Antibody-drug-conjugates comprising novel anthracycline-derivatives for cancer treatment

Abstract

The present invention relates to novel anthracycline derivatives comprising two oxazolidine cycles and the preparation of said anthracycline derivatives. Furthermore, antibody-drug conjugates (ADCs) comprising the novel anthracycline derivatives and the use of such ADCs in the prevention and treatment of cancer are disclosed.

Claims

1. Process for production of 9-amino-anthracycline-derivatives at least comprising two oxazolidine moieties at the anthracycline, at least comprising the steps of: a) Suspending 9-amino-anthracycline according to the following Formula 1 in an aprotic solvent ##STR00016## wherein R1 is selected from the group consisting of H, OH, OMe or F and R2 is selected from the group consisting of methyl, ethyl or propyl; b) Reacting formaldehyde and the two 1,2 amino-alcohol-moieties of the 9-amino- anthracycline to form an anthracycline according to the following Formula 2 comprising two oxazolidine moieties ##STR00017##

2. Process according to claim 1, wherein in step b) the molar ratio of formaldehyde to 9-amino-anthracycline of formula I (formaldehyde:anthracycline) is 0.1 and 10.

3. Process according to claim 1, wherein the concentration of the 9-amino-anthracycline of formula I in the solvent in step a) is 5 mM and 50 mM.

4. Process according to claim 1, wherein formaldehyde is used in form of paraformaldehyde and the molar ratio of formaldehyde to 9-amino-anthracycline of formula I (formaldehyde:anthracycline) is 1.0 and 2.0.

5. Process according to claim 1, wherein the aprotic solvent is HCC13.

6. Process according to claim 1, wherein in an additional step c) anthracycline according to Formula 2 is reacted with linker molecules L to form a reaction product according to at least one of the following formula 3a-c ##STR00018##

7. Process according to claim 6, wherein the linker L is maleimidocaproyl-L-valine-L-citrulline-p-aminobenzoyl-carbamate ##STR00019##

8. Process according to claim 6, wherein in an additional step d) the not linker-protected oxazolidine ring in formula 3a or 3b is hydrolyzed to yield the respective 1,2 amino-alcohol moiety according to the following formula 4a or 4b ##STR00020##

9. Process according to claim 6 or claim 8, wherein in an additional step e) an antibody is attached to the linker L of formulae 3a-c or attached to the linker L of formulae 4a-b to form an Antibody-Drug-Conjugate (ADC).

10. Anthracycline at least comprising two oxazolidine moieties at the anthracycline core according to the following Formula 2 ##STR00021## wherein R1 is selected from the group consisting of H, OH, OMe or F and R2 is selected from the group consisting of methyl, ethyl or propyl.

11. Anthracyclines according to claim 10, wherein R2=methyl.

12. Antibody-drug-conjugate comprising an antibody, one or two linker-groups and a drug, characterized in that the drug is a 9-amino-anthracycline and the anthracycline comprises two oxazolidine moieties or one oxazolidine moiety and an 1,2 amino-alcohol-moiety at the anthracycline.

13. A pharmaceutical composition, at least comprising the Antibody-drug-conjugate according to claim 12 in a pharmaceutical acceptable carrier.

14. A method for the treatment of cancer in a subject, the method comprising administering to the subject the pharmaceutical composition according to claim 13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 to 4 display

(2) FIG. 1 a schematic overview of one inventive route of synthesis

(3) FIG. 2 a .sup.1H-NMR of the starting material before reacting the 9-amino-anthracycline with formaldehyde;

(4) FIG. 3 .sup.1H-NMR after 24 hours reaction time;

(5) FIG. 4 .sup.1H-NMR after 120 hours reaction time.

(6) FIG. 1 is a schematic diagram of a possible route of synthesis. The overall process is displayed using Amrubicinone as starting material. The process starts with a glycosylation in step 1. A sugar is used for the glycosylation, wherein the functional groups of the 1,2 amino-alcohol moiety are protected. In addition in 9-position of the anthracycline a keto-group is present. Consequently, the keto-group is reduced to the respective alcohol in step 2, followed by de-protection of the functional amino-alcohol-moieties in step 3. In step 4 both 1,2 amino-alcohol-moieties are reacted with formaldehyde to yield the anthracycline comprising two oxazolidine cycles. If necessary the modified anthracycline can be reacted with suitable linker groups. In this step either both oxazolidine cycles may be reacted with linker molecules or only one of the oxazolidine cycles. Furthermore, it is possible to hydrolyse one of the oxazolidine cycles to the respective 1,2 amino-alcohol-structure at or prior linking to an antibody.

(7) FIGS. 2-4 shows the .sup.1H-NMR spectra in the range of 4.26 up to 4.74 ppm as a function of anthracycline and formaldehyde reaction time (according to the described experimental example, step 4). The signal for the (CH.sub.2) groups of the oxazolidine cycle appear in this ppm-range. If a bicyclic oxazolidine anthracycline is present it will be present in form of two diastereomers, because the carbon C13 is racemic (the cycle attached to the C9). Consequently, in that case two sets of signals are visible in the NMR spectra. .sup.1H-NMR shifts for the (CH.sub.2) groups generated by oxazolidine cyclisation can be attributed to signals at 4.30/4.69 ppm (two doublets, at the sugar moiety) and at 4.31/4.68; 4.59/4.65 ppm (four doublets, ring at the C9-position), respectively.

(8) FIG. 2 exhibits a .sup.1H-NMR spectrum in the range of 4.26 up to 4.74 ppm prior a reaction of the 9-amino-anthracycline with formaldehyde. In the displayed range no signals are visible, indicating that no oxazolidine cycles are present in the educt.

(9) FIG. 3 displays a .sup.1H-NMR-spectrum after 24 h reaction time. It is clearly visible that both oxazolidine cycles are formed simultaneously.

(10) FIG. 4 exhibits the .sup.1H-NMR-spectrum after 100 h reaction time. Also here it can be deduced from the peak intensities that by the inventive process two oxazolidine cycles are generated.

EXPERIMENTAL EXAMPLES

(11) 1. StepGlycosylation

(12) ##STR00011##

(13) The starting amrubicinone 100 mg (0.273 mmol) and 1,4-di-O-acetyl-N-trifluoroacetyl--L-daunosamine 178 mg (0.545 mmol, 2 eq.) were dissolved in 20 ml dry THF under an argon atmosphere. 4 molecular sieves (161 mg) were added, followed by dropwise addition of 25 ml of diethyl ether to the stirred reaction mixture. The reaction mixture was cooled to 33 C.-30 C. and then 119 mg (0.545 mmol, 2 eq.) trimethylsilyl trifluoromethanesulfonate (TMSOTf) was added. After addition of TMSOTf, the reaction mixture was stirred for 1-1.5 h at 33 C.-30 C. and then stirring was continued at 25 C.-20 C. The conversion was monitored by TLC. After the reaction ceased, the reaction mixture was poured under stirring into a pre-cooled (0 C.) mixture of 25 ml saturated NaHCO.sub.3 aq. solution and 30 ml of ethyl acetate. The phases were separated and the aqueous phase was extracted once with ethyl acetate (15 ml). The organic phases were combined, washed with brine and dried over Na.sub.2SO.sub.4. The solvent was evaporated and the crude product was purified by column chromatography on silica gel (eluent: diethyl ether/ethyl acetate). 83 mg (48%) of target compound was obtained.

(14) 2. StepReduction of the Keto-Moiety

(15) ##STR00012##

(16) To a solution of 50 mg (0.0788 mmol) of the coupling product from step 1 in 3 ml dry ethanol was added 35 mg (2.1 eq) NaHB(OAc).sub.3. The reaction mixture was stirred for 1 h and the consumption of the starting material was confirmed by TLC monitoring. The solvent was evaporated and 3 ml water was added to the residue. The target product was extracted by diethyl ether (310 ml), organic phases were combined, the solvent was evaporated under reduced pressure and the crude product was dried in vacuo. The product was purified by preparative TLC (Eluent DCM/MeOH 19/1) Rf=0.25-0.3. 18 mg (36%) of pure product was obtained.

(17) 3. StepDe-Protection

(18) ##STR00013##

(19) The reduced product prepared according to step 2 (77 mg, 0.121 mmol) was dissolved in 12 ml mixture THF/MeOH/H.sub.2O 2/2/1 and then 51 mg (10 eq) of LiOH*H.sub.2O was added at 0 C. The reaction mixture was stirred for 15 h at 0 C. After this period, 20 ml water was added to the violet-colored mixture and the mixture was neutralized to pH 8.2-8.3 by dropwise addition of HCl 10%-aq. solution. The target product was extracted by 3*30 ml chloroform. The combined organic extracts were dried over Na.sub.2SO.sub.4 and the solvent was evaporated under reduced pressure. The crude product was triturated with diethyl ether and the formed precipitate was separated by centrifugation. 20 mg (33%) of pure material was obtained.

(20) 4. StepRing Formation

(21) ##STR00014##

(22) 20 mg of de-protected 9 amino anthracycline from step 3 was suspended in 5 ml of dry chloroform. 1.2 mg (1.9 eq.) of solid paraformaldehyde was added and the mixture was stirred for 3 days at room temperature. Paraformaldehyde and unreacted starting material were removed by filtration. The filtrate was concentrated and triturated with diethyl ether. The solid red precipitate was separated, 12.5 mg of product was obtained. The presence of two oxazolidine cycles at the anthracycline was verified by NMR (see FIGS. 2-4). The product had acceptable purity according to NMR spectra and was used without further purification.

(23) 5. StepLinker Attachment

(24) ##STR00015##

(25) 10 mg of the di-oxazolidine product from the previous step was dissolved in 1 ml of dry DMSO, 14 mg (19 mol, 1 eq.) of maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (MC-Val-Cit-PABC-PNP), 2.9 mg 1-hydroxybenzotriazole and 6.6 l of diisopropylethylamine were added to the mixture. The reaction mixture was stirred for 24 h at room temperature. The reaction mixture was poured into 10% solution of citric acid, the red precipitate was separated by filtration and the crude product was purified by preparative HPLC.

(26) 6 mg (28%) of the target 9-aminoanthracycline conjugated to MC-Val-Cit-PABC linker was obtained. The product is suitable for conjugation to primary antibodies.