-methacryloyl~
amino-2-hydroxypropane and N-(methylencarbonyl-Phe Le~
Gly 4-nitrophenoxy)methacrylamide, prepared according to -~
J~ Kopecek et al., Makromol ChPm 177, p. 2833 (1976j, in ~.
15 ml of anhydrous dimethylformamide were added 100 mg of ~-,paclitaxel and 15 mg of dimethylaminopyridine.
The yellow solution was stirred for 8 hours at room temperature under anhydrous conditions. Then 2-hydroxy . .
propylamine (0.2 ml) was droppecl into rea~tion flask, and ~hen the whole was stirred for 30 minutes.
The reaction solution was quenched with 0.3 ml glacial acetic acid, concentrated uhder vacuum to a smal~ volume, and then poured into 200 ml of acetone. ~i Ater 30' mixing, the precipitate was filtered and washed with acetone to yleld 1.25 g of the title compound. ~;
The paclitaxel content was 4.5% (evaluated by enzymatic hydrolysis and HPLC analysis). -Unreact~d paclitaxel was recovered from acetone solu~ion.
~xample 2 2'(N-trityl-phenylalanyl-leUCyl glycyl)paclitaxel ~
To a solution of 170 mg of paclitaxel in 16 ml of ;`
acetonitrile were added 24 mg of dimethylamino-pyridine and 150 mg of N-trityl-phenylanyl-leucyl-glycine 4-W094/00156 2 ~ 1 2 4 8 2 PCT/EP93/01433 nitrophenyl ester.
The yellow solution was stirred for 20 hours at room temperature, and then evaporated under vacuum to dryness.
The residue was cromatographed on silica gel with ethyl- . ;
acetate-hexane 35:25 as eluant, affording 3~0 mg of the :~
title compound. t ' ' ~ ' H-NMR(400 MHz, CDCl3)~
0.82 (d, J-6.4 Hz, 3H, ~-Leu) 0.~5 (d, ~=6.7 Hz, 3H, ~-Leu) 1.15 (s, 3H, 16) 1.26 (s, 3~, 17) ~ 1.6 (m. 3H, ~+~+~-Leu~ .
, - .
1.69 (s, 3H, l9) ;., .
1.85 (s, lH, OH~
15 ~ 1.89 (m, lH, 6~
1.9~ (d, J-1.2 Hz, 3H, 18):
2.14 (dd, J=5.9:Hz~:, J=l3.5 Hz, lH~ Phe);~
~ ~ 2.24 (s, 3H, CH3CO-10)~
: ~ ~ 2.2-2.7 (m, 5H, CH2-l4+0H-7+6:~+~Phe+NH~Ph;e) : 2.47 (s, 3H, CH3CO-4) 3.50 (m, lH, ~-Phe) ;;~
3.74 (dd, J=4.7 Hz, J=18.2 Hz, lH, ~-Gly) 3.~0 (~j lH, ~-Leu):~
3.83 (d, J=7.0~Hz,~lH, 3:) ;
The present invention is directed to polymer-bound paclitaxel and polymer-bound paclitaxel derivatives endowed with antitumor activity, to a method for their preparation and to pharmaceutical compositions containing them.
Paclitaxel (also named Taxol in several publications) is a member of the ~axane family of diterpenes, isolated and characterized from an extract of bark of Taxus brevifolia L.; other analogues of paclitaxel are also known and were prepared by semisynthesis starting from 10-deacetyl baccatin III, extracted from the needles o~
Taxus baccata L., see Wain et al. in JACS, 93, 2325 " .. ,..,,;- .
(1971) and Lovelle et al., Proc. Am. Assoc. Cancer Res., 31, p. 417, (1990). These compounds have been shown to possess potent antitumor activity, but they are very slightly soluble in water and some toxic side-effects are associated also with their administration by injection or intravenous infusion using as Garrier cremophor EL ~TM).
The present invention provides a polymer conjugate of the formula I consisting essentially of:
from 90 to 99.9 mol % of units of the formula:
~H2 from 0.1 to 5 mol % units represented by the formula WO 94/00156 P~EP93/01433 2'~ ~4~
R O
~C~
~ ~ :
H Rl-O ~ ~C~CH~ ~ -~6 O
wherein one of R~ and R2 is a copolymer residue of the formula l~2 CH3--C-CC)NH-CH2-CO-A~
and the other one is hydrogen atom; ~ --from O to g;9 mol % of units represented by the formula ~;
l~2 `;- -CH3--C-CONH-CH2-CO-A,-NH-CH2-CHO~}-CH3 ~ .:
'~"", ~ '~
', '~ ' ",` '.:
~. ,` `, .
wherein R is a phenyl or t-butoxy group, R3 is ~ or an :~
acetyl group, A and Al which may be the same or ;;
differ~, represent a chemical slngle ~ond, an amino acid residue or peptide spacer selected from ~ Ala, i-Gly, Phe-Gly, Phe-Phe , Leu Gly, Val-Ala, Phe-Ala, Leu~
Phe, Leu-Ala, Phe-Leu-Gly, Phe-Phe-Leu, Leu-Leu-Gly, Phe~
Tyr-Ala, Phe Gly-Phe, Phe-Phe-Gly, Phe-Leu-Gly-Phe, Gly~
Phe-Leu-Gly-Phe,Gly-~Ala, Phe-Gly-~Ala, Phe-ihe-~Ala, `~
W094/OD156 PCT/EP~3/01433 2 I ~ 2 Leu-Gly-~Ala, Val-Ala-~Ala, Phe-Ala-~Ala, Leu-Phe-~Ala, Leu-Gly-~Ala, Phe-Leu-Gly-~Ala, Phe-Phe-Leu ~Ala, Leu-Leu-Gly-~Ala, Phe ~yr-Ala-~Ala, Phe-Gly-Phe-~, Phe-Phe-Gly-~Ala, Phe-Leu-Gly-Phe-~Ala or Gly Phe-Leu-Gly-Phe-~Ala.
More particularly the preSent invention prcvides pol~mer conjugates of paclitaxel and derivatives of paclitaxel with improved wa~er solubility and decreased toxicity.
Preferably, the mol ~ ~f units containing the paclitaxel and paclitaxel derivatives is from 0.5 to 2, more preferably, the content of paclitaxel in the polymer was from 2 to lO ~ (w/w), most preferred compounds are those characterized by a content of from 4 to 7 % ~w/w). The wavy line denotes that the ~xygen linked at position 7 of lS thP paclitaxel structure may be in both configurations, i.e. ~ (natural) or Preferably R represents a phenyl group, R3 is an acetyl group and A is a Phe-Leu-Gly or Phe-Leu-Gly-~Ala residue.
All the amino acid residues have the natural L-20~ configuration. According to a preferred embodiment, the polymer conjugate is a copolymer of l-methacryloylamino-2-hydroxypropane, ~methacryloyl~lycyl-phenylalanyl~
leucylglycyl~3~amino-2 hydroxypropane an~ 2'~
(methacryloylglycylphenylalanylleucylglycyl-~alanyl) paclitaxel.
The % w/w content was determined after enzymatic hydrolysis by HPLC method analogous to that described in Cancer Treatment Rep. 71 (l), 19~7, p. 53~59.
WO~4/001~6 PC~/EP93/01433 æ
Enzymatic hydrolysis To 1 ml of murine or human plasmas various concen~rations of the polymer-bound paclitaxel were added and at appropriate time (24, 48, 72, g6 h) 100 ~1 were collected and stored at -70~C until further processing.
Extraction procedure Samples were extracted by adding 75 ~1 of Tetra Butyl Ammonium Phospha~e (TBAP3 O. 5 M, 1~50 ~1 CH3C~ and 150 ~1 of NaCl SM and vigorously shacked for 20' a~ 4~C.
After that time samples were spun at 15000 x ~ for 10' and the supernatants were collected and evaporated using a high va~uum centrifuge . Samples were recovered by aclding 500 ~1 o~ MeOH:H20 (75:25 V/~ and injected into HPLC for de~erminlng ~he total paclitaxel percentage content. ~ ~
HPLC sy tem ~ `
CGlumn : Nova Pak C~8 (Waters) 3.~ x 300 mm :~
Flow Rate : 1.5 ml/m -:
Detector : W 231 nm ~ :~
2~ Injection : 20 ~
Mobile Phase : 57.5 % HtO brouqht to pH 2 42.5 % CH3CN ;~
The pr.Psent invention also provides a process for ~`~
preparing a polymer conjugate which process comprises reacting a compound of the formula II
, ` ~, ; ~.
WO~4/001S6 PCr/~P93/01433 2 ~ 2 ~ ~ ~
R~ ~
2 OH , oeou~
~360 ;~
: .. . .:
wherein one of A2 and A3 is a chemical bond and the o her one is A, and A, R and R3 are as defined above, with an activated polymer consisting essentially of from 90 to 99.9 mol % of units representecl by formula 1~H2 -CH3 - C-CO-NH-CH2-CHOH-CH3 ~:
'~
and from 10 to 0.1 mol % of units represented by the formula wherein A, is as de~ined above and the eatin~ the resultant polymer conjugate with 2-hydroxypropylamine.
Preferably the reaction between the compounds of ~he formula II and the activated polymer is carried out in an anhydrous polar organic solvent such as dimethylsulfoxide or dimethylformamide optionally in presence of an organic or inoryanic base such as an alkaline carbonate, dimethylaminopyridine or W~9~/00156 , PCT/~P93/0~433 `~i3~J
triethylamine. The reaction can typically be effected for ~`
from 1 to 24 hours.
The reaction is typically carried out at a temperature of from 15 to 40C, preferably at room temperature. The 5 alkaline carbonate is, for example, an alkali metal carbonate or an alkaline earth metal carbonate. :~
Some of the starting compounds of the formula II are .~
known compounds, i.e. paclitaxel or paclitaxel analogues - ;
or may be prepared starting from known compounds. ~
For example 7-epi derivatives may be prepared by .~.
refluxing in toluene paclitaxel or its analogs in the presence of a base (Na2CO3 or diazabicycloundec~ne~ . -Other compounds of the formula II are new, in particular those in which A3 is ~Ala residue and those in which either A2 or A3 represents di,, tri, or te~ra peptide .
spacer as defined above fQr A and are within the scope of , ,.
the invention. ~:
The compounds of formula II wherein A2 is not a chemical - sinyle bond may be prepared ~y reacting a paclitaxel or a 2~ paclitaxel analog with protected amino acid or peptide in the presence of a condensating reagent, and with or without the additional presence of a catalyst, preferably at room .temperature, followed by the removal of the protecting group with known methods.
The condensation may be also carried out using activated esters such as paranitrophenyl ester of peptide or amino acid. Suitable condensing reagents include carbodiimides - such as dicyclohexyl carbodiimide (DCC).
WO94~00l~fi PCT~EP93/0i433 2~
,~
Suitable catalysts include 4-dimethylamino-pyridine (DM~P~, pyridine or triethylamine.
Various known amino protecting groups can be utilized and commercially available protected amino acid or peptides can be utilized as the starting materials. Amino acids or peptides protected with t-BOC, trityl, FMOC or carbobenzyloxy ~CBZ) can be utilized. Amino Acid or peptides procted wlth t-BOC, trityl or FMOC groups are preferred.
The compounds of the formula II wherein A3 is not a chemical single bond may be prepared by protecting or blocking the 2'-hydroxy group and then esterifying the 7-position hydroxyl and then removing the 2'-protecting or blocking group.
More preferably, the compounds of the formula II wherein A3 is Gly or ~Ala residue are prepared by reacting paclitaxel with 2-3 equivalents of N-prote ted amino acid to produce 2',7-disubstituted paclitaxel, the 2'-position amino acid is cleaved and then the 7-position amino acid 2Q is deprotected.
Reaction of paclitaxel and the protected amino a~id is conducted in the presence of a condensing reagent and a catalyst, like those above defined.
,-- , Cleavage o the 2'-amino acid is conducted by adjusking the pH of the 2'-7-(amino acid) paclitaxel solution to pH
7-7.4 for example by mixture of the 2',7-di(amino acid) paclitaxel in a phosphate buffer at pH 7-7.4 or with a slight excess of NaHCO3. ,.
~.;
W~94/0~1~6 PCI`/EP93/0143 ~ 8 -Deprotection of the amino acid is conducted under a known amino acid deprotection method, such as mild acid treatment with, for example, acetic acid, or by reduction.
Thus, for example, paclitaxel is allowed to react with 2-3 mol. equivalent of N-protected amino acid (t.boc, CBZ
or FMOC pro~ected) in methylene chloride in the presence of DCC and a catalytic amount of 4-dimethylaminopyridine.
In this manner, the protected amino acid is introduced at 2' and 7-position. The 2',7-bis amino acid derivative of paclitaxel is allowed to stand in presence of NaHCO3 in H2O/MeOH for 2-5 hours, whereby selertive deprotection at the 2'-position occurs to yield the 7-substi.tuted derivative of paclitaxel. The protecting groups are removed by appropriate deprotecting agent (e.g., acid, mild base or hydrogenolysis).
The activated polymer is a synthetic, water soluble polymer prepared by the copolymerization of N'-(2~
hydroxypropyl) methacrylamide with p-nitrophenylesters 20~ of N-methacryloyl oligopeptides, as described in US-A~
4062~31 and US-A-4097470.
The polymer conjugates of the formula I and the new paclitaxel derivatives of the formula II exhibit good water solubility, biocompatibility and release the paclitaxel or paclitaxel derivative in the plasma or after internalization into ce~ls by cleaving of the oligopep~ide spacers~
WO ~4/()0156 PCI`/EP93/01433 2 1 ~ 2 ~
g :.-.. ;
Biols:~qical ac~ivity Microtubule a~3sembly and disa~sembly a.~say Calf brain tubulin was prepared by two ~ycles of assembly-disassembly ~Shelanski M.L. Gaskin F. and Cantor C.R., Proc. Natl, Acad.Sci. U.S.A. 70, 765-768, 1973) and stored in liquid nitrogen in MAB (0.1 M MES, 2.5 mM EGTA, O.S mM MgSO4, O.1 mM EDTA, 0.1 mM DTT pH 6.4).
All the experiments were carrie~ ou~ on protein stored for less than 4 weeks.
~efore each experiment, the tuhulin was kept 30 min at 4C.
Ass mbly was moni~ored ~y the method of Gaskin et al s-(Gaskin F., Cantor C.R. Shelanski M.L. J. Molec . Biol 89, 737-758, 1974). The cuvette (1 cm path) containing tubulin ~1 mg/ml) and 1 mM GTP was shifted to 37C a~d continous turbidity measurements were made at 340 nm on Perkin-Elmer 557 Double Wavelenght `Double Beam ~:
Spectrophotometer equipped with an automatic recorder and a termostatically regulated sample chamber.
20~ After 30 minutes 4 mM CaCl2 was added and depolymerisation was measured for 10 minutes as decreased turbidity. At regular intervals of 15 minutes scalar doses of. the tested compounds were added and variations in the turbidity were monitored. Data are espressed as percentage of ripolymerisation induced by the tested compounds. The results are shown in Table I.
W~94/~0156 ~ PCT/EP93/0l433 -- 10 -- ,~
In vitro druq sensitivity assay Exponentially growing B16-F10 murine melanoma cells were seeded (2 x 104/ml) in RPMI 1640 medium were supplemented -with 10% heat-inactivated fetal calf serum and 2 mM
glutamine in 24 well-plates (Costar). Scalar concentrations of tested compounds were added immediately . .
after seeding. The inhibition of cell growth was ;~
evalua~ed by counting cells with a coulter~counter after 72 hrs incubation. For each kested compound:conc~ntration :~-triplicate cultures were used. The antiproliferative activity of the tested compounds was calculated from .
dose-response curves and espressed as IC50 (dose causing 50% inhibition cell growth in treated cultures relative to untreated controls).~The resu~lt are shown in Table I.
~ :~ T~ble I
.
Compound prepared in ~ubulin As~embly :%~ ICso 6 : ~ 0.5~M ~ ;s~
Example l ; ~ o : 15~ 19 ~ :~
. ~ . _ : ~ : :., :: Example 9 : ~ 79 145~ 47 .- ~ - - _ Example 3 46 : ~86 ~ 23~ ;
. . . _. __ _ : _ ~ ., ,- ~ .
Example 4 ~ ~10 ~ ~ .~. .
: ~ ~
Example 6 : ~ 0 ~ ~lO~ 51~ : : ,~ .
~ . ,. . . - -- ~ _ :
Reference compound : : ~ ~ ~ ;
Paclltaxel 41 96 39 v~l~
: ; : ^',:,~''''`",-, '`, ~;
WO94/00156 PCT~EP93/01433 2 1 1 2 ~
The copolymer-paclitaxel prepared in Example 6 was tested in vivo agains~ Bl6 FlO murine melanoma in compariYon with paclitaxel.
Mice C57Bl6 female mice were obtained from Charles River ~ , Italy. -Animals were 8 to lO weeks old at the beginning of the experiment.
, Drugs Because of its limited a~ueous solubility, paclitaxel: was dissolved in a vehicle consisting of polyoxyethylated -~
castor oil (Cremophor EL) 50% and ethanol ~50~ and then:
diluted with glucose 5% solution at the desidered concentrations. The isolution was slightly hazy and I5~ precipitates formation~was observed after hort time.~
The compound o~ example 6 was easily dissolved in gluGo~e 5% and the resultlng solutlon was clear:~for:~long time (more than 2 hours). Fi~al concentrations~were referred to the ~paclitaxel conteDt of the compound (4~ of total).
2~ Tumor : The~murine melanoma Bl~6FlO was used. A suspension of :lO~5~
tumor cells in 0.2 ml was injected subcutaneously in the ~ ~-fla~k of.mice.
Tumor size was measured with caliper and tumor weight was calculated with~the formula~
~: a~+b 2 ~
'., .:
~;~.";.., ' ~'. ',:
:~'''''' -WOg4/n0l56 PCT~EP93/01433 ?, 4~q~
- 12 - ~ .
Druqs administration Paclitaxel was given intraperitoneally because of its ;~
poor solubility and vehicle toxicity. ~ :
The compound of example 6 was injected intravenously.
Both compounds were administered at day 1, 5, 9 after tumor implantation.
The data reported in Table 2 show that the compound of the present invention is more active than paclitaxel~
The dose of the polymer-conjugate is referred to the paclitaxel content.
Table 2 _ _- ~
Compound Do~e Tumor Tox -~:
~mg/~g) Inhibition ~%) ,..... _ ~ .. ".. ". :'"": .
Control _ ~ . _ _ Pacli~axel 14.~ 53 0/10 . ,_. _ ~ .. .. _ 22 3~ 0/10 ........ .. _ .. . . _ . _. .. . .. , . ." , . ~.,,., ~. ,,.:
33 ~ 92 l/7 .
. .. _ . .
15' Compound of ex. 6 14.6 77 0/9 ., _ . _ -. -- 22 ~ 0/10 ~ ~
~, .. . ~. ~,.
TOX: number of mice which died for toxicity. ~ ;`
TOX determination was made when mice died before the .
control or when significant body weight loss and/or spleen and or/or liver size reduction were observed. ~;
From the above data, it can be seen that the polymer . ~
' .":~',' W094/OOlS6 21.1 2 ~ ~ 2 PCT/EP93/01433 conjugates of the present invention exhibit excellent antitumor activity. These compounds, therefore, are useful a~titumor agents due to the lower toxicity and :~
increased water solubility as compared to paclitaxel or its derivative. Examples of tumors that can be treated are for istance, sarcomas, carcinoma, lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor, leukemlas and adenocarcinoma. ~::
The improved solubility a~ decreased toxicity of the`
polymer-conjugates of the present invention means that~
they are suitable for intravenous injectisn or infusion.
The dosage dep~nds upon the age, weight and condition of :~
the patient. The dosage may be from l mg/kg body weight to I g/kg body weight, preferably from 4 to 800 mg/kg ~:~
body wei~ht~ Typical formulations contain a quantity of polymer-bound paclitaxel/polymerbound ~ paclitaxel ~ -derivative equivalent~ to 0.5,~l.5t lO, 20, 25, or~50 mg~~:
: of the active pa;clitaxel/pacIitaxel derivative.
The polymer conjugates ~ may~;~ be formulated ~ as 20- pharmaceutical ~compositions with a pharmaceutically acceptable carrier or diluent. Any approprlate carrier:~or -.. :
diluent may be used. The solutlons for intraYenous injection or infusion may contain as carrier:or di~luent, for example, sterile water or preferably they may be ln :
the form of ster~ile, ^ aqueous: or isotonlc saline~
solutions.
The following:examples illustrate the invention.
.."'`;'~, WO94/~0156 PCT/EP93/01433 q~
9~ - 14 -Example 1 Copolymer of l-methacryloylamino-2-hydroxypropane~
1 (methacryloyl-glycyl-phenylalanyl-leucyl-glycyl)amino- -2-hydroxypropane and 2'(methacryloyl-glycyl-phenyl -alanyl-leucyl-glycyl~paclitaxel. -To a solution of 1.4 g of copolymer of 1-methacryloyl~
amino-2-hydroxypropane and N-(methylencarbonyl-Phe Le~
Gly 4-nitrophenoxy)methacrylamide, prepared according to -~
J~ Kopecek et al., Makromol ChPm 177, p. 2833 (1976j, in ~.
15 ml of anhydrous dimethylformamide were added 100 mg of ~-,paclitaxel and 15 mg of dimethylaminopyridine.
The yellow solution was stirred for 8 hours at room temperature under anhydrous conditions. Then 2-hydroxy . .
propylamine (0.2 ml) was droppecl into rea~tion flask, and ~hen the whole was stirred for 30 minutes.
The reaction solution was quenched with 0.3 ml glacial acetic acid, concentrated uhder vacuum to a smal~ volume, and then poured into 200 ml of acetone. ~i Ater 30' mixing, the precipitate was filtered and washed with acetone to yleld 1.25 g of the title compound. ~;
The paclitaxel content was 4.5% (evaluated by enzymatic hydrolysis and HPLC analysis). -Unreact~d paclitaxel was recovered from acetone solu~ion.
~xample 2 2'(N-trityl-phenylalanyl-leUCyl glycyl)paclitaxel ~
To a solution of 170 mg of paclitaxel in 16 ml of ;`
acetonitrile were added 24 mg of dimethylamino-pyridine and 150 mg of N-trityl-phenylanyl-leucyl-glycine 4-W094/00156 2 ~ 1 2 4 8 2 PCT/EP93/01433 nitrophenyl ester.
The yellow solution was stirred for 20 hours at room temperature, and then evaporated under vacuum to dryness.
The residue was cromatographed on silica gel with ethyl- . ;
acetate-hexane 35:25 as eluant, affording 3~0 mg of the :~
title compound. t ' ' ~ ' H-NMR(400 MHz, CDCl3)~
0.82 (d, J-6.4 Hz, 3H, ~-Leu) 0.~5 (d, ~=6.7 Hz, 3H, ~-Leu) 1.15 (s, 3H, 16) 1.26 (s, 3~, 17) ~ 1.6 (m. 3H, ~+~+~-Leu~ .
, - .
1.69 (s, 3H, l9) ;., .
1.85 (s, lH, OH~
15 ~ 1.89 (m, lH, 6~
1.9~ (d, J-1.2 Hz, 3H, 18):
2.14 (dd, J=5.9:Hz~:, J=l3.5 Hz, lH~ Phe);~
~ ~ 2.24 (s, 3H, CH3CO-10)~
: ~ ~ 2.2-2.7 (m, 5H, CH2-l4+0H-7+6:~+~Phe+NH~Ph;e) : 2.47 (s, 3H, CH3CO-4) 3.50 (m, lH, ~-Phe) ;;~
3.74 (dd, J=4.7 Hz, J=18.2 Hz, lH, ~-Gly) 3.~0 (~j lH, ~-Leu):~
3.83 (d, J=7.0~Hz,~lH, 3:) ;
4.17 (dd, J=7.0 Hz, J=18.:2~Uz, lH, '-Gly)~
4.22~ 4.33 ttwo-d, J=8.5 Hz, 2H,~ CH2-20): :
4.46 (m, lH, 7) 4.97 (dd, J=2.2 Hz, J=9.9 Hz, lH,;5) `~
W094/00156 A ~ PCI~/EP93/01433 5.44 (d, J=2.3 ~z, lH, 2') ~;;;
5.71 ~d, J=7.0 Hz, lH, 2) -.
5.97 (dd, J=4.7 Hz, J=7.0 Hz, lH, NH-Gly) 6.07 (dd, J=2.3 Hz, J=9.4 Hz, lH, 3'~
6.2-6.3 (m. 2H, 13~10) ;-~
6.8-9.2 (m, 30 H, 6 Ph) 6.95 (d, J-6.7 Hz, lH, NH-Leu) : .
8.00 ~d, J=9.4 Hz, lH, NH-4') ~.
Exampl~ 3 .
2'(phenylalanyl-leucyI-glycyl~paclitaxel. .:`~
The compound 2'(N-Trit-Phe-Leu-Gly)paclitaxel (250 mg) .
was dissolved into a mixture of glacial acetic (22 ml) and wateir (6 ml~ and the whole was stirred for 1 hour at room temperature. ;
The solvents were evaporated in vacuum to the dryness, ~;
the residue stirred with: diethylether-hexane 1:1 ~for~ 30 minutes and filtered to obtain 160 mg of ~the title: .:~
compound.
FAB-MS: m/z 1171, M+Hl+; 1112, M-CH3COOH ~ 2H; 1051, :2~ 1024, 911, 603, 569,~ 5~9.
H-NMR(4ooMHzl CDCl~
0.88(d,J=6.4Hz,3H, ~ Leu) 0.9~(d,~J~6.4Hz,3H, ~' Leu) 1.13 (s, 3H, 16) :
1.16 (s, 3H, 17 1.4-2.0(m J 4Hj~+~'+~Leu+6 1.69 (s,3H~ 19) l.91 (d,J=1.2Hz,3H,18) ; '' ~
WO94/001~6 21 12 ~ ~ 2 PCT/EP93/01q33 2.16 (dd, J=6~0 Hz,J=13.8Hz,lH,14) 2.23 (s, 3H, COCH3-lO) 2.4-2.6 (m, 3H, 6~+14+~Phe) 2.53 (s,3H,COCH3-4) 2.90 (dd, JC4.lHZr J=13.5Hz, lH,~'-Phe) 3.49 (dd, J=4.1 Hz, J-9.lHz,lH, ~Phe) 3.82 (d, J=7.3Hz,lH,3) ~
3.9-4.~ (m,2H,~'Gly) 4.2Z, 4.33 (two-d, J=8.7 HZ, 2H, CH2-20) 4.27 (m, lH, ~-Le~
4.44 (dd, J=6.4Hz, J=10.8Hz,7) `
4.98 (dd, J=2.4Hz, J=9.7 Hz,5) 5.61 (d, 3-3.2Hz,lH,2') ~, . . .
5.70 (d, Ja7.3Hz,lH,2) ~ `
. . ..: ..
6.12 (dd,J=3.2Hz,J=9.4Hz,lHt3') 6.21 (m, lH, 13) , 6.2~ (s,lH,lO) ;
6.8-8.2 (m,21H, 4-Ph + NHLeu) 7.87 (d,J=9.4Hz,lH,NH-4') .~.... ~, .
20~ Example 4 Copolymer of 1-methacryloylamino-2-hydroxypropane, 1~
(methacryloyl-glycyl:) amlno-2-hydroxypropane and 2'(methacryloyl-gl~ycyl-ph~enylalanyl-leucy~
glycyl)paclitaxel.
~5 To a solution of 1 g of copolymer o~ l-methacryloylamino~
2-hydroxypropane and N-~methylencarbonyl-4-nitro - ~
phenoxy) methacrylamide, ~ ~ prepared according to P. ;
Rejmanova et al., Makromol. ehem. 178, p. 2159-2168, in .:
'~
, W0~4/nO156 PCT/EP93/01433 , It ~
~ - 18 ~ -10 ml of anhydro dimethyiformamide were added 100 mg of 2' Phe-Leu-Gly-paclitaxel and lO mg of ~
dimethylaminopyridine. ` -`
The yellow solution was stirred for 2 hours at room temperature under anhydrous conditions. Then 2-hydroxy -propylamine (O.lS ml) was added to the reaction solution, and the whole was stirred for 30 minutes. The reaction .
solution was quenched with 0.2 ml of glacial acetic acid, concentrated under vacuum to a small volume and then poured into 200 ml of acetone. ..
The mixture was stirred for 1 hours, the precipitate was .-filtered and washed with acetone to yield 960 mg of the.``-.
title compound. ~.
The paclitaxel content was 6% (evaluated by enzymatic hydrolysis and HPLC cnalysis).~ :
Example 5 Copolymer of 1-methacryloylamino-2~hydroxy -prop~ne, l(methacryloyl-glycyl)amino-2-hydroxypropane -and 2'(methacryloyl-glycyl)paclitaxel. ;~
~ .
2d To a solution of 1.6 g of copolymer of l-methacryloyl amino-2-hydroxypropane and N-(methylen-carbonyl-4-nitro- ~
phenoxy)metha~rylamide in 16 ml of anhydrous dimethyl- ;
formamide were added 100 mg of paclitaxel and 20 mg of dimethylamino-pyridine. The ye~llow solution was stirred ~ .
for 20 hours at room temperature, then 2-hydroxypropylamine~(0.2 ml) was added, and the whole was~
:
stirred for 30 minutes.: The reaction solution was quenched with 0.3 ml of glacial acetic acid, concentrated ~, ~
W~94/00156 21 i 2 ~ 8 2 PCT/EP93/01433 under vacuum to a small volume, and then poured into 200 ml of acetone. The mixture was stirred for 1 hour, the precipitate was filtered and washed with acetone to yield 1440 mg of the title compound. ~ -~
The paclitaxel content was 2 . 75% w/w% . :
Example 6 ~, Copolymer of l-methacryloylamino -2-hydroxypropane,l-(methacryloyl-glycyl:-phenylanyl-leucyl-glycyl)amino-2- ..
hydroxypropane and 2'(methacryloyl-glycyl-phenylalanyl~
leucyl-glycyl-~a1anyl)paclitaxel. ~ .
To a solution of 620 mg of copolymer of 1-metacrylamino- ~-2-hydroxypropane and N-(methylencarbonyl-Phe~Leu-Gly-4-nitro- phenoxy)methacrylamide in 6 ml of anhydrous .. :
dimethyl-formamide were added 62 mg of 2'- ;~
-lS (~alanyl)paclitaxel, prepared according to N. F. Magri et al, J Nat. Products 51 298-306, 1988, and 10 mg of . . .
dimethylamino pyridine. The yellow solution was stirred for 5 hours at room temperature under anhydrous :
conditions. , -~
. .: . .:
20~ Then 2-hydroxypropylamine (0.1 ml) was added and the ~ ~
~.
whole was stirred for 30 mlnutes.
The reaction solution was quenched with 0,15 ml of glacial acetic acid, concentrated under vacuum to a small volume and poured in 150 ml of acetone. The mixure was ~ ~
.:
stirred for 1 hour, the precipitate was filtered and -~
washed with acetone to ~ield 5.85 mg of the title compound~
The paclitaxel content was 4% w/w% ~:
W094/0~156 ~ 6~ ~b~ PCT/EP93/01433 - 20 - ~ ::
~xample 7 `~
2',7-di(carbobenzyloxy-~-alanyl?paclitaxel.
To a solution of 200 mg of paclitaxel în 15 ml of acetonitrile were added 400 mg of N,N'-dicyclohexylcarbodiimide, 200 mg of carbobenzyloxy-~alanine and 6G mg of ~imethylamino-pyridine~ The reaction mixture was stirred for 20 hours, the precipitate was .
filtered and the solvent evaporated under vacuum to .
dryness. ;
The residue was chromatographed on silica gel with ethyl ;``~-acetate-hexane 1:1 as eluant, affording 300 mg of the ti~le compound.
F~B-MS: m/z 1264 M+Hl+, 1204, 1130, 1070 ~, '''~ '`
W~94/00156 2 I 2 ~ 8 ~ PCT/EP93/0l433 Example ~
7-(carbobenzyloxy-~alanyl)paclitaxel ~-To a solution of 171 mg of 2'7-di(carbobenzyloxy-~-..., . ~ .
alanyl)paclitaxel in 60 ml of methanol, were added 30 mg ~ ;
of sodium bicarbonate and 7 ml of water . The reaction mixtur~ was stirred for 3 hours at room temperature. ~:~
The methanol was evapora~ed and ~he product extracted - `~
with ethylacetate~ ~`
The solvent was evaporated under vacuum: to dryness, :
affordin~ 13~ mg of the title compound.
Example g 7-(~alanyl)paclitaxel ::
To a solutîon of 135 mg of 7-(carboben~zyloxy- ;~
~alanyl)paclitaxel in 20~ ml of methan~l and 13 ml of :-:
formic acid, was added 20~ mg of Pd/C 5%.~The reaction ~;
mixture was stlrred for 6 hours at room-temperature.~The catalyst was fil~ered, washed ~with methanol and the ;~ -solvents were evaporated to~ dryness under vacuum.~ The ~
~ residue was dissolved in 8 ml of ~methanol and ~ ~-20 precipitated with lSo ml of diethylether, affording:85 mg of the title compound.
FAB-MS: M/z 925,M~Hl+;; 947, M~Nal+ ; :
H NMR(400MHz, CDCl3)~
~ ~ :
1.14 (s,3H,CH3-16) ~ :
1.20(s,3H,CH3 17) : :~
1.79 (s,3H~CH3~19): ~:
1.85 (s, 3H,CH3-18) WO94/00156 ~ PCT/EP93/01433 ~;
2~17(s,3H,CH3CO-lO) 2.2-2.6(m,6H,CH2-l4+CH2-6~OCOCH~2NH2) `~
2.42(s,3H~CH3CO-4) - .
",'~'';' '~
3.0-3.2(m,2H,OCOCH~g~2~H2) 3.90~d,J-6.8HZ,lH,3) 4.18, 4.31(two d, J=8.2HZ,2H,CH2-20) .
4.80~d,J=3.2Hz,lH,2'~
4.91(d,J=8.5Hz,lH,5) 5.62(dd,J=lO.2Hz, J=7.0HZ~lH~7 5.66 (d,J=6.8HZ,IH,2 5.8l(dd,J=2.9Hz, J=9.lHz,1H,3' ` ;~
6.17 (m,lH,13) 6.19(S,lH,lO) 7.3-8.2 (m,16H,NH-4'+3-Phj -~
; ';~
15 : ExamPl~ lO
Copolymer of 1-methacryloylamino~2-hydroxypropane, :(methacryloyl-glycyl-phenylalanyl-leucyl-glycyl)amino-2-hyroxypropane and 7-(methacryloyl-glycyl-phenylalanyl~
i~ leucyl-glycyl-,~alanyl) paclitaxel .
To a solution of 1~500 mg of ~c~polymer of 1-methacryloyl~
amino 2-hydroxypropane and N-(methylencarbonyl-Phe-Leu- ;~
Gly-4nitrophenoxy)methacrylamide in 13 ~ml of anhydrous : dimethylformamide were added 135~ mg of ~ 7~
; .. ~ ~:
(,Balanyl)paclitaxel and 20 mg of dimethylaminopyridine.
The yellow solution~ was stirred under~ anhydrous conditions for: 5 hours:at room temperature. ; ~ ~ ;
: Then 2-hydr-oxypropylamine ~(0;2 ml) was added , and the "' : . ~
WO94J001~6 PCT/EP~3/01433 21124~
- 23 - ;
whole was stirred for 30 minutes.
The reaction solution was quenched with 0.3 ml of glacial ~.
acetic acid, concentratPd under vacuum to a small volume ~:
and poured in 250 ml of acetone. The mixture was stirred ~ .
for 1 hour, the precipitate was filtered and washed with .
acetone to yield 1520 mg of the titlQ compound. ```
The paclitaxel content was 7.8% w/w~.
;:, . . : - . .
, ,`','' '' '.', -~'' ;:
~ ;",-'"~ '`
.
'~
4.22~ 4.33 ttwo-d, J=8.5 Hz, 2H,~ CH2-20): :
4.46 (m, lH, 7) 4.97 (dd, J=2.2 Hz, J=9.9 Hz, lH,;5) `~
W094/00156 A ~ PCI~/EP93/01433 5.44 (d, J=2.3 ~z, lH, 2') ~;;;
5.71 ~d, J=7.0 Hz, lH, 2) -.
5.97 (dd, J=4.7 Hz, J=7.0 Hz, lH, NH-Gly) 6.07 (dd, J=2.3 Hz, J=9.4 Hz, lH, 3'~
6.2-6.3 (m. 2H, 13~10) ;-~
6.8-9.2 (m, 30 H, 6 Ph) 6.95 (d, J-6.7 Hz, lH, NH-Leu) : .
8.00 ~d, J=9.4 Hz, lH, NH-4') ~.
Exampl~ 3 .
2'(phenylalanyl-leucyI-glycyl~paclitaxel. .:`~
The compound 2'(N-Trit-Phe-Leu-Gly)paclitaxel (250 mg) .
was dissolved into a mixture of glacial acetic (22 ml) and wateir (6 ml~ and the whole was stirred for 1 hour at room temperature. ;
The solvents were evaporated in vacuum to the dryness, ~;
the residue stirred with: diethylether-hexane 1:1 ~for~ 30 minutes and filtered to obtain 160 mg of ~the title: .:~
compound.
FAB-MS: m/z 1171, M+Hl+; 1112, M-CH3COOH ~ 2H; 1051, :2~ 1024, 911, 603, 569,~ 5~9.
H-NMR(4ooMHzl CDCl~
0.88(d,J=6.4Hz,3H, ~ Leu) 0.9~(d,~J~6.4Hz,3H, ~' Leu) 1.13 (s, 3H, 16) :
1.16 (s, 3H, 17 1.4-2.0(m J 4Hj~+~'+~Leu+6 1.69 (s,3H~ 19) l.91 (d,J=1.2Hz,3H,18) ; '' ~
WO94/001~6 21 12 ~ ~ 2 PCT/EP93/01q33 2.16 (dd, J=6~0 Hz,J=13.8Hz,lH,14) 2.23 (s, 3H, COCH3-lO) 2.4-2.6 (m, 3H, 6~+14+~Phe) 2.53 (s,3H,COCH3-4) 2.90 (dd, JC4.lHZr J=13.5Hz, lH,~'-Phe) 3.49 (dd, J=4.1 Hz, J-9.lHz,lH, ~Phe) 3.82 (d, J=7.3Hz,lH,3) ~
3.9-4.~ (m,2H,~'Gly) 4.2Z, 4.33 (two-d, J=8.7 HZ, 2H, CH2-20) 4.27 (m, lH, ~-Le~
4.44 (dd, J=6.4Hz, J=10.8Hz,7) `
4.98 (dd, J=2.4Hz, J=9.7 Hz,5) 5.61 (d, 3-3.2Hz,lH,2') ~, . . .
5.70 (d, Ja7.3Hz,lH,2) ~ `
. . ..: ..
6.12 (dd,J=3.2Hz,J=9.4Hz,lHt3') 6.21 (m, lH, 13) , 6.2~ (s,lH,lO) ;
6.8-8.2 (m,21H, 4-Ph + NHLeu) 7.87 (d,J=9.4Hz,lH,NH-4') .~.... ~, .
20~ Example 4 Copolymer of 1-methacryloylamino-2-hydroxypropane, 1~
(methacryloyl-glycyl:) amlno-2-hydroxypropane and 2'(methacryloyl-gl~ycyl-ph~enylalanyl-leucy~
glycyl)paclitaxel.
~5 To a solution of 1 g of copolymer o~ l-methacryloylamino~
2-hydroxypropane and N-~methylencarbonyl-4-nitro - ~
phenoxy) methacrylamide, ~ ~ prepared according to P. ;
Rejmanova et al., Makromol. ehem. 178, p. 2159-2168, in .:
'~
, W0~4/nO156 PCT/EP93/01433 , It ~
~ - 18 ~ -10 ml of anhydro dimethyiformamide were added 100 mg of 2' Phe-Leu-Gly-paclitaxel and lO mg of ~
dimethylaminopyridine. ` -`
The yellow solution was stirred for 2 hours at room temperature under anhydrous conditions. Then 2-hydroxy -propylamine (O.lS ml) was added to the reaction solution, and the whole was stirred for 30 minutes. The reaction .
solution was quenched with 0.2 ml of glacial acetic acid, concentrated under vacuum to a small volume and then poured into 200 ml of acetone. ..
The mixture was stirred for 1 hours, the precipitate was .-filtered and washed with acetone to yield 960 mg of the.``-.
title compound. ~.
The paclitaxel content was 6% (evaluated by enzymatic hydrolysis and HPLC cnalysis).~ :
Example 5 Copolymer of 1-methacryloylamino-2~hydroxy -prop~ne, l(methacryloyl-glycyl)amino-2-hydroxypropane -and 2'(methacryloyl-glycyl)paclitaxel. ;~
~ .
2d To a solution of 1.6 g of copolymer of l-methacryloyl amino-2-hydroxypropane and N-(methylen-carbonyl-4-nitro- ~
phenoxy)metha~rylamide in 16 ml of anhydrous dimethyl- ;
formamide were added 100 mg of paclitaxel and 20 mg of dimethylamino-pyridine. The ye~llow solution was stirred ~ .
for 20 hours at room temperature, then 2-hydroxypropylamine~(0.2 ml) was added, and the whole was~
:
stirred for 30 minutes.: The reaction solution was quenched with 0.3 ml of glacial acetic acid, concentrated ~, ~
W~94/00156 21 i 2 ~ 8 2 PCT/EP93/01433 under vacuum to a small volume, and then poured into 200 ml of acetone. The mixture was stirred for 1 hour, the precipitate was filtered and washed with acetone to yield 1440 mg of the title compound. ~ -~
The paclitaxel content was 2 . 75% w/w% . :
Example 6 ~, Copolymer of l-methacryloylamino -2-hydroxypropane,l-(methacryloyl-glycyl:-phenylanyl-leucyl-glycyl)amino-2- ..
hydroxypropane and 2'(methacryloyl-glycyl-phenylalanyl~
leucyl-glycyl-~a1anyl)paclitaxel. ~ .
To a solution of 620 mg of copolymer of 1-metacrylamino- ~-2-hydroxypropane and N-(methylencarbonyl-Phe~Leu-Gly-4-nitro- phenoxy)methacrylamide in 6 ml of anhydrous .. :
dimethyl-formamide were added 62 mg of 2'- ;~
-lS (~alanyl)paclitaxel, prepared according to N. F. Magri et al, J Nat. Products 51 298-306, 1988, and 10 mg of . . .
dimethylamino pyridine. The yellow solution was stirred for 5 hours at room temperature under anhydrous :
conditions. , -~
. .: . .:
20~ Then 2-hydroxypropylamine (0.1 ml) was added and the ~ ~
~.
whole was stirred for 30 mlnutes.
The reaction solution was quenched with 0,15 ml of glacial acetic acid, concentrated under vacuum to a small volume and poured in 150 ml of acetone. The mixure was ~ ~
.:
stirred for 1 hour, the precipitate was filtered and -~
washed with acetone to ~ield 5.85 mg of the title compound~
The paclitaxel content was 4% w/w% ~:
W094/0~156 ~ 6~ ~b~ PCT/EP93/01433 - 20 - ~ ::
~xample 7 `~
2',7-di(carbobenzyloxy-~-alanyl?paclitaxel.
To a solution of 200 mg of paclitaxel în 15 ml of acetonitrile were added 400 mg of N,N'-dicyclohexylcarbodiimide, 200 mg of carbobenzyloxy-~alanine and 6G mg of ~imethylamino-pyridine~ The reaction mixture was stirred for 20 hours, the precipitate was .
filtered and the solvent evaporated under vacuum to .
dryness. ;
The residue was chromatographed on silica gel with ethyl ;``~-acetate-hexane 1:1 as eluant, affording 300 mg of the ti~le compound.
F~B-MS: m/z 1264 M+Hl+, 1204, 1130, 1070 ~, '''~ '`
W~94/00156 2 I 2 ~ 8 ~ PCT/EP93/0l433 Example ~
7-(carbobenzyloxy-~alanyl)paclitaxel ~-To a solution of 171 mg of 2'7-di(carbobenzyloxy-~-..., . ~ .
alanyl)paclitaxel in 60 ml of methanol, were added 30 mg ~ ;
of sodium bicarbonate and 7 ml of water . The reaction mixtur~ was stirred for 3 hours at room temperature. ~:~
The methanol was evapora~ed and ~he product extracted - `~
with ethylacetate~ ~`
The solvent was evaporated under vacuum: to dryness, :
affordin~ 13~ mg of the title compound.
Example g 7-(~alanyl)paclitaxel ::
To a solutîon of 135 mg of 7-(carboben~zyloxy- ;~
~alanyl)paclitaxel in 20~ ml of methan~l and 13 ml of :-:
formic acid, was added 20~ mg of Pd/C 5%.~The reaction ~;
mixture was stlrred for 6 hours at room-temperature.~The catalyst was fil~ered, washed ~with methanol and the ;~ -solvents were evaporated to~ dryness under vacuum.~ The ~
~ residue was dissolved in 8 ml of ~methanol and ~ ~-20 precipitated with lSo ml of diethylether, affording:85 mg of the title compound.
FAB-MS: M/z 925,M~Hl+;; 947, M~Nal+ ; :
H NMR(400MHz, CDCl3)~
~ ~ :
1.14 (s,3H,CH3-16) ~ :
1.20(s,3H,CH3 17) : :~
1.79 (s,3H~CH3~19): ~:
1.85 (s, 3H,CH3-18) WO94/00156 ~ PCT/EP93/01433 ~;
2~17(s,3H,CH3CO-lO) 2.2-2.6(m,6H,CH2-l4+CH2-6~OCOCH~2NH2) `~
2.42(s,3H~CH3CO-4) - .
",'~'';' '~
3.0-3.2(m,2H,OCOCH~g~2~H2) 3.90~d,J-6.8HZ,lH,3) 4.18, 4.31(two d, J=8.2HZ,2H,CH2-20) .
4.80~d,J=3.2Hz,lH,2'~
4.91(d,J=8.5Hz,lH,5) 5.62(dd,J=lO.2Hz, J=7.0HZ~lH~7 5.66 (d,J=6.8HZ,IH,2 5.8l(dd,J=2.9Hz, J=9.lHz,1H,3' ` ;~
6.17 (m,lH,13) 6.19(S,lH,lO) 7.3-8.2 (m,16H,NH-4'+3-Phj -~
; ';~
15 : ExamPl~ lO
Copolymer of 1-methacryloylamino~2-hydroxypropane, :(methacryloyl-glycyl-phenylalanyl-leucyl-glycyl)amino-2-hyroxypropane and 7-(methacryloyl-glycyl-phenylalanyl~
i~ leucyl-glycyl-,~alanyl) paclitaxel .
To a solution of 1~500 mg of ~c~polymer of 1-methacryloyl~
amino 2-hydroxypropane and N-(methylencarbonyl-Phe-Leu- ;~
Gly-4nitrophenoxy)methacrylamide in 13 ~ml of anhydrous : dimethylformamide were added 135~ mg of ~ 7~
; .. ~ ~:
(,Balanyl)paclitaxel and 20 mg of dimethylaminopyridine.
The yellow solution~ was stirred under~ anhydrous conditions for: 5 hours:at room temperature. ; ~ ~ ;
: Then 2-hydr-oxypropylamine ~(0;2 ml) was added , and the "' : . ~
WO94J001~6 PCT/EP~3/01433 21124~
- 23 - ;
whole was stirred for 30 minutes.
The reaction solution was quenched with 0.3 ml of glacial ~.
acetic acid, concentratPd under vacuum to a small volume ~:
and poured in 250 ml of acetone. The mixture was stirred ~ .
for 1 hour, the precipitate was filtered and washed with .
acetone to yield 1520 mg of the titlQ compound. ```
The paclitaxel content was 7.8% w/w~.
;:, . . : - . .
, ,`','' '' '.', -~'' ;:
~ ;",-'"~ '`
.
'~
Claims (10)
1. A polymer conjugate consisting essentially of: from 90 to 99.9 mol % of units represented by the formula ;
from 0.1 to 5 mol % of units represented by the formula wherein one of R1 and R2 is a copolymer residue of the formula and the other one is hydrogen atom;
from 0 to 9.9 mol % of units represented by the formula wherein R is a phenyl or t-butoxy group, R3 is H or an acetyl group, A and A1 which may be the same or different, represent a chemical single bond, an amino acid residue or peptide spacer selected from .beta. Ala, Gly, Phe-Gly, Phe-Phe-, Leu-Gly, Val-Ala, Phe-Ala, Leu-Phe, Leu-Ala, Phe-Leu-Gly, Phe-Phe-Leu, Leu-Leu-Gly, Phe-Tyr-Ala, Phe-Gly-Phe, Phe-Phe-Gly, Phe-Leu-Gly-Phe, Gly-Phe-Leu-Gly-Phe,Gly-.beta.Ala, Phe-Gly-.beta.Ala, Phe-Phe-.beta.Ala, Leu-Gly-.beta.Ala, Val-Ala-.beta.Ala, Phe-Ala-.beta.Ala, Leu-Phe-.beta.Ala, Leu-Gly-.beta.Ala, Phe-Leu-Gly-.beta.Ala, Phe-Phe-Leu .beta.Ala, Leu-Leu-Gly-.beta.Ala, Phe-Tyr-Ala-.beta.Ala, Phe-Gly-Phe-.beta. Phe-Phe-Gly .beta.Ala, Pùe-Leu-Gly-Phe-.beta.Ala or Gly-Phe-Leu-Gly-Phe-.beta.Ala.
from 0.1 to 5 mol % of units represented by the formula wherein one of R1 and R2 is a copolymer residue of the formula and the other one is hydrogen atom;
from 0 to 9.9 mol % of units represented by the formula wherein R is a phenyl or t-butoxy group, R3 is H or an acetyl group, A and A1 which may be the same or different, represent a chemical single bond, an amino acid residue or peptide spacer selected from .beta. Ala, Gly, Phe-Gly, Phe-Phe-, Leu-Gly, Val-Ala, Phe-Ala, Leu-Phe, Leu-Ala, Phe-Leu-Gly, Phe-Phe-Leu, Leu-Leu-Gly, Phe-Tyr-Ala, Phe-Gly-Phe, Phe-Phe-Gly, Phe-Leu-Gly-Phe, Gly-Phe-Leu-Gly-Phe,Gly-.beta.Ala, Phe-Gly-.beta.Ala, Phe-Phe-.beta.Ala, Leu-Gly-.beta.Ala, Val-Ala-.beta.Ala, Phe-Ala-.beta.Ala, Leu-Phe-.beta.Ala, Leu-Gly-.beta.Ala, Phe-Leu-Gly-.beta.Ala, Phe-Phe-Leu .beta.Ala, Leu-Leu-Gly-.beta.Ala, Phe-Tyr-Ala-.beta.Ala, Phe-Gly-Phe-.beta. Phe-Phe-Gly .beta.Ala, Pùe-Leu-Gly-Phe-.beta.Ala or Gly-Phe-Leu-Gly-Phe-.beta.Ala.
2. A polymer conjugate according to clalm 1, in which R represents a phenyl group, R3 is an acetyl group and A
is a Phe-Leu-Gly or Phe-Leu-Gly-.beta.Ala residue.
is a Phe-Leu-Gly or Phe-Leu-Gly-.beta.Ala residue.
3. A polymer conjugate according claim 1 or 2, which is a copolymer of 1-methacryloylamino-2-hydroxypropane, (methacryloylglycyl-phenylalanylleucylglycyl)3-amino-2 hydroxypropane and 2'-(methacryloylglycylphenylalanyl-leucylglycyl-.beta.alanyl) paclitaxel.
4. A process for preparing a polymer conjugate as defined in claim 1, which process comprises reacting a compound of the formula II
(II) wherein one of A2 and A3 is a chemical bond and the other one is A, and A, R and R3 are as defined in claim 1, with an activated polymer consisting essentially of from 90 to 99.9 mol % of units represented by formula and from 10 to 0.1 mol % of units represented by the formula wherein Al is as defined in claim 1 and then treating the resultant polymer conjugate with 2-hydroxy-propylamine.
(II) wherein one of A2 and A3 is a chemical bond and the other one is A, and A, R and R3 are as defined in claim 1, with an activated polymer consisting essentially of from 90 to 99.9 mol % of units represented by formula and from 10 to 0.1 mol % of units represented by the formula wherein Al is as defined in claim 1 and then treating the resultant polymer conjugate with 2-hydroxy-propylamine.
5. A process according to claim 4, in which the reaction is carried out in an anhydrous polar solvent at a temperature of from 15 to 40°C for from 1 to 24 hours, in the presence of an organic or inorganic base.
6. A compound of the formula II as defined in claim 4 wherein A2 represents di, tri or tetra peptide spacer as defined in claim 1 for A.
7. A process for preparing a compound of the formula II as defined in claim 6, which process comprises reacting a paclitaxel or a paclitaxel analog with desired protected amino acid or peptide in the presence of a condensating reagent or using activated esters and with or without the additional presence of a catalyst and then removing the protecting groups.
8. A compound of formula II as defined in claim 4 wherein A3 represents .beta.Ala or di, tri or tetra peptide spacer as defined in claim 1 for A.
9.. A process for preparing a compound of the formula II as defined in claim 8, which process comprises either (i) protecting or blocking the 2'-hydroxy group of paclitaxel and esterifying the 7-position hydroxyl and then removing the 2'-protecting or blocking group, or (ii) reacting paclitaxel with 2-3 moles of the desired N-protected amino acid to produce 2',7-disubstituted taxol, cleaving the 2'-position amino acid and then deprotecting the 7-position amino acid.
10. A pharmaceutical composition comprising, as active ingredient, a polymer conjugate as defined in claim 1, 6 or 8 and a pharmaceutically acceptable diluent or carrier.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9213077.2 | 1992-06-19 | ||
| GB929213077A GB9213077D0 (en) | 1992-06-19 | 1992-06-19 | Polymerbound taxol derivatives |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2112482A1 true CA2112482A1 (en) | 1994-01-06 |
Family
ID=10717411
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002112482A Abandoned CA2112482A1 (en) | 1992-06-19 | 1993-06-07 | Polymer-bound paclitaxel derivatives |
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| US (4) | US5362831A (en) |
| EP (1) | EP0600062B1 (en) |
| JP (1) | JP3693340B2 (en) |
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| CN (1) | CN1041281C (en) |
| AT (1) | ATE205725T1 (en) |
| AU (2) | AU659750B2 (en) |
| CA (1) | CA2112482A1 (en) |
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| DE (1) | DE69330776T2 (en) |
| ES (1) | ES2164663T3 (en) |
| FI (1) | FI940733A0 (en) |
| GB (1) | GB9213077D0 (en) |
| HU (2) | HUT67914A (en) |
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| PL (1) | PL173898B1 (en) |
| RU (1) | RU2130462C1 (en) |
| SG (1) | SG49248A1 (en) |
| TW (1) | TW266201B (en) |
| UA (1) | UA39926C2 (en) |
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| CS173846B1 (en) * | 1974-04-23 | 1977-03-31 | ||
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