CONJUGATES OF HETEROAROMATIC NITROGEN-COMPRISING COMPOUNDS

Abstract

The present invention relates to conjugates of π-electron-pair-donating heteroaromatic nitrogen-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 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 π-electron-pair-donating heteroaromatic N of a moiety -D and wherein the linkage between -D and -L.sup.1- is reversible and wherein a moiety -L.sup.2- is conjugated to Z, wherein each -D is independently a π-electron-pair-donating heteroaromatic N-comprising moiety of a drug D-H; 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 of formula (I): ##STR00047## wherein the dashed line marked with an asterisk indicates the attachment to -L.sup.2-; the unmarked dashed line indicates the attachment to the π-electron-pair-donating heteroaromatic N of -D; —Y— is selected from the group consisting of —N(R.sup.3)—, —O— and —S—; R.sup.1, —R.sup.2 and —R.sup.3 are independently selected from the group consisting of —H, -T, 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.4, 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.5)—, —S(O).sub.2N(R.sup.5)—, —S(O)N(R.sup.5)—, —S(O).sub.2—, —S(O)—, —N(R.sup.5)S(O).sub.2N(R.sup.5a)—, —S—, —N(R.sup.5)—, —OC(OR.sup.5)(R.sup.5a)—, —N(R.sup.5)C(O)N(R.sup.5a)— and —OC(O)N(R.sup.5)—; 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.4, which are the same or different; wherein —R.sup.4, —R.sup.5 and —R.sup.5a 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 is selected from the group consisting of small molecule, medium size, peptide and protein drug moieties.

3. The conjugate or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein -D is a small molecule drug moiety.

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

6. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 5, 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.

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

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

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

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

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

12. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 11, wherein —Y— is —N(R.sup.3)—.

13. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 12, wherein —R.sup.3 is —H.

14. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 11, wherein —Y— is —O—.

15. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 14, wherein —R.sup.1 is —H.

16. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 15, wherein —R.sup.2 is C.sub.1-6 alkyl.

17. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 16, wherein -L.sup.2- is a spacer moiety.

18. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 17, wherein -L.sup.2- has a molecular weight in the range of from 14 g/mol to 750 g/mol.

19. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 18, wherein the linkage between Z and -L.sup.2- is stable.

20. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 19, 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.4)(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.5R.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.5R.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.

21. A pharmaceutical composition comprising the conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 20.

22. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 20 or the pharmaceutical composition of claim 21 for use as a medicament.

23. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 20 or the pharmaceutical composition of claim 21 for use in a method of treating a disease that can be treated with D-H.

24. A method of preventing a disease or treating a patient suffering from a disease that can be prevented or treated with D-H, comprising administering an effective amount of the conjugate or the pharmaceutically acceptable salt thereof of any one of claims 1 to 20 or the pharmaceutical composition of claim 21 to the patient.

Description

EXAMPLES

[0246] Materials and Methods

[0247] All materials were commercially available except where stated otherwise.

[0248] Reactions

[0249] Reactions were performed with dry solvents (CH.sub.2Cl.sub.2, DMF, THF) stored over molecular sieves purchased from Sigma-Aldrich Chemie GmbH, Munich, Germany. Generally, reactions were stirred at room temperature and monitored by LCMS.

[0250] Flash Chromatography

[0251] 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.

[0252] RP-HPLC Purification

[0253] 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.

[0254] UPLC-MS Analysis

[0255] 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 1

[0256] ##STR00017##

[0257] The commercially available materials methyl 2-(4-formylphenyl)acetate (1.0 eq) and tripropyl orthoformate (3.0 eq) are combined in EtOH with a catalytic amount of TsOH (0.05 eq) and heated at reflux for 4 h. After removing the volatiles in vacuo, the material is purified by normal-phase flash chromatography.

[0258] In an alternative synthesis route for 1 tripropyl orthoformate is replaced by triethyl orthoformate.

Example 2: Synthesis of Compound 2

[0259] ##STR00018##

[0260] Under N.sub.2, a solution of 1 (1.0 eq) in anhydrous THF is cooled in an ice-bath before addition of thionyl chloride (0.2 eq) and acetic acid (1.5 eq). The solution is warmed to rt before being heated at reflux for 1 h. The volatiles are removed in vacuo.

Example 3: Synthesis of Compound 3b

[0261] ##STR00019##

[0262] Under a N.sub.2 atmosphere, eletriptan (3a) (1.0 eq) is dissolved in anhydrous DMF before addition of sodium hydride (1.2 eq). After stirring for 30 min, compound 2 (1.2 eq) is added and the mixture stirred at rt for 16 h. The solvent is removed in vacuo and the crude material purified by prep-HPLC.

Example 4: Synthesis of Compound 4b

[0263] ##STR00020##

[0264] Compound 3b (1.0 eq) is stirred in 2 M aq. LiOH for 18 h, before acidification with 1 M aq. HCl. After 3× extraction into DCM, the combined organics are dried over MgSO.sub.4 and the volatiles removed in vacuo to give 4a.

[0265] Compound 4a (1.0 eq) is combined with bis(pentafluorophenyl)carbonate (2.0 eq) in DMF. To the mixture is added DIPEA (6.0 eq) before stirring for 2 h. The solvent is removed in vacuo. Purification is performed via prep-HPLC and only non-buffered solvents are used and the pure fractions freeze-dried immediately upon collection to give 4b.

Example 5: Synthesis of Compound 5b

[0266] ##STR00021##

[0267] The amine-functionalized PEG-based hydrogel 5a is synthesized as described in WO 2011/012715 A1, Example 3. The hydrogel is swollen in 1% DIPEA in DMF in a syringe reactor with frit and washed three times with a 1% DIPEA/DMF solution. 4b (2.0 eq per hydrogel amine) is dissolved in 1% DIPEA in DMF. The solution is drawn into the hydrogel-containing reactor and shaken for 16 h at rt. The syringe is drained, the hydrogel washed several times with DMF, then washed several times with pH 5.5 20 mM sodium succinate aqueous buffer. A hydrogel suspension in pH 5.5 aqueous buffer is obtained.

Example 6: Synthesis of Compound 6

[0268] ##STR00022##

[0269] To benzaldehyde diethylacetal (51 μl, 0.28 mmol) was added acetyl chloride (100 μl, 1.4 mmol) and thionyl chloride (20 μl, 0.28 mmol) under an argon atmosphere and the mixture was heated to 65° C. for 90 min. The heating was stopped, and the mixture was concentrated under reduced pressure. Benzimidazole (32.8 g, 0.28 mmol) was dissolved in THF (250 μL) and DMF (200 μl), the mixture was cooled to 0° C., and n-butyl lithium (2.5 M in hexane, 122.1 μl, 0.31 mmol) was added dropwise. After complete addition the cooling bath was removed, and the mixture was stirred at ambient temperature for 30 min. The crude benzaldehyde diethylacetal product was dissolved in THF (250 μl) and added dropwise. After complete addition the reaction mixture was stirred at ambient temperature for 3 h 15 min. The reaction mixture was quenched with saturated Na.sub.2CO.sub.3 (1 ml) and diluted with H.sub.2O (3 ml). The mixture was extracted with DCM (3×1 ml). The combined organic extracts were dried with Na.sub.2SO.sub.4, filtrated and concentrated under reduced pressure. 6 was purified using flash chromatography.

[0270] Yield: 16 mg (22.9%)

[0271] MS: m/z 253.20=[M+H].sup.+, (calculated=253.13).

Example 7: Synthesis of Compound 7

[0272] ##STR00023##

[0273] A solution of ethyl 2-(4-formylphenyl)acetate (100 mg, 0.52 mmol) in triethyl orthoformate (500 μl) was cooled to 0° C. Amberlyst 15 (hydrogen form, 25 mg) was added, and the mixture was stirred at 0° C. for 5.5 h. The cooling bath was removed, and the reaction mixture was stirred for 18 h. The reaction mixture was diluted with ethyl acetate (20 ml) filtrated and concentrated under reduced pressure. 7 was purified using flash chromatography.

[0274] Yield: 110 mg (75.4%)

[0275] NMR: .sup.1H-NMR (43 MHz, CHCl.sub.3-d.sub.1): δ=1.24 (t, J=7.0 Hz), 3.34-4.40 (m), 7.34-7.38 (m).

Example 8: Synthesis of Compound 8

[0276] ##STR00024##

[0277] Compound 7 (110 mg, 0.39 mmol) was dissolved in acetyl chloride (200 μl, 2.8 mmol) and thionyl chloride (40 μl, 0.55 mmol) under an argon atmosphere and the mixture was heated to 65° C. for 90 min. The mixture was concentrated under reduced pressure and the crude material was dried under high vacuum for 20 min. The obtained material was dissolved in THF (400 μl) and used directly in the next step.

Example 9: Synthesis of Compound 9

[0278] ##STR00025##

[0279] A solution of benzimidazole (23.2 mg, 196.2 μmol) in THF (250 μl) and DMF (200 μl) was cooled to 0° C. and n-butyl lithium (2.5 M in hexane, 86.3 μl, 215.8 μmol) was added dropwise. After complete addition the cooling bath was removed, and the mixture was stirred at ambient temperature for 50 min. Compound 8 (50.4 mg, 196.2 μmol) was added dropwise as a solution in THF (200 μl). After complete addition the reaction mixture was stirred for 2 h 40 min. The reaction mixture was quenched by addition of saturated NaHCO.sub.3 (1 ml) and extracted with DCM (3×1.5 ml). The combined organic layers were dried with Na.sub.2SO.sub.4, filtrated and concentrated under reduced pressure. 9 was purified using flash chromatography.

[0280] Yield: 11.9 mg (17.9%)

[0281] MS: m/z 339.13=[M+H].sup.+, (calculated=339.17).

Example 10: Synthesis of Compound 10

[0282] ##STR00026##

[0283] To a solution of compound 9 (5 mg, 13.9 μmol) in THF (200 μl) and water (100 μl) was added LiOH (1.75 mg, 41.7 μmol) and the mixture was stirred at ambient temperature for 1 h 20 min. 10 was purified using RP-HPLC purification without additional TFA.

[0284] Yield: 3.8 mg (88.2%) MS: m/z 311.16=[M+H].sup.+, (calculated=311.14).

Example 11: Synthesis of Compound 11

[0285] ##STR00027##

[0286] Compound 11 was prepared according to the synthesis procedure for compound 6.

[0287] Yield: 13 mg (18.6%)

[0288] MS: m/z 275.19=[M+Na].sup.+, (calculated=275.12).

Example 12: In Vitro Release Kinetics

[0289] The cleavage rate of the reversible bond from conjugates 6, 10 and 11 was monitored at pH 7.4 and 37° C. in aqueous buffer (pH 7.4 48 mM sodium phosphate, 20% acetonitrile). The 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  6 8.0 d 1H-benzo[d]imidazole 10  10 h 1H-benzo[d]imidazole 11 5.7 d indazole

Example 13: Synthesis of Compound 12b

[0290] ##STR00028##

[0291] The commercially available material 3-chloro-4-formylbenzeneacetic acid (1.0 eq) is dissolved in anhydrous DCM and absolute EtOH. Thionyl chloride (2 eq) is added and the reaction mixture is refluxed for 16 h. The solution is cooled and concentrated in vacuo. The obtained crude material is dissolved in DCM and washed with saturated NaHCO.sub.3. The organic layer is dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give 12a.

[0292] To a solution of 12a (1.0 eq) in absolute EtOH is added triethyl orthoformate (3.5 eq) and a catalytic amount of concentrated HCl. The mixture is allowed to reflux for 18 h. The solution is cooled and concentrated in vacuo. The obtained crude material is taken up in diethyl ether, washed with 2M NaOH, the organic layer is dried with Na.sub.2SO.sub.4 and concentrated in vacuo. Compound 12b is purified by flash chromatography.

Example 14: Synthesis of Compound 13

[0293] ##STR00029##

[0294] Compound 13 is prepared from compound 12b according to the synthesis procedure for example 8.

Example 15: Synthesis of Compound 14

[0295] ##STR00030##

[0296] Starting from commercially available material 3-fluoro-4-formylbenzeneacetic acid, compound 14 is prepared according to the procedure for example 13.

Example 16: Synthesis of Compound 15

[0297] ##STR00031##

[0298] Compound 15 is prepared from compound 14 according to the synthesis procedure for example 8.

Example 17: Synthesis of Compound 16

[0299] ##STR00032##

[0300] Compound 16 is synthesized according to the procedure described by Wu et al. in Organic Letters 2016, 18, 5564-5567.

Example 18: Synthesis of Compound 17

[0301] ##STR00033##

[0302] Compound 17 is prepared from compound 16 according to the synthesis procedure for example 8.

Example 19: Synthesis of Compound 18b

[0303] ##STR00034##

[0304] Under a N.sub.2 atmosphere, mibefradil (18a, 1 eq) is dissolved in anhydrous THF before addition of sodium hydride (1.2 eq). After stirring for 30 min, a solution of compound 8 (1.1 eq) in anhydrous THF is added, and the mixture is stirred for 16 h. The solvent is removed in vacuo and the crude material purified by flash chromatography to give 18b.

Example 20: Synthesis of Compound 19b

[0305] ##STR00035##

[0306] To a solution of compound 18b (1.0 eq) in THF/water (2:1) is added LiOH (1.2 eq), and the mixture is stirred at rt. The solvent is removed in vacuo to give 19a as the lithium salt. Compound 19a (1.0 eq) is combined with bis(pentafluorophenyl)carbonate (2.0 eq) in DMF. To the mixture is added DIPEA (6.0 eq) before stirring for 2 h. The solvent is removed in vacuo. Purification is performed via flash chromatography to give 19b.

Example 21: Synthesis of Compound 20

[0307] ##STR00036##

[0308] 2,2′-dithiodipyridine (1.5 eq) is dissolved in anhydrous methanol and a solution of 6-amino-1-hexanethiol hydrochloride (1.0 eq) in anhydrous methanol is added dropwise. After complete addition the mixture is stirred at rt for 3 h. The solvent is removed in vacuo and the material is purified by flash chromatography to give 20.

Example 22: Synthesis of Compound 21b

[0309] ##STR00037##

[0310] To a solution of compound 19a (1 eq) in anhydrous DMF are added DIPEA (2.4 eq), PyBOP (1.2 eq) and compound 20 (1.2 eq) successively, and the reaction mixture is stirred at rt for 18 h. The solvent is removed in vacuo and the mixture is purified by flash chromatography to give 21a.

[0311] Compound 21a (1.0 eq) is dissolved in anhydrous DMF and a solution of tris(2-carboxyethyl)phosphine hydrochloride (4.0 eq) and DIPEA (4.0 eq) in DMSO is added. After complete addition the mixture is stirred at rt for 30 min. The solvent is removed in vacuo and the material is purified by flash chromatography to give 21b.

Example 23: Synthesis of Compound 22

[0312] ##STR00038##

[0313] The amine-functionalized PEG-based hydrogel 5a is synthesized as described in WO 2011/012715 A1, Example 3. The hydrogel is swollen in 1% DIPEA in DMF in a syringe reactor with frit and washed three times with a 1% DIPEA/DMF solution. 19b (2.0 eq per hydrogel amine) is dissolved in 1% DIPEA in DMF. The solution is drawn into the hydrogel-containing reactor and shaken for 16 h at rt. The syringe is drained, the hydrogel washed several times with DMF, then washed several times with phosphate-buffered saline. A hydrogel suspension in pH 7.4 aqueous buffer is obtained.

Example 24: Synthesis of Compound 23

[0314] ##STR00039##

[0315] A solution of compound 21b (1.1 eq) in acetonitrile/water is mixed with 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 purification without additional TFA to give compound 23.

Example 25: Synthesis of Compound 24

[0316] ##STR00040##

[0317] A solution of compound 21b (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 purification without additional TFA to give compound 24.

Example 26: Synthesis of Compound 25

[0318] ##STR00041##

[0319] The synthesis of compound 25 is performed according to the procedures described for example 19 and example 20, starting from compound 13.

Example 27: Synthesis of Compound 26

[0320] ##STR00042##

[0321] The synthesis of compound 26 is performed according to the procedure described for compound 22, using compound 25.

Example 28: Synthesis of Compound 27

[0322] ##STR00043##

[0323] The synthesis of compound 27 is performed according to the procedures described for example 19 and example 20, starting from compound 15.

Example 29: Synthesis of Compound 28

[0324] ##STR00044##

[0325] The synthesis of compound 28 is performed according to the procedure described for compound 22, using compound 27.

Example 30: Synthesis of Compound 29

[0326] ##STR00045##

[0327] The synthesis of compound 29 is performed according to the procedures described for example 19 and example 20, starting from compound 17.

Example 31: Synthesis of Compound 30

[0328] ##STR00046##

[0329] The synthesis of compound 30 is performed according to the procedure described for example 19 and example 20, using compound 29.

Example 32: Release of Mibefradil In Vitro

[0330] Release of mibefradil from compounds 22, 23, 24, 26, 28 and 30 is effected by hydrolysis in 60 mM sodium phosphate buffer at pH 7.4 and 37° C. Unmodified mibefradil is released as assessed by LCMS.

Abbreviations

[0331] DCM—dichloromethane [0332] DIPEA—diisopropylethylamine [0333] DMF—dimethylformamide [0334] eq—equivalent [0335] EtOH—ethanol [0336] HPLC—high performance liquid chromatography [0337] LCMS—liquid chromatography-coupled mass spectrometry [0338] prep-HPLC—preparative high-performance liquid chromatography [0339] PyBOP—benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [0340] RP—reversed phase [0341] rt—room temperature [0342] THF—tetrahydrofuran [0343] TsOH—para-toluene sulfonic acid