Colloid bonded medicinal compounds

Abstract

The invention relates to colloids bound medicinal compounds or fluorescent markers, to a process for the preparation thereof, and to a pharmaceutical formulation containing such compounds.

Claims

1. A compound of formula (I) ##STR00022## wherein X is a colloid-active compound, characterized in that said colloid-active compound X is selected from the group consisting of amyloses, amylopectins, acemannans, arabinogalactans, galactomannans, galactoglucomannans, xanthans, carrageenan, hyaluronic acid, deacetylated hyaluronic acid, starch and modified starch; L.sup.1 is a first linker by means of which X and the phosphate group are covalently linked together and wherein L.sup.1 is selected from the group consisting of a single bond, alkandiyl, alkendiyl and alkyndiyl; L.sup.2 is a second linker by means of which the phosphate group and A are covalently linked together; A is a medicinally active substance selected from the group of consisting of antibiotics, chemotherapeutics, cytostatic agents, antigens, oligonucleotides, mediators, false metabolic substrates, analgetics and cytotoxic substances or a fluorescence marker; Y is either H or OH; n is an integer of at least 1.

2. The compound according to claim 1, characterized in that said modified starch is selected from the group consisting of hydroxyalkyl starches, esterified starches, carboxyalkyl starches, hydroxyalkyl carboxyalkyl starch, aminated hydroxyalkyl starch, aminated hydroxyalkyl carboxyalkyl starch and aminated carboxyalkyl starch.

3. The compound according to claim 2, characterized in that said modified starch is selected from hydroxyethyl starch or aminated hydroxyethyl starch.

4. The compound according to claim 1 characterized in that said colloid-active compound has an average molecular weight of from 20,000 to 800,000 daltons.

5. The compound according to claim 1, characterized that the degree of substitution, DS, of the modified starch is from 0.2 to 0.8.

6. The compound according to at least claim 1, characterized in that said medicinally active compound A is selected from formula (II) to (IV) ##STR00023## wherein R.sup.1 is H or a C.sub.1-C.sub.28 chain which may be branched or linear and which may be saturated or unsaturated and which may optionally be interrupted and/or substituted by one or more hetero atom(s) and/or functional group(s); or R.sup.1 is a C.sub.3-C.sub.28 moiety which comprises at least one cyclic structure and which may be saturated or unsaturated and which may optionally be interrupted and/or substituted by one or more hetero atom(s) and functional group(s); R.sup.2 is H or an organic moiety comprising 1 to 30 carbon atoms; R.sup.3 and R.sup.4 represent independently from each other H or a C.sub.1-C.sub.28-alkyl moiety which may optionally be substituted or interrupted by one or more heteroatom(s) and/or functional group(s); or R.sup.3 and R.sup.4 form a ring having at least 5 members, wherein the ring may be substituted or interrupted by one or more hetero atom(s) and/or functional group(s); R.sup.5 and R.sup.6 represent independently from each other H or a C.sub.1-C.sub.28-alkyl moiety which may optionally be substituted or interrupted by one or more heteroatom(s) and/or functional group(s); or R.sup.5 and R.sup.6 form a ring having at least 5 members, wherein the ring may be substituted or interrupted by one or more hetero atom(s) and/or functional group(s); R.sup.7 is a hydrogen atom or —O—R.sup.8; R.sup.8 is H or C.sub.1-C.sub.28 chain which may be branched or linear and which may be saturated or unsaturated and which may optionally be interrupted and/or substituted by one or more hetero atom(s) and/or functional group(s).

7. The Compound according to claim 6 wherein R.sup.1 is selected from H, ##STR00024## substituted or unsubstituted cyclic terpene moieties, wherein R.sup.9 and R.sup.19 are independently selected from C.sub.1 to C.sub.30 alkyl, b is an integer ranging 1 to 4; and a is an integer ranging from 1 to 20.

8. The compound according claim 1, characterized in that said fluorescence marker is selected from the group consisting of fluorescein isothiocyanate (FITC), phycoerythrin, rhodamide, 2-aminopyridine and coumarine dyes.

9. The compound according to claim 1, wherein said X is hydroxyethylstarch and said medicinally active substance A is 5-fluorouracil (5-fluoro-1H-pyrimidine-2,4-dione) or a derivative thereof.

10. The compound according to claim 1 which is represented by the formula (V) ##STR00025## wherein HES is hydroxyethylstarch.

11. A pharmaceutical formulation comprising the compound according to claim 1.

12. The pharmaceutical formulation according to claim 11, characterized in that said formulation is aqueous and injectable.

13. A process for preparing a compound of general formula (I) according to claim 1 by linking a compound of formula (VI) ##STR00026## with a compound X-L.sup.1-OH or X which comprise at least one hydroxyl group, wherein X is a colloid active compound, and subsequently oxidizing and hydrolyzing the linked product so as to form a phosphor acid diester.

14. A process according to claim 13, wherein the process comprises the following steps: i) Activation of compound of formula (VI), wherein the activation is carried out by an activating agent selected from the group consisting of 4,5-dicyano-imidazole (DCI), saccharin 1-methylimidazole (SMI) and acidic azoles; ii) Linking the activated compound of step i) with a compound X-L.sup.1-OH or X which comprises at least one hydroxyl group; iii) oxidizing and hydrolyzing the linked product of step ii) so as to form a phosphor acid diester.

15. A process according to claim 13, wherein the process is carried out as a one-pot reaction.

16. A process according to claim 14 wherein the linking reaction of step ii) is carried out for time ranging from 10 to 50 minutes.

17. A process according to claim 13 wherein the reaction temperature is between 15° and 40° C.

Description

EXAMPLES

(1) It has been found that medicinally active substances or fluorescence marker can be coupled to colloid active compounds comprising at least one hydroxy group, such as hydroxyethyl starch (HES). In Scheme 3 a reaction sequence is shown for a corresponding 5-fluorouridine derivative. For this purpose, 5-fluorouridine is first lipophilized at the O-2′,3′ position by a cyclic ketal moiety; the ring size is variable. The ketal moiety can be for example obtained by the synthesis disclosed in E. Malecki, H. Rosemeyer, Helv. Chim. Acta 2010, 93, 1500. An example is shown in the following formula representing compound 6 wherein n=14.

(2) ##STR00018##
The ketal moiety further acts as a protecting group. A phosphoramidite 2 with n=14 (hereinafter 2a) can be prepared as follows:

Preparation of Phosporamidite 2a

5-Fluoro-1-[(4′R,6′R)-2′,3′,4′,5′-tetrahydro-6′-(hydroxymethyl)spiro[cyclopentadecane-1,2′-furo[3,4-d][1,3]dioxol]-4′-yl]pyrimidine-2,4(1H,3H)-dione 2-Cyanoethyldiisopropylphosphoramidite (2a)

(3) ##STR00019##

(4) Anhydrous compound 6 (256 mg, 0.45 mmol) was 5′-phosphitylated under an nitrogen atmosphere using ethyldiisopropylamine (Hünig's base, 147 μl, 0.85 mmol) and (chloro)(2-cyanoethoxy)(diisopropylamino)phosphine (181 μl, 0.80 mmol) The reaction mixture was stirred for 15 min at room temperature, and then an ice-cold 5% aq. NaHCO.sub.3 solution (12 ml) was added. The mixture was extracted three times with cold CH.sub.2Cl.sub.2, the combined organic layers were dried (Na.sub.2SO.sub.4), filtered and evaporated on a rotary evaporator (bath temperature, 25° C.). Chromatography (silica gel, column: 2×8 cm, CH.sub.2Cl.sub.2/MeOH, 8:2, v/v) gave one main zone from which compound 2a (208 mg, 60%) was obtained as colourless oil. TLC (CH.sub.2Cl.sub.2/MeOH, 8:2, v/v): R.sub.f 0.95. .sup.31P-NMR (CDCl.sub.3): 149.56, 149.41.

(5) From Scheme 3 it can be seen that the synthesis of the end product 3 implies—after the coupling of the P(III) derivative 2 to HES—the oxidation of 2 with I.sub.2/H.sub.2O, followed by a cleavage of the cyanoethyl protecting group in concentrate aqueous ammonia. The reactions are preferably performed under strict exclusion of moisture (Ar atmosphere) and using thoroughly dried hydroxyethyl starch (HES). The advantage of a product such as compound 3 is its enzymatic cleavability by phosphodiesterases.

(6) Scheme 4 shows another route for the preparation of a compound of the invention.

(7) Phosphoramidite 4 which is based on 5-(propyn-1-yl)-2′-deoxyuridine a virostatic compound, active against Herpes simplex viruses (A. L. Andronova et al. Russ. J. Bioorg. Chem. 2003, 29, 262-266) has been coupled to HES.

(8) ##STR00020##

(9) HES-40 (1 g) was dried by repeated (7 times) lyophilisation (freeze drying) from anhydrous acetonitrile (MeCN) (20 ml, each) during one week. The HES (500 mg) was transferred into a reactor and purged with anhydrous MeCN (10 ml). Subsequently, the solid was purged with a 0.25 M 4,5-dicyano-imidazole (DCI) activator solution in MeCN (10 ml). Simultaneously, 5-(propin-1-yl)-2′-deoxyuridine 2-(cyanoethyl)(diisopropyl)phosphoramidite (4, 1 g) was dissolved in the DCI activator solution and diluted with MeCN to a total volume of 10 ml. After injection into the reactor, the suspension was slightly agitated for 10 min at room temperature. Then, the product was washed twice with anhydrous MeCN (10 ml, each), and, next, an oxidizer solution (0.02 M I.sub.2, THF, pyridine, 10 ml) was injected. After 1 min of agitation within the reactor, the material was washed 4 times with anhydrous MeCN (20 ml, each). For drying, N.sub.2 gas was purged through the reactor for several minutes, and the material was removed from the column. .sup.31P-NMR (D.sub.6)DMSO: −1.25 ppm.

(10) In order to optimize the coupling reaction and to determine the ideal reaction conditions different activating agents as well as different oxidizing agents have been employed in the formation of the compound according to the present invention. The results are summarized in Table 1. The outcome of the reaction has been determined by .sup.31P-NMR spectroscopy.

(11) The reactions were carried out in form of a one-pot reaction, i.e. the entire reaction sequence was carried out in one reaction vessel by subsequently adding the respective reaction component without isolation of the intermediates.

(12) In a first step the phosphoramidite (4) and the respective activating agent were mixed together and stirred at ambient temperature under a N.sub.2-atmosphere. After 2 hours, hydroxyl ethyl starch (HES) was added and the reaction mixture was stirred for 30 minutes at 20° C. Next, the oxidizing agent was added. Depending on the nature of the oxidizing agent as well as its concentration, the time to completion varied. The respective reaction times are summarized in Table 1. After completion, the solvent was evaporated and the product was purified by washing with MeCN and tetrahydrofurane (THF).

(13) The following activating agents and oxidizing agent were used:

(14) DCI=4,5-dicyano-imidazole

(15) SMI=saccharin 1-methylimididazole

(16) CSO=(1S)-(+)-(10-camphorsulfonyl)-oxaziridine

(17) I.sub.2=Iodine

(18) TABLE-US-00001 colloid active reaction time time of coupling reaction entry compound activating agent [h] [min] oxidizing agent time [min] .sup.31P-signal of ligand 1 HES-40 DCI 2 30 I.sub.2 1 + (500 mg) (0.25 M, 10 ml) (0.02 M, 22 ml) 2 HES-40 1H-tetrazole 2 30 I.sub.2 7 + (500 mg) (0.5 M, 5 ml) (0.02 M, 9 ml) 3 HES-40 1H-tetrazole 2 30 I.sub.2 1 + (250 mg) (0.5 M, 2.5 ml) (0.1 M, 4.5 ml) 4 HES-40 1H-tetrazole 2 30 I.sub.2 1 + (250 mg) (0.5 M, 2.5 ml) (0.02 M, 9 ml) 5 HES-40 1H-tetrazole 2 30 I.sub.2 7 + (250 mg) (0.5 M, 2.5 ml ) (0.02 M, 4.5 ml) 6 HES-40 SMI 2 30 I.sub.2 7 + (250 mg) (0.45 M, 5.6 ml) (0.02 M, 4.5 ml)

(19) As can be depicted from Table 1, all activating agents employed (DCI, 1H-tetrazole, SMI) were sufficient to activate the phosphoramidite compound and the coupling of the phosphoramidite to HES was successfully carried out.

(20) As can be seen from FIG. 1, which shows the .sup.31P-NMR spectra of a coupling product of compound (4) to HES, both, DCI as well as 1H-tetrazole, let to the desired coupling product which was detected via .sup.31P-NMR. Although both activating agent were sufficient the use of 1H-tetrazole lead to fewer side products (spectrum C in comparison to spectrum A wherein DCI was used as activating agent and spectrum B which shows the compound of spectrum A after further purification).

(21) NMR studies of the coupling of compound (4) to HES revealed that the use of 1H-tetrazole as activating agent, without further oxidization, lead to intermediate (6) in which the phosphor is directly bonded to the proton. The proton-coupled .sup.31P-NMR spectrum shows a split of the main peak into two triplets. The coupling constant was estimated to be around 587 Hz, which indicates a J.sup.1(P,H)-coupling. An additional oxidation step leads to diester (7). A possible reaction mechanism is shown in Scheme 5 wherein the moiety “base” represents the 5′-fluorouracil moiety.

(22) ##STR00021##

(23) It is believed that, in a first step, the phosphoramidite (4) is activated by 1H-tetrazole, forming a 1H-tetrazole complex which enables the nucleophilic attack of the HES and the formation of a covalent bond. It is believed that the resulting compound (5) in a next step reacts with water that is still present in the HES, leading to intermediate (6) which, after further oxidization, renders the diester (7).

(24) It has surprisingly been found that in cases where SMI was employed as activator, the intermediate (6) could not be detected. FIG. 3 shows the .sup.31P-NMR spectra of a reaction mixture wherein SMI was used. The proton decoupled recording A shows a new main peak at around −2.4 ppm. The proton coupled spectra B does not show the expected splitting of the main peak. In fact, the coupling constant J was estimated to around 7 Hz which corresponds to a J.sup.2(H,P)-coupling, i.e. a coupling of the phosphor atom to the proton of the hydroxyl group of compound (7).

(25) In order to show that the phosphoramidite is covalently bonded to the HES, a proton coupled .sup.31P-DOSY-NMR spectrum of compound (6) was recorded. FIG. 4 shows the recorded spectrum plotted against the determined diffusion coefficient of compound (6). The coefficient was determined to −10.5 m.sup.2/s, which is a typical value for HES as known in the literature. Thus, it can be concluded that the bond between the phosphoramidite moiety and the HES is covalent.