CONJUGATES OF AN ELECTRON-DONATING NITROGEN OR TERTIARY AMINE COMPRISING COMPOUNDS

Abstract

The present invention relates to conjugates of an electron-donating heteroaromatic nitrogen or tertiary amine comprising drugs and pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising said conjugates and the use of said conjugates as medicaments.

Claims

1. A conjugate or a pharmaceutically acceptable salt thereof comprising at least one moiety -D.sup.+ conjugated via at least one moiety -L.sup.1-L.sup.2- to at least one moiety Z, wherein a moiety -L.sup.1- is conjugated to a N* of a moiety -D.sup.+ and wherein the linkage between -D.sup.+ and -L.sup.1- is reversible and wherein a moiety -L.sup.2- is conjugated to Z, wherein each -D.sup.+ is independently an electron-donating heteroaromatic N.sup.+-comprising moiety or a quaternary ammonium cation comprising moiety of a drug D, wherein each D comprises an electron-donating heteroaromatic N or a tertiary amine; each -L.sup.2- is independently a single bond or a spacer moiety; each Z is independently a polymeric moiety or a C.sub.8-24 alkyl; each -L.sup.1- is independently a linker moiety of formula (I): ##STR00060## wherein the dashed line indicates the attachment to the N.sup.+ of -D.sup.+; t is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6; A- is a ring selected from the group consisting of monocyclic or bicyclic aryl and heteroaryl, provided that -A- is connected to —Y and —C(R.sup.1)(R.sup.1a)— via carbon atoms; wherein said monocyclic or bicyclic aryl and heteroaryl are optionally substituted with one or more —R.sup.2, which are the same or different; —R.sup.1, —R.sup.1a and each —R.sup.2 are independently selected from the group consisting of —H, —C(O)OH, -halogen, —NO.sub.2, —CN, —OH, C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl; wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally substituted with one or more —R.sup.3, which are the same or different; and wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.4)—, —S(O).sub.2N(R.sup.4)—, —S(O)N(R.sup.4)—, —S(O).sub.2—, —S(O)—, —N(R.sup.4)S(O).sub.2N(R.sup.4a)—, —S—, —N(R.sup.4)—, —OC(OR.sup.4)(R.sup.4a)—, —N(R.sup.4)C(O)N(R.sup.4a)— and —OC(O)N(R.sup.4)—; each -T- is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl, wherein each -T- is independently optionally substituted with one or more —R.sup.3, which are the same or different; wherein —R.sup.3 is selected from the group consisting of —H, —NO.sub.2, —OCH.sub.3, —CN, —N(R.sup.4)(R.sup.4a), —OH, —C(O)OH and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; wherein —R.sup.4 and —R.sup.4a are independently selected from the group consisting of —H and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; —Y is selected from the group consisting of: ##STR00061##  and a peptidyl moiety; wherein the dashed line marked with an asterisk indicates the attachment to -A-; —Nu is a nucleophile; —Y.sup.1— is selected from the group consisting of —O—, —C(R.sup.10)(R.sup.10a)—, —N(R.sup.11)— and —S—; ═Y.sup.2 is selected from the group consisting of ═O, ═S and ═N(R.sup.12); —Y.sup.3— is selected from the group consisting of —O—, —S— and —N(R.sup.13)—; -E- is selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl and -Q-; wherein C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl are optionally substituted with one or more —R.sup.14, which are the same or different; —R.sup.5, —R.sup.6, each —R.sup.7, —R.sup.8, —R.sup.9, —R.sup.10, —R.sup.10a, —R.sup.11, —R.sup.12 and —R.sup.13 are independently selected from the group consisting of C.sub.1-20 alkyl, C.sub.2-20 alkenyl, C.sub.2-20 alkynyl and -Q; wherein C.sub.1-20 alkyl, C.sub.2-20 alkenyl and C.sub.2-20 alkynyl are optionally substituted with one or more —R.sup.14, which are the same or different; and wherein C.sub.1-20 alkyl, C.sub.2-20 alkenyl and C.sub.2-20 alkynyl are optionally interrupted by one or more groups selected from the group consisting of Q, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.15)—, —S(O).sub.2N(R.sup.15)—, —S(O)N(R.sup.15)—, —S(O).sub.2—, —S(O)—, —N(R.sup.15)S(O).sub.2N(R.sup.15a)—, —S—, —N(R.sup.1)—, —OC(OR.sup.15)R.sup.15a—, —N(R.sup.15)C(O)N(R.sup.15a)— and —OC(O)N(R.sup.15)—; each Q is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl, wherein each Q is independently optionally substituted with one or more —R.sup.14, which are the same or different; wherein —R.sup.14, —R.sup.1 and —R.sup.15a are independently selected from the group consisting of —H and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and each -L.sup.1- is substituted with -L.sup.2- and optionally further substituted.

2. The conjugate or pharmaceutically acceptable salt thereof of claim 1, wherein -D.sup.+ is selected from the group consisting of small molecule, medium molecule, oligonucleotide, peptide and protein drug moieties.

3. The conjugate or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein Z is a polymeric moiety.

4. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 3, wherein Z is a water-insoluble polymeric moiety.

5. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 4, wherein Z is a water-insoluble polymeric moiety comprising a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolic acids), polybutylene terephthalates, poly(caprolactones), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters), poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines), poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides), poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines), poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic acids), poly(methacrylamides), poly(methacrylates), poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl amines), poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses, carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other carbohydrate-based polymers, xylans, and copolymers thereof.

6. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 5, wherein Z is a hydrogel.

7. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 6, wherein Z is a PEG-based or hyaluronic-acid based hydrogel.

8. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, wherein Z is a PEG-based hydrogel.

9. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, wherein Z is a hyaluronic-acid based hydrogel.

10. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 3, wherein Z is a water-soluble polymeric moiety.

11. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 10, wherein —Y is selected from the group consisting of ##STR00062## wherein —Nu, -E-, —Y.sup.1—, ═Y.sup.2, —Y.sup.3—, —R.sup.5, —R.sup.7, —R.sup.8 and —R.sup.9 are as defined in claim 1.

12. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 11, wherein —Y is ##STR00063## and —Nu, -E-, —Y.sup.1—, ═Y.sup.2 and —Y.sup.3— are as defined in claim 1.

13. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 12, wherein —Nu is a nucleophile selected from the group consisting of primary, secondary or tertiary amine and amide.

14. The conjugate or pharmaceutically acceptable salt thereof of claim 13, wherein —Nu is a secondary amine.

15. The conjugate or pharmaceutically acceptable salt thereof of claim 13, wherein —Nu is a tertiary amine.

16. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 15, wherein -E- is C.sub.1-6 alkyl.

17. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 16, wherein —Y.sup.1— is —N(R.sup.11)— and —R.sup.11 is as defined in claim 1.

18. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 17, wherein ═Y.sup.2 is ═O.

19. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 18, wherein —Y.sup.3— is —O—.

20. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 19, wherein -L.sup.2- is a spacer moiety.

21. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 20, wherein -L.sup.2- has a molecular weight in the range of from 14 g/mol to 750 g/mol.

22. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 21, wherein the linkage between Z and -L.sup.2- is a stable linkage.

23. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 22, wherein -L.sup.2- is a spacer moiety selected from the group consisting of -T′-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y1)—, —S(O).sub.2N(R.sup.y1)—, —S(O)N(R.sup.y1)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y1)S(O).sub.2N(R.sup.y1a)—, —S—, —N(R.sup.y1)—, —OC(OR.sup.y1)(R.sup.y1a)—, —N(R.sup.y1)C(O)N(R.sup.y1a)—, —OC(O)N(R.sup.y1)—, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T′-, C.sub.1-50 alkyl, C.sub.2-50 alkenyl and C.sub.2-50 alkynyl are optionally substituted with one or more —R.sup.y2, which are the same or different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T′-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y3)—, —S(O).sub.2N(R.sup.y3)—, —S(O)N(R.sup.y3)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y3)S(O).sub.2N(R.sup.y3a)—, —S—, —N(R.sup.y3)—, —OC(OR.sup.3)(R.sup.y3a)—, —N(R.sup.y3)C(O)N(R.sup.y3a)—, and —OC(O)N(R.sup.y3)—; wherein —R.sup.y1 and —R.sup.y1a are independently selected from the group consisting of —H, -T′, C.sub.1-50 alkyl, C.sub.2-50 alkenyl and C.sub.2-50 alkynyl; wherein -T′, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more —R.sup.y2, which are the same or different, and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T′-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.y4)—, —S(O).sub.2N(R.sup.y4)—, —S(O)N(R.sup.y4)—, —S(O).sub.2—, —S(O)—, —N(R.sup.y4)S(O).sub.2N(R.sup.y4a)—, —S—, —N(R.sup.y4)—, —OC(OR.sup.y4)(R.sup.y4a)—, —N(R.sup.y4)C(O)N(R.sup.y4a)—, and —OC(O)N(R.sup.y4)—; each T′ is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl and 8- to 30-membered heteropolycyclyl; wherein each T′ is independently optionally substituted with one or more —R.sup.y2, which are the same or different; each —R.sup.y2 is independently selected from the group consisting of halogen, —CN, oxo (═O), —COOR.sup.y5, —OR.sup.y5, —C(O)R.sup.y5, —C(O)N(R.sup.y5R.sup.y5a), —S(O).sub.2N(R.sup.y5R.sup.y5a), —S(O)N(R.sup.y5R.sup.y5a), —S(O).sub.2R.sup.y5, —S(O)R.sup.y5, —N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), —SR.sup.y5, —N(R.sup.y5R.sup.y5a), —NO.sub.2, —OC(O)R.sup.y5, —N(R.sup.y5)C(O)R.sup.y5a, —N(R.sup.y5)S(O).sub.2R.sup.y5a, —N(R.sup.y5)S(O)R.sup.y5a, —N(R.sup.y5)C(O)OR.sup.y5a, —N(R.sup.y5)C(O)N(R.sup.y5aR.sup.y5b), —OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and each —R.sup.y3, —R.sup.y3a, —R.sup.y4, —R.sup.y4a, —R.sup.y5, —R.sup.y5a and —R.sup.y5b is independently selected from the group consisting of —H and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

24. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 23, wherein one hydrogen given by —R.sup.1a is replaced by -L.sup.2- and -L.sup.1- is of formula (Ia): ##STR00064## wherein the unmarked dashed line indicates the attachment to the N.sup.+ of -D.sup.+, the dashed line marked with an asterisk indicates the attachment to -L.sup.2-; and —R.sup.1, -A-, —Y, each —R.sup.2 and t are defined as in formula (I) of claim 1.

25. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 23, wherein one hydrogen given by —R.sup.2 is replaced by -L.sup.2- and -L.sup.1- is of formula (Ib): ##STR00065## wherein the unmarked dashed line indicates the attachment to the N.sup.+ of -D.sup.+, the dashed line marked with an asterisk indicates the attachment to -L.sup.2-; —R.sup.1, —R.sup.1a, -A-, —Y and each —R.sup.2 are defined as in formula (I) of claim 1; and t′ is selected from the group consisting of 0, 1, 2, 3, 4 and 5.

26. A pharmaceutical composition comprising the conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 25.

27. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 25 or the pharmaceutical composition of claim 26 for use as a medicament.

28. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 25 or the pharmaceutical composition of claim 26 for use in a method of treating a disease that can be treated with D.

29. A method of preventing a disease or treating a patient suffering from a disease that can be prevented or treated with D, comprising administering an effective amount of the conjugate or the pharmaceutically acceptable salt thereof of any one of claims 1 to 25 or the pharmaceutical composition of claim 26 to the patient.

Description

EXAMPLES

[0401] Materials and Methods

[0402] All materials were commercially available except where stated otherwise.

[0403] Reactions

[0404] Reactions were performed with dry solvents (CH.sub.2Cl.sub.2, DMF, THF) purchased from Sigma-Aldrich Chemie GmbH, Munich, Germany. Generally, reactions were stirred at room temperature and monitored by LCMS.

[0405] RP-HPLC Purification

[0406] Preparative RP-HPLC purifications were performed with a Waters 600 controller with a 2487 Dual Absorbance Detector or an Agilent Infinity 1260 preparative system using a Waters XBridge BEH300 Prep C18 10 μm, 150×30 mm column as stationary phase. Products were detected at 215 nm, 320 nm or 360 nm. Linear gradients of solvent system A (water containing 0.1% TFA v/v) and solvent system B (acetonitrile containing 0.1% TFA v/v) were used. HPLC fractions containing product were pooled and lyophilized if not stated otherwise.

[0407] Flash Chromatography

[0408] Flash chromatography purifications were performed on an Isolera One system or an Isolera Four system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges. Products were detected at 254 nm or 280 nm.

[0409] UPLC-MS Analysis

[0410] Analytical ultra-performance LC (UPLC)-MS was performed on a Waters Acquity system or an Agilent 1290 Infinity II equipped with a Waters BEH300 C18 column (2.1×50 mm, 1.7 μm particle size or 2.1×100 mm, 1.7 μm particle size); solvent A: water containing 0.04% TFA (v/v), solvent B: acetonitrile containing 0.05% TFA (v/v) coupled to a Waters Micromass ZQ or coupled to an Agilent Single Quad MS system.

Example 1: Synthesis of Compound 1b

[0411] ##STR00044##

[0412] Compound 1a is synthesized as described in WO 2005/099768 A2 for compound 57b. Compound 1a (1.0 eq) and 3-nitro-2-pyridinesulfenyl chloride (1.5 eq) are stirred for 2 h under nitrogen and the volatiles removed in vacuo. This is purified to give compound 1b as the TFA salt.

Example 2: Synthesis of Compound 2

[0413] ##STR00045##

[0414] Compound 1b (1.0 eq) is dissolved in DMF and under cooling in an ice-bath, SOCl.sub.2 (0.2 eq) in DCM is added dropwise. The reaction mixture is allowed to warm to rt and stirred for 30 minutes. The reaction mixture is quenched by addition of water before extraction into ethyl acetate. The organics are dried over MgSO.sub.4, filtered and the solvents are removed in vacuo.

Example 3: Synthesis of Compound 3b

[0415] ##STR00046##

[0416] Compound 2 (1.0 eq) is combined with topotecan (3a) (1.0 eq) in DMF. Under stirring at rt, TBAI (0.2 eq) is added before addition of DIPEA (2.0 eq) before stirring at rt for 18 h. The solvent is then removed in vacuo and the product is purified by prep-HPLC.

Example 4: Synthesis of Compound 4

[0417] ##STR00047##

[0418] Compound 3b is stirred in 10 mM DTT and 25 mM acetate buffer at pH 4.2 for 30 minutes at 4° C. before purification by prep-HPLC.

Example 5: Synthesis of Compound 5b

[0419] ##STR00048##

[0420] The maleimide functionalized hydrogel beads 5a are synthesized as described in WO 2014/056923 A1, Example 5. Hydrogel beads 5a and 4 are combined in 10 mM sodium phosphate, 5 mM Na.sub.2EDTA, 0.01% Tween 20, pH 6.5 buffer to give compound 5b.

Example 6: Synthesis of Compound 6b

[0421] ##STR00049##

[0422] 4-(1-Hydroxyethyl)phenol (100 mg) was dissolved in 1.2 ml THF and DIPEA (0.38 mL) was added with stirring. 4-nitrophenyl chloroformate (160 mg) was dissolved in 0.3 ml of THF and added drop-wise to the reaction. The reaction was stirred for 1 h. tert-Butyl N-methyl-N-[3-(methylamino)propyl]carbamate (190 mg) was dissolved in 0.3 ml of THF and added drop-wise to the reaction. The reaction was stirred for 40 min. The reaction was quenched with 0.38 ml TFA and diluted with water (2 ml) and 6a was purified by RP-HPLC.

[0423] Yield: 193 mg (73%)

[0424] MS: m/z 367.12=[M+H].sup.+, (calculated=367.22).

[0425] Chlorotriazine (79 mg, 0.43 mmol) and DMF (87 μL) were stirred in a 10 ml flask for 15 min. DCM (1 mL) was added. Sodium bicarbonate (131 mg, 1.56 mmol) was added. 6a (150 mg, 0.41 mmol) was dissolved in 0.3 ml dichloromethane and added drop-wise. After 2.5 h the reaction was diluted with 10 ml DCM and 8 ml water. The organic phase was washed with saturated bicarbonate, 1 M HCl and brine (1×8 ml, each). The organic phase was dried (MgSO.sub.4) and concentrated in vacuo. The product was purified by flash chromatography using heptane/ethyl acetate as eluent.

[0426] Yield: 77 mg (49%)

Example 7: Synthesis of Compound 7a and 7b

[0427] ##STR00050##

[0428] 6b (17 mg, 0.04 mmol) and either N,N-dimethyl-N-phenethylamine (6 mg; 0.04 mmol) for 7a, or quinoline (26 mg; 0.2 mmol) for 7b, were dissolved in DMF (0.2 mL), and TBAI (7.4 mg, 0.02 mmol) and DIPEA (17.5 μL, 0.10 mmol) were added. The reaction was stirred for 6.5 days (7a) or 4 days (7b) and the product purified by RP-HPLC.

[0429] Yield (7a): 1.5 mg (7%); MS: m/z 498.24=[M].sup.+, (calculated=498.33).

[0430] Yield (7b): 1 mg (5%); MS: m/z 478.23=[M], (calculated=478.27).

Example 8: Synthesis of Compound 8a and 8b

[0431] ##STR00051##

[0432] 7a (1.5 mg) or 7b (1 mg) was dissolved in DCM (0.5 mL), and TFA (0.5 mL) was added with stirring. After 1 h the volatiles were removed in vacuo and the residue dried yielding the TFA salt of 8a or 8b, respectively in quantitative yield.

Example 9: In Vitro Release Kinetics

[0433] The cleavage rate of the reversible bond from conjugates 8a-b was monitored at pH 7.4 and 37° C. in aqueous buffer (pH 7.4, 48 mM sodium phosphate, 20% acetonitrile). Disappearance of the conjugate was determined by LCMS (UV detection) and fitted with curve fitting software to obtain the half-life of the release.

TABLE-US-00001 Compound t.sub.1/2 (pH 7.4) Released product 8a 1.3 h N,N-dimethyl-N-phenethylamine 8b 1.8 h quinoline

Example 10: Synthesis of Compound 10

[0434] ##STR00052##

[0435] Compound 9 is synthesized as described in WO 2005/099768 A2 for compound 55b. Compound 9 (1.0 eq) is dissolved in THF and lithium borohydride (3.0 eq.) is added in an atmosphere of nitrogen. The reaction is stirred for 24 h. The product is purified by flash chromatography to give 10.

Example 11: Synthesis of Compounds 11e and 11f

[0436] ##STR00053##

[0437] To a solution of n-propylamine (1 eq) and NaCNBH.sub.3 (1.1 eq) in methanol is added 2,4,6-trimethoxybenzaldehyde (0.92 eq) portion wise. The mixture is stirred at rt for 90 min, acidified with 3 M HCl and stirred for a further 15 min. The reaction mixture is added to saturated bicarbonate solution and extracted 5× with DCM. The combined organic phases are dried over sodium sulfate and the solvents are evaporated in vacuo and the product further dried yielding 11a.

[0438] The synthesis of 11b is performed accordingly to 11a using sec-butylamine instead of n-propylamine.

[0439] 11a (1 eq.) and Cbz-sarcosine (1.1 eq) are dissolved in DMF and PyBOP (1.1 eq) and DIPEA (3 eq) are added. After 2 h the reaction is diluted with ethyl acetate. The organic phase is washed with sodium bicarbonate, 1 M HCl, and brine, and the product purified by flash chromatography to give 11c.

[0440] The synthesis of 11d is performed accordingly to 11c using 11b instead of 11a. 11c (1 eq) is dissolved in THF. 10% Palladium on activated charcoal (0.1 eq w/w) is added and the reaction stirred in an atmosphere of hydrogen. After 3 h the reaction is filtered through celite, and the filtrate concentrated in vacuo to give 11e.

[0441] The synthesis of 11f is performed accordingly to 11e using 11d instead of 11c.

Example 12: Synthesis of Compounds 12a-c

[0442] ##STR00054##

[0443] (1 eq) is dissolved in THF and DIPEA (5 eq) is added with stirring. 4-nitrophenyl chloroformate (1.1 eq) is dissolved in THF and added drop-wise to the reaction while stirring. After 1 h 11e (1.5 eq) is added to the reaction while stirring. After 1 h the reaction is quenched with acetic acid, and the product is purified by flash chromatography to give 12b.

[0444] The synthesis of 12c is performed accordingly to 12b using 11f instead of 11e.

[0445] The synthesis of 12a is performed accordingly to 12b using tert-butyl N-methyl-N-[3-(methylamino)propyl]carbamate instead of 11e.

Example 13: Synthesis of Compounds 13a-c

[0446] ##STR00055##

[0447] Chlorotriazine (1.05 eq) and DMF (2.7 eq) are stirred for 15 min. DCM is added. Sodium bicarbonate (3.8 eq) is added. 12a (1 eq) is dissolved in DCM and added drop-wise. After 3 h the reaction is diluted with DCM and water. The organic phase is washed with saturated bicarbonate, 1 M HCl and brine. The organic phase is dried (MgSO.sub.4) and concentrated in vacuo. The product is purified by flash chromatography giving 13a.

[0448] The synthesis of 13b is performed accordingly to 13a using 12b instead of 12a.

[0449] The synthesis of 13c is performed accordingly to 13a using 12c instead of 12a.

Example 14: Synthesis of Compounds 14a-c

[0450] ##STR00056##

[0451] Compound 13a (1.2 eq) is combined with milrinone (1.0 eq) in DMF. Under stirring at rt, TBAI (0.2 eq) is added before addition of DIPEA (2.0 eq) before stirring at rt for 24 h. The solvent is then removed in vacuo and the product is purified by RP-HPLC giving 14a.

[0452] The synthesis of 14b is performed accordingly to 14a using 13b instead of 13a.

[0453] The synthesis of 14c is performed accordingly to 14a using 13c instead of 13a.

Example 15: Synthesis of Compounds 15a-c

[0454] ##STR00057##

[0455] 14a is dissolved in hexafluoroisopropanol/water/triethylsilane (39:1:1) and TFA is added (10% v/v). The reaction is stirred at rt for 3h. The solvent is then removed in vacuo and the product is purified by RP-HPLC giving 15a as TFA salt.

[0456] The synthesis of 15b is performed accordingly to 15a using 14b instead of 14a.

[0457] The synthesis of 15c is performed accordingly to 15a using 14c instead of 14a.

Example 16: Synthesis of Compounds 16a-c

[0458] ##STR00058##

[0459] Hydrogel beads 5a and compound 15a are combined in 10 mM sodium phosphate, 5 mM Na.sub.2EDTA, 0.01% Tween 20, pH 6.5 buffer to give compound 16a.

[0460] The synthesis of 16b is performed accordingly to 16a using 15b instead of 15a.

[0461] The synthesis of 16c is performed accordingly to 16a using 15c instead of 15a.

Example 17: Synthesis of Compound 17

[0462] ##STR00059##

[0463] A solution of compound 16a (4.5 eq) in acetonitrile/water is mixed with 4-arm PEG 20 kDa maleimide (1 eq) and the pH is adjusted to 7.0 by addition of pH 7.4 buffer (50 mM phosphate). The mixture is stirred at rt for 2 h and then purified by RP-HPLC to give compound 17.

Example 18: Release of Milrinone In Vitro

[0464] Release of milrinone from compounds 16a-c and 17 is effected by incubation in 60 mM sodium phosphate buffer at pH 7.4 and 37° C. Unmodified milrinone is released as assessed by LCMS.

Abbreviations

[0465] Boc—tert.-butyloxycarbonyl [0466] Cbz—benzyloxycarbonyl [0467] DCM—dichloromethane [0468] DIPEA—diisopropylethylamine [0469] DMF—dimethylformamide [0470] DTT—dithiothreitol [0471] EDTA—ethylenediaminetetraacetic acid [0472] eq—equivalent [0473] HPLC—high performance liquid chromatography [0474] LC-MS—liquid chromatography-coupled mass spectrometry [0475] Me—methyl [0476] prep-HPLC—preparative high-performance liquid chromatography [0477] PyBOP—benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [0478] RP—reversed phase [0479] rt—room temperature [0480] TBAI—tetrabutylammonium iodide [0481] TFA—trifluoroacetic acid [0482] Tmob—2,4,6-trimethoxybenzyl [0483] Trt—trityl [0484] UPLC—ultra performance liquid chromatography