Prodrugs comprising an aminoalkyl glycine linker

Abstract

The present invention relates to novel prodrugs of primary or secondary amine- or hydroxyl-comprising biologically active moieties and pharmaceutically acceptable salts thereof, prodrug reagents, pharmaceutical compositions comprising these prodrugs and the use of these prodrugs. A disclosed prodrug may include a biologically active moiety reversibly and covalently linked to a specialized protective group; the linker may be reversible (i.e., hydrolytically cleavable in the absence of enzymes under physiological conditions) and, upon cleavage, may release a drug in its unmodified, pharmacologically active form.

Claims

1. A prodrug or a pharmaceutically acceptable salt thereof comprising: a conjugate D-L; wherein: D is a primary or secondary amine- or hydroxyl-comprising biologically active moiety; and L comprises a linker moiety -L.sup.1 represented by formula (I): ##STR00140## wherein: the dashed line indicates attachment to the primary or secondary amine or hydroxyl of the biologically active moiety by forming an amide or ester linkage, respectively; R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, and R.sup.3a are independently of each other selected from the group consisting of —H, —C(R.sup.8R.sup.8aR.sup.8b), —C(═O)R.sup.8, —C≡N, —C(═NR.sup.8)R.sup.8a, —CR.sup.8(═CR.sup.8aR.sup.8b), —C≡CR.sup.8, and -T; R.sup.4, R.sup.5, and R.sup.5a are independently of each other selected from the group consisting of —H, —C(R.sup.9R.sup.9aR.sup.9b), and -T; a1 and a2 are independently of each other 0 or 1; each occurrence of R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a, R.sup.8b, R.sup.9, R.sup.9a, and R.sup.9b is independently selected from the group consisting of —H, halogen, —CN, —COOR.sup.10, —OR.sup.10, —C(O)R.sup.10, —C(O)N(R.sup.10R.sup.10a), —S(O).sub.2N(R.sup.10R.sup.10a), —S(O)N(R.sup.10R.sup.10a), —S(O).sub.2R.sup.10, —S(O)R.sup.10, —N(R.sup.10)S(O).sub.2N(R.sup.10aR.sup.10b), —SR.sup.10, —N(R.sup.10R.sup.10a), —NO.sub.2, —OC(O)R.sup.10, —N(R.sup.10)C(O)R.sup.10a, —N(R.sup.10)S(O).sub.2R.sup.10a, —N(R.sup.10)S(O)R.sup.10a, —N(R.sup.10)C(O)OR.sup.10a, —N(R.sup.10)C(O)N(R.sup.10aR.sup.10b), —OC(O)N(R.sup.10R.sup.10a), -T, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl, wherein: -T, 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.11, which are the same or different; and C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are further 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.12), —S(O).sub.2N(R.sup.12)—, —S(O)N(R.sup.12)—, —S(O).sub.2—, —S(O)—, —N(R.sup.12)S(O).sub.2N(R.sup.12a)—, —S—, —N(R.sup.12)—, —OC(OR.sup.12)(R.sup.12a)—, —N(R.sup.12)C(O)N(R.sup.12a)—, and —OC(O)N(R.sup.12)—; each occurrence of R.sup.10, R.sup.10a, and R.sup.10b is independently selected from the group consisting of —H, -T, C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl, wherein: -T, 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.11, which are the same or different; and C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are further 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.12)—, —S(O).sub.2N(R.sup.12)—, —S(O)N(R.sup.12)——S(O).sub.2—, —S(O)—, —N(R.sup.12)S(O).sub.2N(R.sup.12a)—, —S—, —N(R.sup.12)—, —OC(OR.sup.12)(R.sup.12a)—, —N(R.sup.12)C(O)N(R.sup.12a)—, and —OC(O)N(R.sup.12)—; each occurrence of 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, each occurrence of T being further independently optionally substituted with one or more R.sup.11, which are the same or different; each occurrence of R.sup.11 is independently selected from the group consisting of halogen, —CN, oxo (═O), —COOR.sup.13, —OR.sup.13, —C(O)R.sup.13, —C(O)N(R.sup.13R.sup.13a), —S(O).sub.2N(R.sup.13R.sup.13a), —S(O)N(R.sup.13R.sup.13a), —S(O).sub.2R.sup.13, —S(O)R.sup.13, —N(R.sup.13)S(O).sub.2N(R.sup.13aR.sup.13b), —SR.sup.13, —N(R.sup.13R.sup.13a), —NO.sub.2, —OC(O)R.sup.13, —N(R.sup.13)C(O)R.sup.13a, —N(R.sup.13)S(O).sub.2R.sup.13a, —N(R.sup.13)S(O)R.sup.13a, —N(R.sup.13)C(O)OR.sup.13a, —N(R.sup.13)C(O)N(R.sup.13aR.sup.13b), —OC(O)N(R.sup.13R.sup.13a), and C.sub.1-6 alkyl, which C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; each occurrence of R.sup.12, R.sup.12a, R.sup.13, R.sup.13a, R.sup.13b, and R.sup.9b is independently selected from the group consisting of —H and C.sub.1-6 alkyl, which C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; optionally, one or more of the pairs R.sup.1/R.sup.1a, R.sup.2/R.sup.2a, R.sup.3/R.sup.3a, R.sup.6/R.sup.6a and R.sup.7/R.sup.7a are joined together with the atom to which they are attached to form a C.sub.3-10 cycloalkyl or a 3- to 10-membered heterocyclyl; and optionally, one or more of the pairs R.sup.1/R.sup.2, R.sup.1/R.sup.3, R.sup.1/R.sup.4, R.sup.1/R.sup.5, R.sup.1/R.sup.6, R.sup.1/R.sup.7, R.sup.2/R.sup.3, R.sup.2/R.sup.4, R.sup.2/R.sup.5, R.sup.2/R.sup.6, R.sup.2/R.sup.7, R.sup.3/R.sup.4, R.sup.3/R.sup.5, R.sup.3/R.sup.6, R.sup.3/R.sup.7, R.sup.4/R.sup.5, R.sup.4/R.sup.6, R.sup.4/R.sup.7, R.sup.5/R.sup.6, R.sup.5/R.sup.7, and R.sup.6/R.sup.7 are joined together with the atoms to which they are attached to form a ring A, where: each ring A 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; and wherein -L.sup.1 is substituted with one to five -L.sup.2-Z moieties, where: each -L.sup.2- is independently a single chemical bond or a spacer moiety; and each —Z is independently a carrier moiety which comprises a polymer with a molecular weight of at least 5 kDa.

2. The prodrug of claim 1; wherein D is a small molecule biologically active moiety, oligonucleotide moiety, peptide nucleic acid moiety, peptide moiety, or protein moiety.

3. The prodrug of claim 1; wherein D is a primary or secondary amine-containing biologically active moiety.

4. The prodrug of claim 1; wherein R.sup.1, R.sup.1a, R.sup.7, and R.sup.7a are independently of each other selected from the group consisting of —H, methyl, ethyl, propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methybutyl, 1-methylbutyl, 1-ethylpropyl, ##STR00141## ##STR00142## wherein: dashed lines indicate attachment to the remainder of -L.sup.1.

5. The prodrug of claim 1; wherein a1 is 0.

6. The prodrug of claim 1; wherein R.sup.2, R.sup.2a, R.sup.6, and R.sup.6a are independently of each other selected from the group consisting of —H, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methybutyl, 1-methylbutyl, and 1-ethylpropyl.

7. The prodrug of claim 1; wherein a2 is 0.

8. The prodrug of claim 1; wherein R.sup.3 and R.sup.3a are independently of each other selected from the group consisting of —H, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methybutyl, 1-methylbutyl, and 1-ethylpropyl.

9. The prodrug of claim 1; wherein R.sup.4 is selected from the group consisting of —H, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methybutyl, 1-methylbutyl, and 1-ethylpropyl.

10. The prodrug of claim 1; wherein R.sup.5 is selected from the group consisting of —H, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methybutyl, 1-methylbutyl, and 1-ethylpropyl.

11. The prodrug of claim 1; wherein R.sup.5a is H.

12. The prodrug of claim 1; wherein Z comprises a PEG-based hydrogel comprising at least 10% w/w PEG.

13. The prodrug of claim 1; wherein each occurrence of -L.sup.2- is independently 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 C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are further 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.y3)(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 of each other 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 C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are further 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 occurrence of 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, each occurrence of T being further independently optionally substituted with one or more R.sup.y2, which are the same or different; each occurrence of 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, which C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and each occurrence of 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, which C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

14. The prodrug of claim 1; wherein L.sup.1 is substituted with one -L.sup.2-Z moiety.

15. The prodrug of claim 1; wherein the prodrug is of formula (Ia) or (Ib): ##STR00143## wherein: the unmarked dashed line indicates attachment to -D; the dashed line marked with the asterisk indicates attachment to -L.sup.2-Z; and -D, R.sup.1, R.sup.1a, R.sup.2, R.sup.2a, R.sup.3, R.sup.3a, R.sup.4, R.sup.5, R.sup.5a, -L.sup.2-, and —Z are as defined in claim 1.

16. A pharmaceutical composition comprising: the prodrug of claim 1; and one or more excipients.

17. A medicament comprising: the prodrug of claim 1.

18. A medicament comprising: the pharmaceutical composition of claim 16.

19. The prodrug of claim 1; wherein Z comprises a polymer with a molecular weight of at least 7.5 kDa.

Description

EXAMPLES

Example 1

(1) ##STR00110##

(2) Triphenylmethanethiol (2) (5.00 g; 18.09 mmol; 1.00 eq.) was stirred in ethanol (36.25 ml), yielding a slightly turbid, yellow solution. A solution of sodium hydroxide, NaOH (795.90 mg; 19.90 mmol; 1.10 eq.) in water (5.45 ml) was added. After 15 min of stirring at room temperature 1,6-dibromohexane (1) (4.17 ml; 27.14 mmol; 1.50 eq.) in ethanol (7.25 ml) was added dropwise. A slightly yellowish solid precipitated from the solution. The reaction mixture was stirred at room temperature overnight.

(3) The ethanol was evaporated and the residue was taken up in CH.sub.2Cl.sub.2 (36 ml). The solution was washed with water (15 ml) and brine (15 ml). The organic layer was dried over MgSO.sub.4, filtered and the solvent was evaporated. The residue was dried under high vacuum for 2 hours.

(4) The yellow, oily residue was dissolved in CH.sub.2Cl.sub.2 (7 ml) and hexane (18 ml) was added. A yellowish precipitate was observed after the solution was kept at −20° C. for 40 h. The supernatant was decanted and the residue was washed with hexane and dried to yield a first product batch. The mother liquor and the hexane from the washing step were combined, evaporated and dried for 2 h at high vacuum. The residue was taken up in dichloromethane (2 ml) and hexane (15 ml). The solution was kept at −20° C. over the weekend. The formed yellowish precipitate was isolated and dried at high vacuum for 2 hours to yield a second batch. The batches were similar in purity and therefore combined.

(5) Yield: 4.57 g, 57%

Example 2

(6) ##STR00111##

(7) [6-bromohexylsulfanyl(diphenyl)methyl]benzene (3) (500.00 mg; 1.14 mmol; 1.00 eq.) was dissolved in anhydrous acetonitrile (10.00 ml) and N-Boc-N-methylethylenediamine (4) (1.02 ml; 5.69 mmol; 5.00 eq.) was added.

(8) The reaction mixture was stirred at room temperature overnight. A white precipitate was observed. An UPLC chromatogram showed full conversion to the product. The crude material was purified by preparative HPLC and the product containing fractions were lyophilized.

(9) Et.sub.2O (20 ml) and sat. NaHCO.sub.3 (20 ml) were added to the isolated TFA-salt (227 mg). The mixture was stirred under evolution of gas until all solids were dissolved. The phases were separated and the aqueous phase was extracted two additional times with Et.sub.2O (20 ml each). The organic solutions were combined and dried over MgSO.sub.4, filtered and concentrated.

(10) Yield: 185.00 mg; 31%

(11) MS: m/z=533.85 [M+H].sup.+ (calculated: 533.32)

Example 3

(12) ##STR00112##

(13) tert-butyl N-methyl-N-[2-(6-tritylsulfanylhexylamino)ethyl]carbamate (5) (185.00 mg; 0.35 mmol; 1.00 eq.) was dissolved in anhydrous acetonitrile (3.70 ml). Methyl bromoacetate (6) (65.74 μl; 0.69 mmol; 2.00 eq.) and N,N-diisopropylethylamine (604.82 μl; 3.47 mmol; 10.00 eq.) were added.

(14) After an UPLC chromatogram showed full conversion to the product, the reaction mixture was filtered and the filter cake was washed with acetonitrile (2 ml). The solvent was evaporated and the residue was dissolved in 2 ml of dichloromethane. The crude material was purified by column chromatography.

(15) Yield: 97.00 mg; 46%

(16) MS: m/z=605.99 [M+H].sup.+ (calculated: 605.34)

Example 4

(17) ##STR00113##

(18) Methyl 2-[2-[tert-butoxycarbonyl(methyl)amino]ethyl-(6-tritylsulfanylhexyl)amino]acetate (7) (97.00 mg; 0.16 mmol; 1.00 eq.) was dissolved in methanol (7.62 ml). A 1 N solution of sodium hydroxide in water (5.08 ml; 1.00 mol/l; 5.08 mmol; 31.67 eq.) and water (4 ml) were added and the reaction mixture was stirred until full conversion was observed by UPLC-MS.

(19) The reaction was quenched by the addition of 1 N hydrochloric acid (5.08 ml; 1.00 mol/l; 5.08 mol; 31.67 eq.). The reaction mixture was stored at 4° C. overnight whereupon an emulsion was formed. The supernatant was carefully removed and the second phase was diluted with water, frozen and lyophilized. The residue was triturated with 3 ml of dichloromethane. The solution was filtered and the solvent evaporated. The residue was dried under high vacuum for 1 hour.

(20) Yield: 94.00 mg; 99%

(21) MS: m/z=591.84 [M+H].sup.+ (calculated: 591.33)

Example 5

(22) ##STR00114##

(23) 2-[2-[Boc(methyl)amino]ethyl-(6-tritylsulfanylhexyl)amino]acetic acid (8) (21.50 mg; 0.04 mmol; 1.20 eq.) and TSTU (12.78 mg; 0.04 mmol; 1.40 eq.) were dissolved in DMF (1 ml) and DIPEA (11.76 mg; 0.09 mmol; 3.00 eq.) was added. The reaction mixture was stirred for 15 min at room temperature.

(24) Insulin (9) (176.13 mg; 0.03 mmol; 1.00 eq.) was dissolved in reaction buffer (2.5 ml, 4:6.sub.(v/v) borate buffer (0.375 M sodium borate, pH 8.50): DMF). The activated linker solution was added and the reaction mixture was stirred at room temperature for 45 min. A mixture of unmodified insulin, two different mono-adducts and a bis-adduct was observed by UPLC-MS. The two mono-adducts were separated by preparative HPLC. The fractions containing the major mono-adduct isomer were pooled and lyophilized.

(25) Yield: 39.60 mg; 17%

(26) MS: m/z=1597.22 [M+4H].sup.4+ (calculated: 1594.99)

Example 6

(27) ##STR00115##

(28) The protected insulin-linker conjugate 10 (39.60 mg; 0.01 mmol; 1.00 eq.) was dissolved in HFIP (2 ml) to yield a yellow solution. TFA (200 μl) and TES (50 μl) were added (solution turns colorless) and the solution was stirred at room temperature for 3 h. Full conversion was observed and the crude reaction mixture was concentrated. The resulting crude product was used without further purification in the next step.

(29) MS: m/z=1511.28 [M+4H].sup.4+ (calculated: 1509.45)

Example 7

(30) ##STR00116##

(31) A stock solution of 20 mg/ml maleimide functionalized PEG 12 in 1:1 MeCN/H.sub.2O (18.5 μmol/ml) was prepared.

(32) The deprotected insulin-linker conjugate 11 (ca. 5.7 μmol, 1.00 eq., crude) was dissolved in 2.5 ml of 1:1 MeCN/H.sub.2O and 616 μl of the PEG stock solution (2 eq, 11.4 μmol) were added. The reaction was started by the addition of buffer (600 μl, 0.5 M phosphate, pH 7.5). The pH was checked with pH paper (ca. pH 7.5) and stirred for 15 min. The reaction was quenched by the addition of 10% AcOH.sub.aq (200 μl). The pH was checked with pH paper (ca. pH 4.0).

(33) The resulting solution was purified by preparative HPLC. The product containing fractions were pooled and lyophilized.

(34) Yield: 35 mg; 79% over 2 steps

(35) MS: m/z=1780.07 [M+4H].sup.4+ (calculated: 1780.81)

Example 8

(36) ##STR00117##

(37) The hydrolysis kinetics of PEGylated linker-insulin conjugate 13 were studied in buffer at 37° C. Therefore, the starting material was dissolved in buffer (60 mM phosphate, 3 mM EDTA, pH 7.4) and mixed for 15 min. The resulting solution was incubated in a water bath at 37° C. At given points in time samples were withdrawn, quenched and analyzed by UPLC-MS.

(38) Insulin was released with a half-life of 28 d.

Example 9

(39) ##STR00118##

(40) N-Boc-N-methylethylenediamine (4a, 576.13 μl; 3.22 mmol; 5.00 eq.) and N-(6-bromohexyl)-phthalimide (14, 200.00 mg; 0.64 mmol; 1.00 eq.) were dissolved in acetonitrile and stirred at room temperature overnight. The reaction mixture was diluted with water and purified by preparative HPLC. Product containing fractions were pooled and lyophilized to yield amine 15a.

(41) Yield: 139 mg; 42%

(42) MS: m/z=404.20 [M+H].sup.+

(43) Phthalimide 15b was synthesized accordingly, starting from amine 4b.

Example 10

(44) ##STR00119##

(45) Amine 15a (139.00 mg; 0.27 mmol; 1.00 eq.) was dissolved in acetonitrile (2.50 ml) and DIPEA (93.56 μl; 0.54 mmol; 2.00 eq.) was added. Bromide 16 (51.55 μl; 0.35 mmol; 1.30 eq.) was added and the reaction mixture was stirred at room temperature for 2 h.

(46) The solvent was evaporated and the residue was dissolved in 1 ml of heptane/ethyl acetate, filtered and purified by flash chromatography to yield 17a.

(47) Yield: 116 mg; 83%

(48) MS: m/z=518.33 [M+H].sup.+

(49) Amine 17b was synthesized accordingly, starting from amine 15b.

Example 11

(50) ##STR00120##

(51) Hydrazine hydrate (33.00 μl; 0.68 mmol; 3.04 eq.) was added to a solution of phthalimide 17a (115.70 mg; 0.22 mmol; 1.00 eq.) in ethanol (2.00 ml). The reaction mixture was heated under reflux (oil bath 93° C.) for 2 h. A white precipitate formed.

(52) The reaction mixture was cooled to room temperature. The precipitate was filtered off and washed with cold EtOH (2×1 ml). The filtrate was concentrated to yield a white residue (63 mg). It was redissolved in chloroform (1.3 ml) and stirred for 1 h. The precipitate was filtered off through a small bed of Celite, washed with chloroform (0.5 ml) and the organic phase was concentrated and dried under vacuum to yield amine 18a.

(53) Yield: 63 mg; 72%

(54) MS: m/z=388.31 [M+H].sup.+

(55) Amine 18b was synthesized accordingly, starting from amine 17b.

Example 12

(56) ##STR00121##

(57) Amine 18a (62.50 mg; 0.16 mmol; 1.00 eq.) and DIPEA (84.27 μl; 0.48 mmol; 3.00 eq.) were dissolved in acetonitrile (1.00 ml). NHS ester 19 (51.52 mg; 0.19 mmol; 1.20 eq.) was added and the reaction mixture was stirred at room temperature for 15 min. Analysis by LCMS showed full conversion of the starting material. The reaction mixture was stored at −20° C. overnight.

(58) The reaction was brought to room temperature and quenched by the addition of TFA (36 μl). A drop of water was added to dissolve the DIPEA salts. The crude mixture was purified by preparative HPLC. Product containing fractions were pooled and lyophilized to yield maleimide 20a.

(59) Yield: 84 mg; 80%

(60) MS: m/z=539.31 [M+H].sup.+

(61) Maleimide 20b was synthesized accordingly, starting from amine 18b.

Example 13

(62) ##STR00122##

(63) Maleimide 20a (83.90 mg; 0.13 mmol; 1.00 eq.) was dissolved in dichloromethane (1.00 ml) and TFA (1.00 ml; 13.07 mmol; 101.66 eq.) was added. The reaction mixture was stirred at room temperature for 3.5 h. The volatiles were removed in a stream of nitrogen and the residue was dried carefully under vacuum.

(64) Product 21a was used immediately (without further purification) in the next step.

(65) Yield: 78 mg; 100%

(66) MS: m/z=383.21 [M+H].sup.+

(67) Product 21b was synthesized accordingly, starting from amine 20b.

Example 14

(68) ##STR00123##

(69) p-Hydroxyacetophenone (22, 680.74 mg; 5.00 mmol; 1.00 eq.) was dissolved in THF (27 ml). DIPEA (1.75 ml; 10.00 mmol; 2.00 eq.) and 2 min later hexanoyl chloride (23, 908.54 μl; 6.50 mmol; 1.30 eq.) were added dropwise under stirring. After 15 min the reaction mixture was analyzed by LCMS and showed complete conversion to the product.

(70) The solution was filtered and the filtrate was diluted with diethyl ether (100 ml) and washed once with saturated NaHCO.sub.3-solution (100 ml). The aqueous phase was extracted with diethyl ether (50 ml) and the combined organic phases were washed twice with 100 ml of a 0.1 M HCl-solution. The organic phase was dried over Na.sub.2SO.sub.4, filtered and the solvents were removed under reduced pressure.

(71) Yield: 1.2 g; 100%

(72) MS: m/z=235.03 [M+H].sup.+

Example 15

(73) ##STR00124##

(74) Ketone 24 (1.29 g; 5.00 mmol; 1.00 eq.) was dissolved in acetonitrile (16 ml). Sodium borohydride (378.32 mg; 10.00 mmol; 2.00 eq.) was added in portions. At the end ethanol (820.01 μl) was added. The reaction mixture was stirred overnight at room temperature. An LCMS chromatogram after 19 hours showed full conversion to the product.

(75) The reaction mixture was diluted with 150 ml of diethyl ether. The organic layer was washed twice with 100 ml of water. The organic layer was dried over MgSO.sub.4, filtered and concentrated under reduced pressure.

(76) Yield: 951 mg; 80%

(77) MS: m/z=237.02 [M+H].sup.+

Example 16

(78) ##STR00125##

(79) Benzyl alcohol 25 (951.00 mg; 4.02 mmol; 1.00 eq.) was dissolved in acetonitrile (20 ml) and cooled to 0° C. in an ice bath. Bis(pentafluorophenyl) carbonate (3.97 g; 10.06 mmol; 2.50 eq.), DMAP (122.91 mg; 1.01 mmol; 0.25 eq.) and DIPEA (3.50 ml; 20.12 mmol; 5.00 eq.) were added. The reaction mixture was stirred at room temperature for 1 hour.

(80) An LCMS chromatogram showed complete conversion of the starting material.

(81) The reaction mixture was diluted with 75 ml of diethyl ether. The organic layer was washed twice with 80 ml of water. The organic layer was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography.

(82) Yield: 1.13 g; 63%

Example 17

(83) ##STR00126##

(84) Building block 21a (78.50 mg; 0.13 mmol; 1.00 eq.) was dissolved in acetonitrile (1.00 ml) and DIPEA (134.38 μl; 0.77 mmol; 6.00 eq.) was added. PFP-carbonate 26 (68.87 mg; 0.15 mmol; 1.20 eq.) and DMAP (3.14 mg; 0.03 mmol; 0.20 eq.) were added and the reaction mixture was stirred at room temperature. The reaction was quenched with TFA (77 μl) after 40 min and purified by preparative HPLC to yield carbamate 27a.

(85) Yield: 56 mg; 59%

(86) MS: m/z=645.42 [M+H].sup.+

(87) Product 27b was synthesized accordingly, starting from amine 21b.

Example 18

(88) ##STR00127##

(89) Carboxylic acid 27a (10.00 mg; 0.01 mmol; 1.10 eq.), DIPEA (5.40 μl; 0.03 mmol; 2.59 eq.), HOSu (1.38 mg; 0.01 mmol; 1.00 eq.) and N-cyclohexylcarbodiimide-N-methyl polystyrene (18.92 mg; 0.04 mmol; 3.00 eq.) were shaken with anhydrous dichloromethane (0.40 ml) and 40 μl of THF in a syringe reactor overnight.

(90) The resin was filtered off and washed with dry DCM (2×0.5 ml). The solvent was evaporated in a stream of argon and dried under vacuum for 2 h to yield 14.6 mg of product 28a as oil. The residue was dissolved in dry DMSO (600 μl) and filtered through a syringe filter (22 μm) to yield a 20 mM solution. LCMS analysis after derivatization of a sample with Boc-ethylenediamine in DMSO showed a ratio of 6:94 carboxylic acid/NHS ester. The solution was stored at −20° C.

(91) Product 28b was synthesized accordingly, starting from amine 27b.

Example 19

(92) ##STR00128##

(93) 400 μL of an insulin solution in DMSO (50 mg/mL; 3.44 μmol; 1.00 eq) were mixed with 400 μL DMSO and 400 μL borate buffer (0.375 M boric acid, adjusted to pH 8.1 with tetrabutylammoniumhydroxide). A solution of 27b in DMSO was added (68.8 μL; 0.05 mol/L; 3.44 μmol; 1.00 eq). The mixture was agitated for 15 min at ambient temperature and diluted under cooling on an ice bath with 2 mL 10 vol % AcOH and 6 mL water. The solution was purified by preparative HPLC. The pure fractions were combined, frozen and lyophilized to yield protected insulin-linker conjugate 29.

(94) Yield: 0.8 mg; 4%

(95) MS: m/z=1605.01 [M+4H].sup.4+

Example 20

(96) ##STR00129##

(97) 0.8 mg (0.125 μmol, 1.00 eq) of protected insulin-linker conjugate 29 were dissolved in 0.5 mL 20 mM succinate buffer, pH 5.0. Lipase B was added (0.1 mg) and the suspension was incubated at ambient temperature for 1 h. The solution was filtered through a 0.22 μm PVDF filter, diluted with 0.5 mL water and purified by preparative HPLC. The pure fractions were combined, frozen and lyophilized to yield deprotected insulin-linker conjugate 30.

(98) Yield: 0.5 mg; 65%

(99) MS: m/z=1231.79 [M+5H].sup.5+

Example 21

(100) ##STR00130##

(101) 11.9 g tritylsulfide 32 (43.1 mmol, 1.02 eq) were dissolved in 50 mL DMSO. 7.30 mL DBU (48.8 mmol; 1.14 eq) and 13.3 g phthalimide 31 followed by 15 mL DMSO were added. The mixture was stirred for 12 min at ambient temperature. 700 mL ethyl acetate and 200 mL 0.1 N HCl were added. The mixture was stirred until both solvent layers were clear. The layers were separated. The aqueous phase was extracted with ethyl acetate (3×, 50 mL each). The organic solutions were combined, washed with 80 mL sat. NaHCO.sub.3 and 80 mL brine, dried over Na.sub.2SO.sub.4 and filtered. The solvent was removed in vacuo. The residue was recrystallized from 240 mL ethanol under heating. The suspension was stored for 1 h at −18° C. The trityl protected product 33 was filtered off, washed 2× with ethanol and dried under high vacuum.

(102) Yield: 19.6 g; 90%

(103) MS: m/z=528.07 [M+Na].sup.+

Example 22

(104) ##STR00131##

(105) 19.5 g phthalimide 33 (38.6 mmol; 1.00 eq) was suspended in 300 mL abs. ethanol and 5.74 mL hydrazine hydrate (116.8 mmol; 3.03 eq) were added. The mixture was heated under reflux for 2 h and subsequently cooled to −18° C., the precipitate was filtered off and washed 2× with cold ethanol. The filtrate was concentrated in vacuo and stored for 2 days at −18° C. 234 mL CHCl.sub.3 was added, stirred for 2 h at ambient temperature, and stored at −18° C. for 2 days. The precipitate was filtered off and washed 2× with cold CHCl.sub.3. The filtrate was washed with 250 mL H.sub.2O, 250 mL brine, dried over anhydrous MgSO.sub.4, and filtered. The solvent was removed in vacuo. Amine 34 was dried under high vacuum.

(106) Yield: 13.1 g; 91%

(107) MS: m/z=376.26 [M+H].sup.+

Example 23

(108) ##STR00132##

(109) 114 mg m-dPEG 37-NHS ester 35 (0.06 mmol; 1.00 eq) were dissolved in 1.00 mL anhydrous DCM. 35.9 mg 6-tritylmercaptohexylamine 34 (0.10 mmol; 1.50 eq) and 22.2 μL N,N-diisopropylethylamine (0.13 mmol; 2.00 eq) were added. The mixture was stirred for 1 h at ambient temperature. The solvent was evaporated in a stream of nitrogen. The residue was dissolved in a mixture of 2.00 ml 9:1 MeCN/H.sub.2O+0.1% TFA, 1.00 ml H.sub.2O+0.05% TFA and 0.80 mL MeCN and purified by preparative HPLC (eluents: MeCN+0.1% TFA, H.sub.2O+0.1% TFA). MeCN was removed from the product fractions in vacuo. The aqueous layer was extracted 6 times with DCM. The combined organic fractions were dried over anhydrous MgSO.sub.4 and concentrated in vacuo. 0.2 mL TES and 0.75 mL TFA were added. The mixture was stirred for 1 h at ambient temperature. 500 mL diethyl ether containing 10 vol % n-pentane were added and the mixture was left standing at −20° C. over night. The precipitated thiol 36 was filtered off and dried under high vacuum.

(110) Yield: 32.5 mg; 28%

Example 24

(111) ##STR00133##

(112) Deprotected insulin-linker conjugate 30 (0.50 mg; 0.08 μmol; 1.00 eq) was dissolved in 0.5 mL 1:1 MeCN/H.sub.2O+0.1% TFA. 100 μL of a solution of PEG thiol 36 (2.2 mg/mL; 0.22 mg; 0.12 μmol; 1.50 eq) in 1:1 MeCN/H.sub.2O+0.1% TFA and 100 μL of citrate buffer (0.5 M citric acid, pH 5.0) were added. The mixture was agitated for 1 h at ambient temperature. 100 μL PEG thiol 36 (2.2 mg/mL; 0.22 mg; 0.12 μmol; 1.50 eq) in 1:1 MeCN/H.sub.2O+0.1% TFA and 100 μL phosphate buffer (0.5 M sodium phosphate, pH 6.1) were added. The mixture was agitated for 1.5 h at ambient temperature. 10 μL TFA were added and the product was isolated by preparative HPLC. The pure fractions were combined, frozen and lyophilized to yield PEG conjugate 37.

(113) Yield: 0.2 mg; 31%

(114) MS: m/z=1327.33 [M+6H].sup.6+

Example 25

(115) Two samples of 0.1 mg insulin-linker PEG conjugate 37 each were dissolved in 0.5 mL pH 7.4 buffer (60 mM NaH.sub.2PO.sub.4, 3 mM EDTA, 0.03% (w/v) Tween20, 0.06 mg/mL pentafluorophenol) and 0.5 mL pH 5.5 buffer (60 mM succinic acid, 3 mM EDTA, 0.03% (w/v) Tween 20, 0.01 mg/mL pentafluorophenol) respectively. The samples were incubated at 37° C. in a temperature controlled water bath. At different time points samples were withdrawn and analyzed by RP-HPLC/ESI MS. The amount of released insulin for each time point was calculated from the peak areas of the PEG-conjugate and the peak areas of released insulin. Curve-fitting software was applied to estimate the corresponding halftime of release. Halftimes of 2.9 days (pH 7.4) and 9.3 days (pH 5.5) for the insulin release were determined.

Example 26

(116) ##STR00134##

(117) Sodium methanethiosulfonate (688 mg, 5.13 mmol, 1.00 eq.) was added to a solution of 6-bromohexanoic acid (38, 1.00 g, 5.13 mmol, 1.00 eq.) in DMF (8 ml) at room temperature and under argon atmosphere. The mixture was heated to 80° C. and stirred at this temperature for 3 hours under argon atmosphere. The oil bath was removed and the reaction mixture was cooled to room temperature. An IPC by LCMS confirmed full conversion of the starting materials.

(118) Water (20 ml) was added to the reaction mixture and the product was extracted with diethyl ether (3×40 ml). The combined organic solutions were washed with brine (1×60 ml), dried over MgSO.sub.4, filtered and concentrated. The crude product (988 mg) was dried under high vacuum over the weekend. The crude material was dissolved in diethyl ether (30 ml) and precipitated by adding the solution to 400 ml of heptane. Product 39 was filtered (pore 3 filter) and the colorless solid was dried under high vacuum for 2 h.

(119) Yield: 944 mg; 81%

(120) MS: m/z=249.06 [M+Na].sup.+

Example 27

(121) ##STR00135##

(122) 6-MTS-hexanoic acid (39; 1.04 g; 4.59 mmol; 1.05 eq.) and PyBOP (2.39 g; 4.59 mmol; 1.05 eq.) were dissolved in dichloromethane (50 ml) and N,N-diisopropylethylamine (2.50 ml; 14.35 mmol; 3.28 eq.) was added. The reaction mixture was stirred at room temperature for 30 min. H.sub.2N-PEG(12)-COOH (2.70 g; 4.37 mmol; 1.00 eq.) was added and the solution was stirred at room temperature for additional 30 min. An IPC LCMS confirmed full conversion of the amino acid.

(123) The reaction was quenched by addition of TFA (1.1 ml) and concentrated to a total volume of about 10 ml. 50 ml of cold MTBE were added to the slightly yellow solution, which turned turbid. The mixture was stored at −20° C. overnight. A white precipitate formed. The suspension was decanted and the solids were washed with 50 ml of cold MTBE. The white, solid residue was dried, whereupon it melted to yield a yellowish oil. The crude product was taken up in 1:1 MeCN/H.sub.2O+0.1% TFA and purified by preparative HPLC. The pure fractions were combined, frozen and lyophilized to yield carboxylic acid 40.

(124) Yield: 2.59 g; 72%

(125) MS: m/z=826.45 [M+Na].sup.+

Example 28

(126) ##STR00136##

(127) Carboxylic acid 40 (3.16 g; 3.83 mmol; 1.00 eq.), HOSu (528 mg; 4.59 mmol; 1.20 eq.), DMAP (46.7 mg; 0.38 mmol; 0.10 eq.) and DCC (947 mg; 4.59 mmol; 1.20 eq.) were dissolved in dichloromethane (37 ml). The reaction mixture was stirred at room temperature for 30 min. The urea was filtered off with a syringe reactor and DCM was evaporated. The residue was dissolved in 1:1 MeCN/H.sub.2O+0.1% TFA and purified by preparative HPLC. The pure fractions were combined, frozen and lyophilized to yield MTS-PEG(12)-NHS handle 41.

(128) Yield: 2.88 g; 81%

(129) MS: m/z=923.27 [M+H].sup.+

Example 29

(130) ##STR00137##

(131) Dry amino functionalized hydrogel 42 (100 mg, 13.8 μmol amino groups) as described in WO2015/067791 (example 3a) is filled into a syringe equipped with a filter frit. The hydrogel is suspended in 5 mL NMP/2% DIPEA. The solvent is discarded and the hydrogel is washed five times with 5 mL NMP/2% DIPEA, the solvent is discarded. 31.8 mg (2.5 eq in respect to the amine content of the hydrogel, 34.5 μmol) of MTS-PEG(12)-NHS handle 41 is dissolved in 1.5 mL NMP and drawn into the syringe. The suspension is allowed to incubate for 2 hours at ambient temperature under gentle agitation. The solvent is discarded and the hydrogel is washed five times with each time 5 mL NMP, the solvent is each time discarded. The hydrogel is washed five times with each time 5 mL 0.1% HOAc, 0.01% Tween 20, the solvent is each time discarded. An aqueous solution containing 0.1% HOAc, 0.01% Tween 20 is added to obtain suspension 43 containing 10 mg/mL hydrogel based on initial weight.

Example 30

(132) ##STR00138##

(133) 5 mL (50 mg hydrogel based on initial weight; 6.9 μmol) of hydrogel suspension 43 are transferred to a syringe equipped with a filter frit. The solvent is discarded and 10 mL of a 50 mM TCEP solution in water is drawn into the syringe. The resulting hydrogel suspension is incubated at ambient temperature for 15 min. The solvent is discarded and the hydrogel is washed twice with each time 10 mL of the TCEP solution. The solvent is each time discarded. The hydrogel is washed ten times with each time 10 mL of an aqueous solution of 20 mM succinate, 0.01% Tween 20, pH 4.0, the solvent is each time discarded. 20 mM succinate, 0.01% Tween 20, pH 4.0 is added to obtain suspension 44 containing 10 mg/mL hydrogel based on initial weight.

Example 31

(134) ##STR00139##

(135) 5 mL (50 mg hydrogel based on initial weight; 6.9 μmol; 1.00 eq) of hydrogel suspension 44 are transferred to a syringe equipped with a filter frit. The solvent is discarded. The hydrogel is washed ten times with sodium succinate buffer (pH 3.0, 20 mM; 1 mM EDTA, 0.01% Tween 20). The solvent is each time discarded. Deprotected insulin-linker conjugate 30 (55 mg; 8.3 μmol; 1.20 eq) is dissolved in 3.0 mL 1:1 MeCN/H.sub.2O+0.1% TFA. The solution is drawn into the syringe. 1.00 mL phosphate buffer (0.5 M sodium phosphate, pH 6.1) is drawn into the syringe. The suspension is incubated at ambient temperature for 1 hour. The solvent is discarded and the hydrogel is washed ten times with sodium succinate buffer (pH 3.0, 20 mM; 1 mM EDTA, 0.01% Tween 20) and ten times with sodium acetate buffer (pH 5.0, 10 mM; 130 mM NaCl, 0.01% Tween 20). The insulin content of hydrogel suspension 45 is determined by quantitative amino acid analysis after total hydrolysis under acidic conditions.

Example 32

(136) Hydrogel 45 (containing approx. 1 mg insulin) is suspended in 2 ml 60 mM sodium phosphate, 3 mM EDTA, 0.01% Tween 20, pH 7.4, and incubated at 37° C. The suspension is centrifuged at time intervals and the supernatant is analyzed by RP-HPLC at 280 nm and ESI-MS. UV-signals correlating to liberated insulin are integrated and plotted against incubation time.

(137) Curve-fitting software is applied to estimate the corresponding halftime of release.

Abbreviations

(138) abs. absolute

(139) AcOH acetic acid

(140) aq. aqueous

(141) Boc tert-butyloxycarbonyl

(142) DCC N,N′-dicyclohexylcarbodiimide

(143) DCM dichloromethane

(144) DIPEA diisopropylethylamine

(145) DMAP 4-(dimethylamino)-pyridin

(146) DMF dimethylformamide

(147) DMSO dimethylsulfoxide

(148) EDTA ethylendiaminetetraacetic acid

(149) eq stoichiometric equivalent

(150) ESI Elektrospray ionization

(151) Et.sub.2O diethyl ether

(152) HFIP hexafluoroisopropanol

(153) HOSu N-hydroxysuccinimide

(154) HPLC high performance liquid chromatography

(155) IPC in process control

(156) LCMS liquid chromatography with mass spectroscopy

(157) MeCN acetonitrile

(158) MS mass spectrum/mass spectrometry

(159) MTBE methyl tert-butyl ether

(160) MTS methanethiosulfonate

(161) NHS N-hydroxysuccinimide

(162) NMP N-methyl-2-pyrrolidone

(163) PEG poly(ethylene glycol)

(164) PFP pentafluorophenol

(165) PVDF polyvinylidene fluoride

(166) SEC size exclusion chromatography

(167) Su succinimide

(168) TCEP tris-(2-carboxyethyl)-phosphine

(169) TES triethylsilane

(170) TFA trifluoroacetic acid

(171) THF tetrahydrofurane

(172) TLC thin layer chromatography

(173) TSTU O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate

(174) Trt trityl

(175) UPLC ultra performance liquid chromatography