Carrier-Linked Treprostinil Prodrugs

Abstract

The present invention relates to prodrugs or a pharmaceutically acceptable salt thereof comprising a covalent treprostinil carrier conjugate as well as pharmaceutical composition comprising said compounds. The compounds may be used as medicaments, especially for diseases or disorders which can be treated by treprostinil, such as pulmonary arterial hypertension (PAH).

Claims

1. A method of treating, controlling, delaying, or preventing in a mammalian patient in need of the treatment of one or more conditions, comprising: administering by inhalation to said patient a diagnostically and/or therapeutically effective amount of a carrier-linked treprostinil prodrug of formula (II), or a pharmaceutical salt thereof: ##STR00135## wherein each T is independently selected from structures (i) to (v): ##STR00136## ##STR00137## wherein: dashed lines indicating attachment to the rest of the molecule; y is an integer ranging of from 1 to 64; R.sup.a1 is selected from the group consisting of: unsubstituted alkyl, substituted alkyl, unsubstituted phenyl, substituted phenyl, unsubstituted naphthyl, substituted naphthyl, unsubstituted indenyl, substituted indenyl, unsubstituted indanyl, substituted indanyl, unsubstituted tetralinyl, substituted tetralinyl, unsubstituted C.sub.3-10 cycloalkyl, substituted C.sub.3-10 cycloalkyl, unsubstituted 4- to 7-membered heterocyclyl, substituted 4- to 7-membered heterocyclyl, unsubstituted 9- to 11-membered heterobicyclyl, and substituted 9- to 11-membered heterobicyclyl; R.sup.a2 is selected from the group consisting of: H, unsubstituted alkyl, and substituted alkyl; R.sup.a3 and R.sup.a4 are independently selected from the group consisting of: H, unsubstituted alkyl, and substituted alkyl; n is 0 or 1; Q is a spacer moiety; optionally, R.sup.a1 and R.sup.a3 are joined together with the atoms to which they are attached to form a ring A; A is selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4- to 7-membered aliphatic heterocyclyl, and 9- to 11-membered aliphatic heterobicyclyl, wherein A is unsubstituted or substituted; and Z.sup.1 is a carrier comprising a covalently bound polymer; and wherein R.sup.a2 and R.sup.a4 are absent if A is an aromatic ring.

2. The method of claim 1; wherein R.sup.a2 is H.

3. The method of claim 1; wherein R.sup.a4 is selected from H, C.sub.1-6 alkyl or substituted C.sub.1-6 alkyl.

4. The method of claim 1; wherein R.sup.a1 and R.sup.a3 are joined together with the atoms to which they are attached to form a ring A; wherein A is selected from the group consisting of: phenyl; naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4- to 7-membered aliphatic heterocyclyl, and 9- to 11-membered aliphatic heterobicyclyl; and wherein A is unsubstituted or substituted.

5. The method of claim 1; wherein T is selected from structure (iii).

6. The method of claim 1; wherein y is 4, 6, 8, 10, or 12.

7. The method of claim 1; wherein the carrier-linked treprostinil prodrug has the structure of formula (II-A): ##STR00138## wherein: each T is independently selected from structures (i) to (v): ##STR00139## ##STR00140## wherein: dashed lines indicating attachment to the rest of the molecule; y is an integer ranging of from 1 to 64; R.sup.a2 is selected from H, unsubstituted alkyl, and substituted alkyl; R.sup.a4 is selected from the group consisting of: H, unsubstituted alkyl, and substituted alkyl; A is selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4- to 7-membered aliphatic heterocyclyl, and 9- to 11-membered aliphatic heterobicyclyl, wherein A is unsubstituted or substituted; Q is a spacer moiety; and Z.sup.1 is a carrier comprising a covalently bound polymer.

8. The method of claim 1; wherein Q in formula (II) is selected from the group consisting of: COOR.sup.a9, OR.sup.a9, C(O)R.sup.a9, C(O)N(R.sup.a9R.sup.a9a), S(O).sub.2N(R.sup.a9R.sup.a9a), S(O)N(R.sup.a9R.sup.a9a), S(O).sub.2R.sup.a9, S(O)R.sup.a9, N(R.sup.a9)S(O).sub.2N(R.sup.a9aR.sup.a9b), SR.sup.a9, N(R.sup.a9R.sup.a9a), OC(O)R.sup.a9, N(R.sup.a9)C(O)R.sup.a9a, N(R.sup.a9)S(O).sub.2R.sup.a9a, N(R.sup.a9)S(O)R.sup.a9a, N(R.sup.a9)C(O)OR.sup.a9a, N(R.sup.a9)C(O)N(R.sup.a9aR.sup.a9b), OC(O)N(R.sup.a9R.sup.a9a), W; C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein W 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.a10, which are the same or different; 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: W, C(O)O, O, C(O), C(O)N(R.sup.a11), S(O).sub.2N(R.sup.a11), S(O)N(R.sup.a11), S(O).sub.2; S(O), N(R.sup.a11)S(O).sub.2N(R.sup.a11a), S, N(R.sup.a11), OC(O)R.sup.a11, N(R.sup.a11)C(O), N(R.sup.a11)S(O).sub.2, N(R.sup.a11)S(O), N(R.sup.a11)C(O)O, N(R.sup.a11)C(O)N(R.sup.a11a), and OC(O)N(R.sup.a11R.sup.a11a); R.sup.a9, R.sup.a9a, and R.sup.a9b are independently selected from the group consisting of: H, W, and C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein W, 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.a10, which are the same or different; 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: W, C(O)O, O, C(O), C(O)N(R.sup.a11), S(O).sub.2N(R.sup.a11), S(O)N(R.sup.a11), S(O).sub.2, S(O), N(R.sup.a11)S(O).sub.2N(R.sup.a11a), S, N(R.sup.a11), OC(O)R.sup.a11, N(R.sup.a11)C(O), N(R.sup.a11)S(O).sub.2, N(R.sup.a11)S(O), N(R.sup.a11)C(O)O, N(R.sup.a11)C(O)N(R.sup.a11a), and OC(O)N(R.sup.a11R.sup.a11a); W is selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 4- to 7-membered heterocyclyl, and 9- to 11-membered heterobicyclyl; wherein W is optionally substituted with one or more R.sup.a10, which are the same or different; R.sup.a10 is selected from the group consisting of: halogen, CN, oxo (O), COOR.sup.a12, OR.sup.a12, C(O)R.sup.a12, C(O)N(R.sup.a12R.sup.a12a), S(O).sub.2N(R.sup.a12R.sup.a12a), S(O)N(R.sup.a12R.sup.12a), S(O).sub.2R.sup.a12, S(O)R.sup.a12, N(R.sup.a12)S(O).sub.2N(R.sup.a12aR.sup.a12b), SR.sup.a12; N(R.sup.a12R.sup.a12a), NO.sub.2, OC(O)R.sup.a12, N(R.sup.a12)C(O)R.sup.a12a, N(R.sup.a12)S(O).sub.2R.sup.a12a, N(R.sup.a12)S(O)R.sup.a12a, N(R.sup.a12)C(O)OR.sup.a12a, N(R.sup.a12)C(O)N(R.sup.a12aR.sup.a12b), OC(O)N(R.sup.a12R.sup.a12a), 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 R.sup.a11, R.sup.a11a, R.sup.a12, R.sup.a12a, and R.sup.a12b 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.

9. The method of claim 8; wherein each -Q- is independently -Q.sup.1a-Q.sup.1-*, wherein the asterisk indicates the connection to Z.sup.1; wherein: Q.sup.1a is a bond selected from the group consisting of: C(O)O, O, C(O), C(O)N(R.sup.a9a), S(O).sub.2N(R.sup.a9a), S(O)N(R.sup.a9a), S(O).sub.2, S(O), N(R.sup.a9a)O(O).sub.2N(R.sup.a9b), S, N(R.sup.a9a), OC(O), N(R.sup.a9a)C(O), N(R.sup.a9a)S(O).sub.2, N(R.sup.a9a)S(O), N(R.sup.a9a)C(O)O, N(R.sup.a9a)C(O)N(R.sup.a9b), OC(O)N(R.sup.a9a), and W. Q.sup.1 is selected from the group consisting of: C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl which are optionally substituted with one or more R.sup.a10, which are optionally interrupted, provided that Q.sup.1 is at least C.sub.2, by one or more groups selected from the group consisting of: C.sub.3-7 cycloalkyl, 4- to 7-membered heterocyclyl, ##STR00141## wherein each of said group may individually be present one or more times; and which C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl may optionally be terminated at the end connected to Z.sup.1 by a group selected from C.sub.3-7 cycloalkyl, 4- to 7-membered heterocyclyl, ##STR00142## wherein: R.sup.a9a is as defined in claim 8; R.sup.a10 is selected from the group consisting of: halogen, CN, oxo (O), COOR.sup.a12, OR.sup.a12, C(O)R.sup.a12, C(O)N(R.sup.a12R.sup.a12a), S(O).sub.2N(R.sup.a12R.sup.a12), S(O)N(R.sup.a12R.sup.a12a), S(O).sub.2R.sup.a12, S(O)R.sup.a12, N(R.sup.a12)S(O).sub.2N(R.sup.a12aR.sup.a12b), SR.sup.a12, N(R.sup.a12R.sup.a12a), NO.sub.2, OC(O)R.sup.a12, N(R.sup.a12)C(O)R.sup.a12a, N(R.sup.a12)S(O).sub.2R.sup.a12a, N(R.sup.a12)S(O)R.sup.a12a, N(R.sup.a12)C(O)OR.sup.a12a, N(R.sup.a12)C(O)N(R.sup.a12aR.sup.a12b), OC(O)N(R.sup.a12R.sup.a12a), 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; R.sup.a12, R.sup.a12a and R.sup.a12b 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 R.sup.13 and R.sup.13a are independently selected from the group consisting of: H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, and C.sub.2-6 alkynyl.

10. The method of claim 1; wherein the carrier-linked treprostinil prodrug is of formula (IIaa): ##STR00143## wherein: each T is independently selected from structures (i) or (iii): ##STR00144## wherein: dashed lines indicate attachment to the rest of the molecule; y is an integer ranging of from 1 to 64; R.sup.a2 is selected from H, unsubstituted alkyl, and substituted alkyl; R.sup.a4 is selected from H, unsubstituted alkyl, and substituted alkyl; ring A.sup.1 is a C.sub.3-10 cycloalkyl; 4- to 7-membered aliphatic heterocyclyl; or 9- to 11-membered aliphatic heterobicyclyl; wherein A.sup.1 is unsubstituted or substituted; Q.sup.1 is selected from C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl which are optionally substituted with one or more R.sup.a10, which are optionally interrupted, provided that Q.sup.1 is at least C.sub.2, by one or more groups selected from the group consisting of: C.sub.3-7 cycloalkyl, 4- to 7-membered heterocyclyl, ##STR00145## wherein each of said group may individually be present one or more times; and which C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.1-50 alkynyl may optionally be terminated at the end connected to Z.sup.1 by a group selected from C.sub.3-7 cycloalkyl, 4- to 7-membered heterocyclyl, ##STR00146## wherein: R.sup.a10 is selected from the group consisting of: halogen, CN, oxo (O), COOR.sup.a12, OR.sup.a12, C(O)R.sup.a12, C(O)N(R.sup.a12R.sup.a12a), S(O).sub.2N(R.sup.a12R.sup.a12a), S(O)N(R.sup.a12R.sup.a12a), S(O).sub.2R.sup.a12, S(O)R.sup.a12, N(R.sup.a12)S(O).sub.2N(R.sup.a12aR.sup.a12b), SR.sup.a12, N(R.sup.a12R.sup.a12a), NO.sub.2, OC(O)R.sup.a12, N(R.sup.a12)C(O)R.sup.a12a, N(R.sup.a12)S(O).sub.2R.sup.a12a, N(R.sup.a12)S(O)R.sup.a12a, N(R.sup.a12)C(O)OR.sup.a12a, N(R.sup.a12)C(O)N(R.sup.a12aR.sup.a12b), OC(O)N(R.sup.a12R.sup.a12a), 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; R.sup.a12, R.sup.a12a and R.sup.a12b 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; R.sup.13 and R.sup.13a are independently selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl; and Z.sup.1 is a carrier comprising a covalently bound polymer.

11. The method of claim 1; wherein the carrier-linked treprostinil prodrug is of formula (IIab): ##STR00147## wherein: each T is independently selected from structures (i) or (iii): ##STR00148## wherein: dashed lines indicate attachment to the rest of the molecule; y is an integer ranging of from 1 to 64; R.sup.a2 is selected from H, unsubstituted alkyl, and substituted alkyl; R.sup.a4 is selected from H, unsubstituted alkyl, and substituted alkyl; ring A.sup.1 is a C.sub.3-10 cycloalkyl; 4- to 7-membered aliphatic heterocyclyl; or 9- to 11-membered aliphatic heterobicyclyl; wherein A.sup.1 is unsubstituted or substituted; Q.sup.1 is selected from C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl which are optionally substituted with one or more R.sup.a10, which are optionally interrupted, provided that Q.sup.1 is at least C.sub.2, by one or more groups selected from the group consisting of: C.sub.3-7 cycloalkyl, 4- to 7-membered heterocyclyl, ##STR00149## wherein each of said group may individually be present one or more times; and which C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl may optionally be terminated at the end connected to Z.sup.1 by a group selected from C.sub.3-7 cycloalkyl, 4- to 7-membered heterocyclyl, ##STR00150## wherein: R.sup.a10 is selected from the group consisting of: halogen, CN, oxo (O), COOR.sup.a12, OR.sup.a12, C(O)R.sup.a12, C(O)N(R.sup.a12R.sup.a12a), S(O).sub.2N(R.sup.a12R.sup.a12a), S(O)N(R.sup.a12R.sup.a12a), S(O).sub.2R.sup.a12, S(O)R.sup.a12, N(R.sup.a12)S(O).sub.2N(R.sup.a12aR.sup.a12b), SR.sup.a12, N(R.sup.a12R.sup.a12a), NO.sub.2, OC(O)R.sup.a12, N(R.sup.a12)C(O)R.sup.a12a, N(R.sup.a12)S(O).sub.2R.sup.a12a, N(R.sup.a12)S(O)R.sup.a12a, N(R.sup.a12)C(O)OR.sup.a12a, N(R.sup.a12)C(O)N(R.sup.a12aR.sup.a12b), OC(O)N(R.sup.a12R.sup.a12a), 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; R.sup.a12, R.sup.a12a and R.sup.a12b 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; R.sup.13 and R.sup.13a are independently selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl; and Z.sup.1 is a carrier comprising a covalently bound polymer.

12. The method of claim 1; wherein the carrier-linked treprostinil prodrug is of formula (IIac) or (IIad): ##STR00151## wherein: each T is independently selected from structures (i) or (iii): ##STR00152## Z.sup.1 is a carrier comprising a covalently bound polymer; y is an integer ranging of from 1 to 64; x is selected from 2, 3, 4, 5, 6, 7 or 8; and X.sub.1 is selected from C.sub.1-15 alkyl, C.sub.2-15 alkenyl and C.sub.2-15 alkynyl, which are optionally substituted or interrupted by one or more groups selected from the group consisting of: C.sub.3-7 cycloalkyl, 4- to 7-membered heterocyclyl, ##STR00153## wherein R.sup.13 and R.sup.13a are independently selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl.

13. The method of claim 1; wherein the carrier-linked treprostinil prodrug is of formula (IIb): ##STR00154## wherein: X.sub.1 is selected from C.sub.1-15 alkyl, C.sub.2-15 alkenyl and C.sub.2-15 alkynyl, which are optionally substituted or interrupted by one or more groups selected from the group consisting of: C.sub.3-7 cycloalkyl, 4- to 7-membered heterocyclyl, ##STR00155## wherein: R.sup.13 and R.sup.13a are independently selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl; y is an integer ranging of from 1 to 64; x is selected from 2, 3, 4, 5, 6, 7 or 8; and Z.sup.1 is a carrier comprising a covalently bound polymer.

14. The method of claim 12; wherein x is 6.

15. The method of claim 1; wherein the carrier-linked treprostinil prodrug has the structure of formula (IIba): ##STR00156## wherein: y is an integer ranging of from 1 to 64; and Z.sup.1 is a carrier comprising a covalently bound polymer.

16. The method of claim 1; wherein the carrier Z.sup.1 has the structure of formula (VII):
BA).sub.n(VII); wherein: B is branching core; A is a poly(ethylene glycol)-based polymeric chain; and n is an integer of from 3 to 32.

17. The method of claim 1; wherein Z.sup.1 represents a moiety (IIca): ##STR00157## wherein: t ranges from 80 to 160; w ranges from 2 to 6; and dashed lines indicate attachment to the rest of the carrier-linked treprostinil prodrug.

18. The method of claim 17; wherein w is 2 or 3.

19. The method of claim 1; wherein the carrier-linked treprostinil prodrug is of formula (IIc), or a pharmaceutically acceptable salt thereof: ##STR00158## wherein y is 4 and Z.sup.1 represents a moiety (IIca): ##STR00159## wherein: dashed lines indicate attachment to the rest of the structure of formula (IIc); t ranges from 80 to 160; and w is 2 or 3.

20. The method of claim 1; wherein R.sup.a1 is C.sub.1-6 alkyl or substituted C.sub.1-6 alkyl.

21. The method of claim 1; wherein R.sup.a3 is H, C.sub.1-6 alkyl or substituted C.sub.1-6 alkyl.

22. The method of claim 1; wherein the carrier-linked treprostinil prodrug, or a pharmaceutically acceptable salt thereof, as claimed in claim 1 is administered as part of a pharmaceutical composition that optionally further comprises one or more pharmaceutically acceptable excipients.

23. The method of claim 22; (i) wherein the carrier-linked treprostinil prodrug is sufficiently dosed in the pharmaceutical composition to provide a therapeutically effective amount of treprostinil for at least 12 hours in one application; and/or (ii) wherein a single dose of the pharmaceutical composition comprises about 2 to about 6 mg the treprostinil moiety T.

24. The method of claim 1; wherein the disease or disorder is pulmonary arterial hypertension.

25. The method of claim 1; wherein R.sup.a1 and R.sup.a3 are joined together with the atoms to which they are attached to form a ring A; and wherein A is selected from the group consisting of: cyclopropane, cyclobutane, cyclopentane, cyclohexane, and cycloheptane.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0710] The invention is illustrated hereinafter by drawings and the working examples, the drawings and working examples serving merely for illustration but not restricting the invention.

[0711] FIG. 1 shows the treprostinil release in buffer and buffered rat plasma at different time points expressed as % treprostinil release compared to total treprostinil content (see Example 29).

[0712] FIG. 2 shows a prolonged duration of circulation of treprostinil conjugate 25 for more than two weeks in monkeys after subcutaneous injection (see Example 30).

[0713] FIG. 3 shows a single dose iv injection of compound 25 and subsequent plasma analysis for total treprostinil and carrier content (see Example 30).

[0714] FIG. 4 shows a single dose iv injection of compound 25 and subsequent plasma analysis for free treprostinil (see Example 32).

[0715] FIG. 5 shows a single dose subcutaneous injection of compound 25 and subsequent plasma analysis for free treprostinil (see Example 32).

OPERATIVE EXAMPLES

[0716] The subject matter of the present invention is elucidated in more detail below, using examples, without any intention that the subject matter of the invention should be confined to these exemplary embodiments.

Materials, Methods and Analytics:

Product Purification

[0717] Normal phase purification was performed on a Biotage Isolera one purification system Biotage AB, Sweden. Biotage KP-Sil silica cartridges. Gradients of Heptane/Ethylacetate or Dichloromethane/Methanol were used. Products were detected and collected at 254 and 280 nm.

[0718] For preparative RP-HPLC, a Waters 600 controller and a 2487 Dual Absorbance Detector was used equipped with a Waters XBridge BEH300 Prep C18 5 m, 15010 mm, flow rate 6 ml/min, or Waters XBridge BEH300 Prep C18 10 m, 15030 mm, flow rate 40 ml/min. Gradients of eluents A (water containing 0.05% TFA v/v or 0.01% HCl v/v) and B (acetonitrile containing 0.05% TFA v/v or 0.01% HCl v/v) were used.

[0719] HPLC fractions containing product were pooled and lyophilized if not stated otherwise.

Automated Flash Chromatography

[0720] Automated Flash Chromatography was performed on a Biotage Isolera one purification system Biotage AB, Sweden, using Biotage KP-Sil silica cartridges. Products were detected and collected at 254 and 280 nm.

LC/MS Analytics

[0721] Analytical RP-HPLC/ESI-MS was performed on waters equipment consisting of a 2695 sample manager, a 2487 Dual Absorbance Detector, and a ZQ 4000 ESI instrument equipped with a 5 m Reprosil Pur 300 ODS-3 column (751.5 mm) (Dr. Maisch, Ammerbuch, Germany; flow rate: 350 l/min, typical gradient: 10-90% MeCN in water, 0.05% TFA over 5 min) or on a Waters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQ Orbitrap Discovery high resolution/high accuracy mass spectrometer equipped with a Waters ACQUITY UPLC BEH300 C18 RP column (2.150 mm, 300 , 1.7 m, flow: 0.25 mL/min; solvent A: UP-H.sub.20+0.04% TFA, solvent B: UP-Acetonitrile+0.05% TFA.

[0722] RP-UPLC/ESI-MS was performed on Waters/Thermo equipment consisting of a Waters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQ Orbitrap Discovery high resolution/high accuracy mass spectrometer equipped with a ACQUITY UPLC BEH300 C18 RP column (Waters Corporation, 2.150 mm, 300 , 1.7 m, Flow: 0.25 mL/min; solvent A: UP-H.sub.20+0.04% TFA, solvent B: UP-MeCN+0.05% TFA. Typical gradients for determination of released treprostinil from TransCon 5 kDa PEG linker treprostinil are: 0.25 mL flow rate, gradient: 30-50% B over 10 min

RP-HPLC Purification:

[0723] For preparative RP-HPLC a Waters 600 controller and a 2487 Dual Absorbance Detector was used equipped with the following columns: Waters XBridge BEH300 Prep C18 5 m, 15010 mm, flow rate 6 ml/min, or Waters XBridge BEH300 Prep C18 10 m, 15030 mm, flow rate 40 ml/min. Linear gradients of solvent system A (water containing 0.05% TFA v/v or 0.01% HCl v/v) and solvent system B (acetonitrile containing 0.05% TFA v/v or 0.01% HCl v/v)

[0724] Typical gradients for purification procedures are: [0725] 6 mL/min flow rate, solvent A: H.sub.20+0.05% TFA, solvent B: MeCN+0.05% TFA, typical gradient: 1-95% B over 14 min [0726] 6 mL/min flow rate, solvent A: H.sub.20+0.05% TFA, solvent B: MeCN+0.05% TFA, typical gradient: 10-80% B over 14 min [0727] 40 mL/min flow rate, solvent A: H.sub.20+0.05% TFA, solvent B: MeCN+0.05% TFA, typical gradient: 40-95% B over 14 min HPLC fractions containing product were pooled and lyophilized if not stated otherwise.

Chemicals and Drug Substances:

[0728] Treprostinil acid was purchased from Shanghai Techwell Biopharmaceutical Co., Ltd., Shanghai, Peoples Republic of China or Chirogate International Inc. Yangmei, Taiwan. 6-(S-Tritylmercapto)hexanoic acid was purchased from Polypeptide, Strasbourg, France. Cis-cyclohexanedicarboxylic anhydride was purchased from Alfa Aesar GmbH & Co KG, Karlsruhe, Germany. 2-Chlorotrityl chloride resin (1%, Novabiochem DVB) was obtained from Merck Biosciences GmbH, Germany. 6-(S-Tritylsulfanyl)-hexaneamine was synthesized according to WO-A 2009/133137. PEGs used in this work were acquired from NOF Europe N.V., Grobbendonk, Belgium. All other chemicals were purchased from Sigma Aldrich GmbH, Taufkirchen, Germany. Water and acetonitrile for analytical RP-HPLC were purchased from Biosolve B.V. and TFA from Thermo scientific.

Example 1

Benzyl Protection of 3-Hydroxybutanoic Acid 1:

[0729] ##STR00105##

[0730] 3-Hydroxybutanoic acid 1 (434 mg, 4.17 mmol) was dissolved in THF (10 mL) and BnBr (700 L, 5.89 mmol) and Cs.sub.2CO.sub.3 (2.5 g, 7.67 mmol) were added. The reaction mixture was refluxed in a sealed tube for 4-6 hours. After cooling down to room temperature the reaction mixture was filtrated and the residue was washed several times with EtOAc. The organic solvents were removed and the product was purified by automated flash chromatography on silica in one portion (SNAP 25 g cartridge, flow 30 ml/min, solvent A: DCM, solvent B: MeOH; gradient: 0-5% B over 19 CV) to remove starting material and obtain desired benzyl protected 3-hydroxybutanoic acid 2 as yellow oil.

[0731] Yield: 361 mg (45%)

[0732] MS: m/z 217.1=[M+Na].sup.+ (MW+Na calculated=217.2).

Example 2

[0733] Coupling Reaction of Benzylated 3-Hydroxybutanoic Acid 2 with Treprostinil:

##STR00106##

[0734] Treprostinil acid (10.5 mg, 0.0268 mmol) was dissolved in DCM (4.5 mL) and DCC (9.4 mg, 0.0455 mmol), HOBT (7.5 mg, 0.0489 mmol) and DMAP (7.5 mg, 0.0613 mmol) were added to the solution. Then benzylated 3-hydroxybutanoic acid 2 (15 mg, 0.0772 mmol) was dissolved in DCM (0.5 mL) and added to the reaction mixture. The mixture was stirred at RT until the consumption was complete (analytical RP-HPLC). Volatile solvents were removed in vacuo and the residue was purified over a small silica column (3 ml silica, DCM/MeOH (100:0)DCM/MeOH (95:5) to obtain the desired linker treprostinil 3 as yellow oil.

[0735] Yield: 8 mg (50%)

[0736] MS: m/z 589.3.sup.=[M+Na].sup.+ (MW+Na calculated=589.7)

Example 3

Hydrogenation Reaction of Benzylester 3:

[0737] ##STR00107##

[0738] Benzylester 3 (13 mg, 0.0229 mmol) was dissolved in EtOAc (4 MS, 2 mL) and 5% palladium on charcoal (5% Pd, 15 mg) was added. Hydrogen was bubbled through the solution for 30 min. The reaction mixture was stirred further 12.5 h under hydrogen atmosphere until the consumption was complete (analytical RP-HPLC). The mixture was filtered over celite and washed several times with EtOAc. Organic solvents were removed in vacuo and the residue was purified using RP-HPLC (solvent A: H.sub.2O with 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 1-95% B over 20 min, flow: 6 mL/min). The product containing fractions were pooled and lyophilized to obtain 4 as white solid.

[0739] Yield: 1.9 mg (29%).

[0740] MS: m/z 499.3=[M+Na].sup.+ (MW+Na calculated=499.6).

Example 4

[0741] Coupling Reaction of Linear PEG 5 kDa Amine with Linker Treprostinil 4:

##STR00108##

[0742] Linker treprostinil 4 (1.9 mg, 3.98 mol) and linear PEG 5 kDa amine (86 mg, 17.2 mol) were dissolved in THF/MeCN (4 MS; 1.5 mL: 0.5 mL) and Et.sub.3N(40 L), a catalytic amount of DMAP and T3P (50% in EtOAc, 50 L, 73.2 mol) were successively added. The reaction mixture was allowed to stir at rt for 12 h. The reaction mixture was diluted with 20 L H.sub.2O and volatile solvents were removed in vacuo. The residue was purified using RP-HPLC (solvent A: H.sub.2O with 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 10-80% B over 20 min, flow: 6 mL/min). The product containing fractions were pooled and lyophilized to obtain TransCon PEG linker treprostinil 5 as white solid.

[0743] Yield: 12.5 mg (58%).

[0744] MS: m/z 1378.6=[M+4H].sup.4+ (calculated=1378.9) for one representative peak in the polymer distribution.

Example 5

Treprostinil Release Kinetics of TransCon PEG Linker Treprostinil 5:

[0745] TransCon PEG linker treprostinil 5 (0.5-1.5 mg) was incubated in pH 7.4 hydrolysis buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05% Tween-20, 1 mL) at 37 C. and aliquots were analyzed by UPLC at various time points for released treprostinil.

Half Life Determination of Hydrolysis Kinetics of TransCon PEG Linker Treprostinil 5:

[0746] The percentage of released treprostinil after incubation at pH 7.4 and 37 C. for a given time period was determined by integrating the corresponding peaks (released material versus conjugate) in the RP-UPLC chromatogram. The data as shown in table 1 were subsequently plotted against time. By using a first order kinetics fit a half life of 4.20 d for the treprostinil release from 5 was obtained.

TABLE-US-00010 TABLE 1 entry Incubation time [d] released treprostinil [%] 1 0.000 2 2 0.83 5 3 1.11 18 4 1.81 27 5 2.06 29 6 5.13 59 7 6.10 64 8 8.80 77 9 11.90 86

Example 6

[0747] Synthesis of Intermediates 6a/6b:

##STR00109##

[0748] 6-(S-Tritylsulfanyl)-hexaneamine (for synthesis see WO-A 2009/133137) (507 mg, 1.35 mmol) was dissolved in DCM (4 ml) and cis-1,2-cyclohexanedicarboxylic anhydride (251 mg, 1.63 mmol) was added to the reaction mixture at RT. DIPEA (0.70 mL, 4.06 mmol) was added and the mixture was stirred at RT until complete consumption of 6-(S-Tritylsulfanyl)-hexaneamine (LC/MS). Volatile solvents were removed in vacuo, the residue was dissolved in H.sub.2O/MeCN (6:1, 18 mL) and the product was purified by RP-HPLC (solvent A: H.sub.2O with 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 40-95% B over 16 min, flow: 40 ml/min). The pooled fractions were neutralized with sat. NaHCO.sub.3 soln. (pH approx. 6) and the organic solvents were removed in vacuo. The remaining aqueous phase was extracted twice with DCM. Combined organic layers were dried with MgSO.sub.4 and the solvent was removed in vacuo obtaining 6a/6b as a racemic mixture.

[0749] Yield: 580 mg (81%).

[0750] MS: m/z 552.23=[M+Na].sup.+ (MW+Na calculated=552.62 g/mol).

Example 7

[0751] Synthesis of Intermediates 7a/7b:

##STR00110##

[0752] N-Boc-1,6-hexanediamine (270 mg, 1.25 mmol) was dissolved in DMF (2 ml) and cis-1,2-cyclohexanedicarboxylic anhydride (231 mg, 1.50 mmol) was added to the reaction mixture at RT. DIPEA (0.65 mL, 3.76 mmol) was added and the mixture was stirred at RT until consumption of N-Boc-1,6-hexanediamine (LC/MS). The reaction mixture was diluted with H.sub.2O/MeCN (9:1) and the product was purified by RP-HPLC (solvent A: H.sub.2O with 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 10-80% B over 16 min, flow: 40 ml/min). The pooled fractions were neutralized with sat. NaHCO.sub.3 soln. (pH approx. 6) and the organic solvents were removed in vacuo. The remaining aqueous phase was extracted several times with DCM. The organic layers were dried with MgSO.sub.4 and the solvent was removed in vacuo obtaining 7a/7b as a racemic mixture.

[0753] Yield: 410 mg (88%).

[0754] MS: m/z 371.39=[M+H].sup.+ (MW+H calculated=371.27 g/mol).

Example 8

Synthesis of Dmob Protected Treprostinil 8:

[0755] ##STR00111##

[0756] Treprostinil (61 mg, 0.156 mmol) was dissolved in toluene (dry, molecular sieve, 2.5 ml) and silylation reagent BSA (0.6 mL, 0.245 mmol) was added. The reaction mixture was stirred for 12 h at RT. Volatile solvents were removed in vacuo and the TMS protected treprostinil was used without further purification.

[0757] TMS protected treprostinil was dissolved in DCM (2.5 mL) and H.sub.2O (60 L). DMAP (76 mg, 0.624 mmol), EDC.HCl (119 mg, 0.624 mmol) and Dmob-alcohol (105 mg, 0.624 mmol) dissolved in DCM (1 ml) were added. The reaction mixture was stirred at RT until reaction was complete (LC/MS). The solution was diluted with DCM and quenched by addition of 0.1 N HCl solution saturated with NaCl. The aqueous phase was extracted several times with DCM. Combined organic layers were dried with MgSO.sub.4 and the solvent was removed in vacuo obtaining crude product 8. Crude product was purified using RP-HPLC (solvent A: H.sub.2O with 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 35-85% B over 16 min, flow: 40 ml/min). Combined HPLC fractions were adjusted to a pH of approx. 7 by adding sat. NaHCO.sub.3 soln. MeCN was removed in vacuo. The remaining H.sub.2O layer was extracted several times with DCM and the combined organic phases were dried with MgSO.sub.4, filtered and the solvent was removed in vacuo obtaining product 8 as colorless solid.

[0758] Yield: 69 mg (82%).

[0759] MS: m/z 563.20 g/mol=[M+Na].sup.+ (MW+Na calculated=563.67 g/mol).

Example 9

Synthesis of Treprostinil Linker Thiol

[0760] ##STR00112##

[0761] Carboxylic acid 6a/6b (147 mg, 0.277 mmol), EDC.HCl (53 mg, 0.277 mmol) and DMAP (34 mg, 0.277 mmol) were dissolved in 0.5 mL DCM. Dmob protected treprostinil 8 (43 mg, 0.08 mmol) was dissolved in 0.5 mL DCM and added to the reaction mixture. The mixture was stirred at RT until consumption of 8 was complete (over night, LC/MS). Volatile solvents were removed in vacuo. The residue was dissolved in HFIP (2 mL), TFA (100 L) and TES(50 L) and stirred for 30 min at RT (LC/MS). Volatiles were removed in vacuo. The residue was dissolved in H.sub.2O/MeCN (9/1, 0.05% TFA, 2 mL) and the mixture of four possible isomers was purified by RP-HPLC (solvent A: H.sub.2O with 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 60-85% B over 16 min, flow: 6 mL/min). Product isomers eluted as three separable peaks. Fractions containing the peak with the shortest elution time (compound 9) were pooled and used in the PEGylation step without further processing. Structural assignment of 9 to the possible isomers 9a, 9b, 9c or 9d was not performed in this experiment. Yield of 9 was determined by using Ellman test.

[0762] Yield: 8.1 mg (26%)

[0763] MS: m/z 682.21 g/mol=[M+Na].sup.+ (MW+Na calculated=682.40 g/mol).

Example 10

Synthesis of Treprostinil Linker Amine

[0764] ##STR00113##

[0765] Carboxylic acid 7a/7b (50 mg, 0.134 mmol), EDC HCl (26 mg, 0.134 mmol) and DMAP (16 mg, 0.134 mmol) were dissolved in DCM (0.3 mL). Dmob protected treprostinil 8 (36 mg, 0.066 mmol) was dissolved in DCM (0.5 mL) and added to the reaction mixture. The mixture was stirred at RT until the consumption was complete (LC/MS). Volatile solvents were removed in vacuo. The residue was dissolved in H.sub.2O/MeCN (9/1, 0.05% TFA, 2 mL) and the mono coupling products (treprostinil coupled to one 7a/7b molecule) were separated from the double coupling products (treprostinil coupled to two 7a/7b molecules) by RP-HPLC: Thermo Fisher Hypersil Gold PFP column, 15010 mm, solvent A: H.sub.2O with 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 35-55% B over 16 min, flow: 6 mL/min. HPLC fractions containing mono coupling products were pooled and lyophilized. Lyophilizate was dissolved in HFIP (0.9 mL), DCM (0.1 mL), TFA (100 L) and TES(20 L) and stirred for 10 min at RT. Volatiles were removed in vacuo, the residue was dissolved in H.sub.2O/MeCN (9/1, 0.05% TFA, 2 mL) and the and the mixture of four possible isomers was purified by RP-HPLC (solvent A: H.sub.2O with 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 35-55% B over 16 min, flow: 6 mL/min). Product isomers eluted as three separable peaks. Fractions containing the peak with the shortest elution time (compound 10) were pooled and used in the PEGylation step without further processing.

[0766] Structural assignment of the 10 to the possible isomers 10a, 10b, 10c or 10d was not performed in this experiment. Yield of 10 was estimated by HPLC by using a treprostinil calibration curve (280 nm).

[0767] Yield: 3.0 mg

[0768] MS: m/z 643.28 g/mol=[M+Na].sup.+ (MW+Na calculated=643.45 g/mol).

Example 11

[0769] PEGylation Reaction of Treprostinil Linker Amine with Linear PEG 5 kDa NHS

##STR00114##

[0770] To treprostinil linker amine 10 (0.6 mg, 1 mol in solution, MeCN/H.sub.2O, 0.05% TFA, 5 mL) linear PEG 5 kDa NHS (23 mg, 4.6 mol) was added. The solution was neutralized by addition of 0.5 M pH 7.4 buffer (0.5 M phosphate, 0.6 mL). H.sub.2O (1 mL) was added for obtaining a clear solution, and reaction mixture was incubated at RT for 1 h. Then the reaction mixture was purified by RP-HPLC (solvent A: H.sub.2O with 0.01% HCl, solvent B: MeCN with 0.01% HCl, gradient: 10-70% B over 16 min, flow: 6 mL/min) to obtain after lyophilization TransCon linear 5 kDa PEG treprostinil 11.

[0771] Yield: 3 mg

Example 12

[0772] PEGylation Reaction of Treprostinil Linker Thiol with Linear PEG 40 kDa Maleimide

##STR00115##

[0773] To the treprostinil linker thiol 9 (6.2 mg, 9.42 mol) solution in MeCN/H.sub.2O (0.05% TFA, 87 mL) linear PEG 40 kDa maleimide (463 mg, 11.3 mol) was added. The solution was neutralized by addition of pH 7.4 buffer (0.5 M phosphate, 4.4 mL). After 1 h incubation time another portion of linear 40 kDa Mal-PEG (73 mg, 178 mol) and H.sub.2O (5 mL) was added and the reaction solution was incubated for another 1.5 h. The reaction mixture was purified by RP-HPLC (solvent A: H.sub.2O with 0.01% HCl, solvent B: MeCN with 0.01% HCl, gradient: 30-50% B over 16 min, flow: 40 mL/min) to obtain after lyophilization TransCon linear 40 kDa PEG treprostinil 12.

[0774] Yield: 321 mg (82%)

Example 13

[0775] PEGylation Reaction of Treprostinil Linker Thiol with 4-Arm PEG 20 kDa Maleimide

##STR00116##

[0776] To the treprostinil linker thiol 9 (2.54 mg, 3.84 mol) solution in MeCN/H.sub.2O (0.05% TFA, 5.7 mL) 4-arm PEG 20 kDa maleimide (21 mg, 0.98 mol) was added. The solution was neutralized by addition of pH 7.4 buffer (0.5 M phosphate, 3.0 mL). H.sub.2O (3 mL) was added until the reaction mixture became a clear solution again. The reaction mixture was incubated at RT for 2 h and then purified by RP-HPLC (solvent A: H.sub.2O with 0.01% HCl, solvent B: MeCN with 0.01% HCl, gradient: 45-85% B over 16 min, flow: 40 mL/min) to obtain after lyophilization TransCon 4-arm PEG 20 kDa treprostinil 13.

[0777] Yield: 14 mg (66%).

Example 14

Treprostinil Release Kinetics of TransCon PEG Linker Treprostinil Compounds 11 and 12:

[0778] Release kinetics were determined according to Example 5. A treprostinil release half life time of 4.3 days (0.7 days) was obtained for compounds 11 and 12.

Example 15

Treprostinil Release Kinetics of TransCon PEG Linker Treprostinil Compound 13

[0779] TransCon PEG linker treprostinil 13 (2.5 mg) was incubated in pH 7.4 hydrolysis buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05% Tween-20, 1 mL) at 37 C. and aliquots were analyzed by UPLC at various time points for released treprostinil. The percentage of released treprostinil was determined in relation to the area of treprostinil after total hydrolysis of an aliquot (50 l hydrolysis solution and 25 l 5 N NaOH were mixed for 20 min. 25 l AcOH was added and the resulting solution was analyzed by LCMS).

[0780] By using a first order kinetics fit, a half life of 5 d for treprostinil release from 13 was obtained.

Example 16

Synthesis of Building Block 14

[0781] Building block 14 was synthesized according to the following scheme:

##STR00117##

[0782] Mmt-chloride (3 g, 9.71 mmol) was dissolved in DCM (20 mL) and added dropwise to a solution of ethylenediamine (6.5 mL, 97.1 mmol) in DCM (20 mL). After two hours the solution was poured into diethyl ether (300 mL) and washed three times with 30/1 (v/v) brine/0.1 M NaOH solution (50 ml each) and once with brine (50 mL). The organic phase was dried over Na.sub.2SO.sub.4 and volatiles were removed under reduced pressure. Mmt-protected amine (3.18 g, 9.56 mmol) was used in the next step without further purification.

[0783] The Mmt-protected amine (3.18 g, 9.56 mmol) was dissolved in anhydrous DCM (30 mL). 6-(S-Tritylmercapto)hexanoic acid (4.48 g, 11.47 mmol), PyBOP (5.96 g, 11.47 mmol) and DIPEA (5.0 mL, 28.68 mmol) were added and the mixture was agitated for 30 min at RT. The solution was diluted with diethyl ether (250 mL) and washed three times with 30/1 (v/v) brine/0.1 M NaOH solution (50 mL each) and once with brine (50 mL). The organic phase was dried over Na.sub.2SO.sub.4 and volatiles were removed under reduced pressure. Amide was purified by flash chromatography eluting with heptane/ethyl acetate containing 0.02% (v/v) diethylmethylamine.

[0784] Yield: 5.69 g (8.07 mmol).

[0785] MS: m/z 705.4=[M+H].sup.+ (MW=705.0).

[0786] Amide (3.19 g, 4.53 mmol) was dissolved in anhydrous THF (50 mL) and BH.sub.3.THF (1 M solution, 8.5 mL, 8.5 mmol) was added. Solution was stirred for 16 h at RT. Further BH.sub.3.THF (1 M solution, 14 mL, 14 mmol) was added and stirred for further 16 h at RT. The reaction was quenched by addition of methanol (8.5 mL). N,N-dimethyl-ethylenediamine (3 mL, 27.2 mmol) was added, the solution was heated to reflux and stirred for 3 h. Reaction mixture was allowed to cool down to RT and was then diluted with ethyl acetate (300 mL), washed with saturated, aqueous Na.sub.2CO.sub.3 solution (2100 mL) and saturated, aqueous NaHCO.sub.3 solution (2100 mL). The organic phase was dried over Na.sub.2SO.sub.4 and volatiles were removed under reduced pressure to obtain crude amine intermediate (3.22 g).

[0787] The amine intermediate (3.22 g) was dissolved in DCM (5 mL). Boc.sub.2O (2.97 g, 13.69 mmol) dissolved in DCM (5 mL) and DIPEA (3.95 mL, 22.65 mmol) were added and the mixture was agitated at RT for 30 min. Boc- and Mmt-protected intermediate was purified by flash chromatography.

[0788] Yield: 3.00 g (3.79 mmol).

[0789] MS: m/z 791.4=[M+H].sup.+, 519.3=[MMmt+H].sup.+ (MW calculated=791.1).

[0790] 0.4 M aqueous HCl (48 mL) was added to a solution of the Boc- and Mmt-protected intermediate in acetonitrile (45 mL). The mixture was diluted with acetonitrile (10 mL) and stirred for 1 h at RT. Subsequently, the pH value of the reaction mixture was adjusted to 5.5 by addition of an aqueous 5 M NaOH solution. Acetonitrile was removed under reduced pressure and the aqueous solution was extracted with DCM (4100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and volatiles were removed under reduced pressure. Crude amine 14 was used without further purification.

[0791] Yield: 2.52 g (3.19 mmol). A MW of 791.1 g/mol of crude amine 14 was assumed

[0792] MS: m/z 519.3=[M+H].sup.+ (MW calculated=519.8 g/mol).

Example 17

Synthesis of Linker Building Blocks 15a, 15b, and 15c

[0793] Linker building block 15a was synthesized according to the following scheme:

##STR00118##

[0794] Amine 14 (503 mg, 0.635 mmol, assuming a MW of 791.1 g/mol of crude 1) was dissolved in 4 mL DMF (anhydrous, mol. sieve). Fmoc-N-Me-Ala-OH (310 mg, 0.953 mmol), COMU (408 mg, 0.953 mmol) and DIPEA (332 l, 1.906 mmol) were added and the reaction was allowed to stir for 3 h at RT. 150 l piperidine and 150 l DBU were added to the mixture and stirring was continued for further 60 min. 400 l acetic acid were added and product was purified by HPLC. HPLC fractions containing product 15a were neutralized with a saturated NaHCO.sub.3 solution and extracted twice with DCM. Combined organic phases were dried over Na.sub.2SO.sub.4 and volatiles were removed under reduced pressure.

[0795] Yield: 203 mg (0.336 mmol).

[0796] MS: m/z 604.1=[M+H].sup.+ (MW calculated=603.9 g/mol).

Linker Building Block 15b

[0797] ##STR00119##

[0798] Linker building block 15b was synthesized as described for 15a except that Fmoc-Aib-OH was used instead of Fmoc-N-Me-Ala-OH.

[0799] Yield: 95 mg (0.161 mmol).

[0800] MS: m/z 604.2=[M+H].sup.+ (MW calculated=603.9 g/mol).

Linker Building Block 15c

[0801] ##STR00120##

[0802] Linker building block 15c was synthesized as described for 15a except that Fmoc-N-Me-Aib-OH was used instead of Fmoc-N-Me-Ala-OH.

[0803] Yield: 149 mg (0.241 mmol).

[0804] MS: m/z 619.0=[M+H].sup.+ (MW calculated=617.9 g/mol).

Example 18

Synthesis of Treprostinil-Linker Thiols 16a, 16b, 16c, 16d, 16e and 16f

[0805] Treprostinil-linker thiols 16a/16b were synthesized according to the following scheme:

##STR00121##

[0806] A 10 mL single use syringe reactor equipped with a PE frit was loaded with 2-chlorotrityl chloride (TCP) resin (153 mg, loading 1.22 mmol/g, 0.186 mmol). A solution of treprostinil (54 mg, 0.138 mmol) and DIPEA (60 l, 0.346 mmol) in DCM (anhydrous, mol. sieve) was drawn into the reactor. Reactor was agitated for 2 h at RT. 200 l methanol were added and reactor was agitated for further 10 min. Solution was dispelled and resin was washed with DCM (5), DMF (5) and DCM (10). Resin was dried under vacuum (1 mbar). Based on weight, a treprostinil loading of 0.72 mmol/g TCP resin was obtained.

[0807] 900 l THF (anhydrous, mol. sieve) and 300 l of a 1 M LiOEt solution in THF (300 mol) were drawn to 30 mg treprostinil loaded TCP resin (21.6 mol) in a single use 2 mL syringe reactor equipped with a PE frit. Reactor was agitated for 40 min at RT. Solution was dispelled and resin was washed with THF (2). A solution of bis(pentafluorophenyl)carbonate (100 mg, 254 mol) in 1 mL THF was drawn into the syringe which was agitated for 90 min at RT. Solution was dispelled and resin was washed with THF (5) and DMF (5). A solution of linker building block 15a (50 mg, 83 mol), DIPEA (50 l, 287 mol) and DMAP (1 mg, 8 mol) in 300 l DMF (anhydrous, mol. sieve) was drawn into the syringe. Syringe was agitated for 3 h at RT. Solution was dispelled and resin was washed with DMF (10) and DCM (10). Product was cleaved from resin by incubation with 500 l of cleavage cocktail HFIP/DCM/TES 90/10/2 v/v/v for 10 min (3). Resin was washed with 500 l DCM (2). TFA (250 L) was added to the combined cleavage and washing solutions and the mixture was incubated at RT for 10 min. Volatiles were removed under reduced pressure. Residue was subjected to HPLC purification which gave thiols 16a/16b as a mixture of the two regioisomers. HPLC eluate was used in the next step without further processing.

[0808] MS: m/z 678.1=[M+H].sup.+ (MW calculated=678.0 g/mol).

Treprostinil Linker Thiols 16c/16d

##STR00122##

[0809] Treprostinil linker thiols 16c/16d were synthesized as described for 16a/16b except that linker building block 15b was used instead of 15a. Thiols 16c/16d were obtained as a mixture of isomers. HPLC eluate was used in the next step without further processing.

[0810] MS: m/z 678.1=[M+H].sup.+ (MW calculated=678.0 g/mol).

Treprostinil Linker Thiols 16e and 16f

[0811] ##STR00123##

[0812] Treprostinil linker thiols 16e and 16f were synthesized as described for 16a/16b except that linker building block 15c was used instead of 15a. Two isomers assigned to structures 16e and 16f were separated by HPLC. HPLC eluates were used in the next step without further processing.

[0813] 15e MS: m/z 693.0=[M+H].sup.+ (MW calculated=692.0 g/mol).

[0814] 15f MS: m/z 693.0=[M+H].sup.+ (MW calculated=692.0 g/mol).

Example 19

[0815] Synthesis of Linker Building Blocks 17a and 17b

[0816] Linker building blocks 17a and 17b were synthesized according to the following scheme:

##STR00124##

[0817] L-Fmoc-Dpr(Boc)-OH (100 mg, 0.234 mmol) was dissolved in 0.5 mL DMF (anhydrous, mol. sieve). 6-(S-Tritylsulfanyl)-hexaneamine (71 mg, 0.189 mmol), COMU (97 mg, 0.227 mmol) and DIPEA (66 l, 0.378 mmol) were added and mixture was stirred for 1 h at RT. Piperidine (50 l, 0.505 mmol) and DBU (40 l, 0.336 mmol) were added and stirring was continued for 10 h. ds-Cyclohexanedicarboxylic anhydride (600 mg, 3.89 mmol) was added and stirring was continued for 1 h. Solution was quenched with water/acetonitrile and acidified with acetic acid Building blocks were purified by RP-HPLC. Structures assignment of the earlier eluting diastereomer 17a and the later eluting diastereomer 17b was done arbitrarily and could also be reverse.

[0818] Yield: 17a 30 mg (0.042 mmol), 17b 42 mg (0.059 mmol)

[0819] MS: m/z 716.2=[M+H].sup.+ (MW calculated=716.0 g/mol).

Example 20

[0820] Synthesis of Treprostinil Linker Thiols 18a/18b

##STR00125##

[0821] Linker building block 17a (11 mg, 12 mol), EDC HCl (7.4 mg, 38.5 mol) and DMAP (4.7 mg, 38.5 mol) were dissolved in 300 l DCM (anhydrous, mol. sieve). Solution was drawn to 15 mg treprostinil loaded TCP resin (10.8 mol, 0.72 mmol/g see Example 3) in a single use 2 mL syringe reactor equipped with a frit. Reactor was agitated for 15 h at RT. Solution was dispelled and resin was washed with DCM (10). Product was cleaved by incubating resin with 500 l HFIP/DCM 30/70 v/v for 10 min (3). Resin was washed with 500 l DCM (2). To the combined cleavage and washing solutions were added 250 l TFA and the mixture was incubated at RT for 10 min. Volatiles were removed under reduced pressure. Residue was subjected to RP-HPLC purification which gave thiols 18a/18b as a mixture of the two regioisomers. HPLC eluate was used in the next step without further processing.

[0822] Yield: 18a/18b 1.5 mg (2 mol) as determined by thiol quantification by Ellman Test.

[0823] MS: m/z 746.2=[M+H].sup.+ (MW calculated=746.0 g/mol).

Example 21

[0824] Synthesis of PEG-Linker-Drug Conjugates 19a/b, 19c/6d, 19e, 19f and 19g/19h

[0825] PEG-linker-drug conjugates were prepared according to the following scheme:

##STR00126##

[0826] To HPLC eluates of treprostinil linker thiols 16a/16b, 16c/16d, 16e, 16f and 18a/18b was given an excess of linear PEG 5 kDa maleimide. Mixtures were neutralized by addition of pH 7.4 buffer (0.5 M phosphate) and incubated at RT. After complete consumption of thiol (approx. 1 h) mixtures were acidified with acetic acid and separated from excess PEG-maleimide by RP-HPLC. HPLC eluates were lyophilized to yield PEG-linker-drug conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h respectively.

Example 22

[0827] Determination of Drug Release Half Life Time from PEG Conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h:

##STR00127##

[0828] PEG-linker-drug conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h were dissolved in pH 7.4 buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05% Tween-20, 1 mL) and incubated at 37 C. At various time points aliquots were analyzed by UPLC to determine the amount of released treprostinil which was plotted against time. Drug release was found to follow first order kinetics. Curve fitting software was used to determine half life time of drug release from the respective conjugates (Table 1)

TABLE-US-00011 TABLE 2 entry PEG-linker-drug conjugate drug release half life time 1 19a/b 31 d 2 19c/19d 17 d 3 19e 24 d 4 19f 37 d 5 19g/19h 35 min

Example 23

Synthesis of Intermediate 20:

[0829] ##STR00128##

[0830] The amino group of 6-(S-Tritylsulfanyl)-hexaneamine was Tmob (2,4,6-Trimethoxybenzyl) protected by dropwise addition of a solution of 2,4,6-trimethoxybenzaldehyde (4.22 g, 21.51 mmol) in 88 mL methanol/DCM 1/1 (v/v) to 6-(S-tritylsulfanyl)-hexaneamine (6.74 g, 17.95 mmol) and sodium cyanoborohydride (1.58 g, 25.14 mmol) in 44 mL methanol. The mixture was stirred for 1.5 h at RT and quenched with 95 mL of 0.4 N aqueous HCl solution. After further stirring at RT for 30 min mixture was extracted with ethyl acetate (4). Combined organic layers were washed with sat. aqueous NaHCO.sub.3 solution (2) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure. Tmob protected amine 20 was purified by flash chromatography eluting with DCM/methanol containing 0.1% (v/v) triethylamine.

[0831] Yield: 5.88 g (55%).

[0832] MS: m/z 556.3.sup.=[M+H].sup.+ (MW calculated=555.79 g/mol).

Example 24

Synthesis of Intermediate 21:

[0833] ##STR00129##

[0834] (1R,2S)-Cyclohexanedicarboxylic acid 1-methyl ester, CAS no. 88335-92-6 (for synthesis see R. Manzano et al. J. Org. Chem. 2010, 75(15), 5417-5420) (506 mg, 2.72 mmol) was dissolved in toluene (11 ml, anhydrous). Thionyl chloride (1.09 mL, 15.0 mmol) was added and mixture was heated for 1 h at 60 C. in a pressure tube. Volatiles were removed in vacuo. A solution of Tmob protected amine 20 (1.66 g, 2.99 mmol) and DIPEA (1.12 mL, 6.43 mmol) in DCM (30 mL, anhydrous) was added and mixture was stirred for 2 h at RT. Ethyl acetate was added and the organic layer was washed with 0.1 N aqueous HCl (2). The organic layer was dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure. Methyl ester 21 was purified by flash chromatography eluting with ethyl acetate/heptane.

[0835] Yield: 1.55 g (79%).

[0836] MS: m/z 746.1=[M+Na].sup.+ (MW calculated=723.98 g/mol).

Example 25

Synthesis of Intermediate 22:

[0837] ##STR00130##

[0838] Methyl ester 21 (3.12 g, 4.31 mmol) was dissolved in isopropanol (10 ml). 35 mL of a 1 M aqueous LiOH solution were added and the mixture was stirred for 5 d at RT. Ethyl acetate was added and the organic layer was washed with 0.05 N aqueous HCl (2) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure. 22 was purified by flash chromatography eluting with ethyl acetate/heptane containing 0.1% formic acid (v/v).

[0839] Yield: 2.41 g (79%).

[0840] MS: m/z 710.1=[M+H].sup.+ (MW calculated=709.95 g/mol).

Example 26

Synthesis of Intermediate 6a:

[0841] ##STR00131##

[0842] Compound 22 (1.23 g, 1.74 mmol) was dissolved in DCM (18 ml). TFA (2 mL) and TES (600 l) were added and the mixture was stirred for 40 min at RT. Volatiles were removed in vacuo. The residue was dissolved in DCM (20 mL) and tritylchloride (728 mg, 2.61 mmol) was added. The mixture was stirred for 2 h at RT. DCM was removed under reduced pressure. Carboxylic acid 6a was purified by flash chromatography using ethyl acetate/heptane containing 0.1% formic acid (v/v) as eluent, followed by RP-HPLC purification.

[0843] Yield: 615 mg (67%).

[0844] MS: m/z 552.2=[M+Na].sup.+ (MW calculated=.sup.5290.7.sup.5 g/mol).

Example 27

Synthesis of Treprostinil Linker Thiol 24a

[0845] ##STR00132##

[0846] Dmob protected treprostinil 8 (100 mg, 0.185 mmol), carboxylic acid 6a (195 mg, 0.368 mmol), EDC.HCl (72 mg, 0.376 mmol) and DMAP (43 mg, 0.352 mmol) were dissolved in DCM (1.8 mL, anhydrous, mol. sieve). The mixture was stirred at RT for 1 d. Ethyl acetate was added and the organic layer was washed with 0.1 N aqueous HCl (3) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure. The residue was dissolved in HFIP (5 mL), TFA (250 L) and TES(250 L) and stirred for 30 min at RT. The precipitate was filtered off and the filtrate was evaporated in vacuo.

[0847] UPLC analysis revealed a 4/1 ratio of regioisomers 24a and 24b (column: Kinetex 1002.1 mm, 1.7 m XB-C18 silica, pore size 100 , Phenomonex Ltd, Aschaffenburg, Germany; flow rate 0.25 mL/min; solvent A: water+0.05% TFA (v/v), solvent B: acetonitrile+0.04% TFA; gradient: 30-58% B (10 min), 58% B isocratic (10 min), 58-80% B (5 min), 80-99% (5 min), wavelength 280 nm). 24a turned out to be identical with compound 9.

[0848] The residue was taken up in acetonitrile/water and 24a was purified by RP-HPLC (solvent A: H.sub.2O+0.01% HCl, solvent B: MeCN+0.01% HCl, gradient: 60-85% B over 16 min). Isomer 24a eluted first, followed by isomer 24b. Fractions containing pure 24a were combined and lyophilized. Mixed fractions containing 24a and 24b were subjected to repurification.

[0849] Yield 24a: 29.5 mg (24%)

[0850] MS: m/z 660.3=[M+H].sup.+ (MW calculated=659.9 g/mol).

[0851] .sup.1H-NMR (CDCl.sub.3, [ppm]): 7.07 (t, 1H), 6.80 (d, 1H), 6.71 (d, 1H), 5.86 (bs, 1H), 4.78-4.63 (m, 3H), 3.53 (bs, 1H), 3.14-3.03 (m, 1H), 3.03-2.83 (m, 2H), 2.82-2.66 (m, 2H), 2.66-2.58 (m, 1H), 2.58-2.46 (m, 4H), 2.46-2.31 (m, 1H), 2.31-2.13 (m, 1H), 2.13-1.92 (m, 2H), 1.92-1.81 (m, 1H), 1.75-1.51 (m, 7H), 1.51-1.21 (m, 21H), 1.21-1.08 (m, 1H), 0.90 (t, 3H).

[0852] .sup.13C-NMR (126 MHz, CDCl.sub.3, [ppm]): 174.7, 174.2, 171.7, 155.2, 140.6, 127.5, 126.3, 121.8, 109.71, 78.8, 72.4, 65.8, 48.3, 43.9, 42.5, 40.4, 39.7, 37.4, 37.3, 35.4, 34.00, 33.0, 32.9, 32.1, 29.3, 28.4, 28.1, 27.0, 26.4, 25.5, 24.6, 24.2, 23.2, 22.8, 14.2.

Example 28

[0853] PEGylation Reaction of Treprostinil Linker Thiol 24a with 4-Arm PEG 20 kDa Maleimide

##STR00133##

[0854] A solution of treprostinil linker thiol 24a (7.5 mg, 11.3 mol) in 2 mL of acetonitrile/water 9/1 (v/v) was mixed with a solution of 4-arm PEG 20 kDa maleimide (53.5 mg, 2.54 mol) in 2 mL of acetonitrile/water 1/1 (v/v). The pH was adjusted to 7.0 by addition of pH 7.4 buffer (50 mM phosphate, 0.8 mL). The reaction mixture was stirred at RT for 1.5 h and then purified by RP-HPLC (solvent A: H.sub.2O with 0.01% HCl, solvent B: MeCN with 0.01% HCl, gradient: 45-85% B over 16 min). Product containing fractions were pooled and acetonitrile was removed under reduced pressure. The solution was neutralized by addition of pH 7.4 buffer (phosphate, 0.5 M). The solution was concentrated and the buffer was exchanged with 10 mM pH 7.0 phosphate containing 46 g/l mannitol by ultrafiltration (Vivaspin centrifugal concentrator, PES membrane with 10 kDa cut off) to obtain 8.5 mL of the final solution of 25. UPLC and SEC analysis revealed a uniform material. The concentration was determined by quantification of treprostinil content after basic hydrolysis: L aliquots were treated with 35 L 0.5 M NaOH. After 30 min incubation at RT 35 L acetic acid was added. The trerostinil content was determined by UPLC by using a treprostinil calibration curve. A total treprostinil content of 2.0 mg was found, corresponding to 30 mg 25. Yield: 50% based on PEG starting material.

Example 29

Treprostinil Release Kinetics of TransCon PEG Linker Treprostinil Compound 25

[0855] Treprostinil release kinetics from 25 was determined as described in example 15 and compared with the results obtained from compound 13. No difference in half life time (5 d) was observed.

Example 30

Treprostinil Release Kinetics of TransCon PEG Linker Treprostinil Compound 13 in Rat Plasma

[0856] 150 l of a pH 7.5 HEPES buffer (1 M HEPES, 3 mM EDTA) were mixed with 1.2 mL rat plasma (WISTAR rat Li heparin plasma, Innovative Research, Novi, Mich., USA). 150 l of a of TransCon PEG linker treprostinil 13 solution (0.15 mg 13 in 1.5 mL 10 mM phosphate 46 g/l mannitol buffer pH 7.0) were added. A pH of 7.4 of the mixture was confirmed by means of a pH electrode. Mixture was incubated at 37 C. At given time points 100 l aliquots were withdrawn. 100 l aliquots were analyzed for released and total treprostinil content.

[0857] For analysis of released treprostinil, 100 l aliquots were spiked with 20 l internal standard (2.8 g/mL tolbutamide in methanol/water 1/1 (v/v)) and transferred to a Ostro 96 well plate (Waters GmbH, Eschborn, Germany). Plasma proteins were precipitated by addition of three volumes of pre-cooled (0-5 C.) acetonitrile containing 1% formic acid. Positive pressure was applied (4 bar, Waters Positive Pressure-96 Processor) and eluate was lyophilized. Lyophilizate was dissolved in 40 l of 10 mM ammonium formiate pH 4.0/acetonitrile 7/3 (v/v). Solution was centrifuged and supernatant was assayed for released treprostinil by UPLC-MS/MS.

[0858] For analysis of total treprostinil content (sum of released and carrier bound treprostinil), 100 l aliquots were spiked with 20 l internal standard (2.8 g/mL tolbutamide in methanol/water 1/1 (v/v)) and 50 l of 0.5 M LiOH were added. Mixture was incubated in a shaker for 2 h at room temperature. After addition 25 L 1 M HCl the mixture was transferred to a Ostro 96 well plate (Waters GmbH, Eschborn, Germany). Plasma proteins were precipitated by addition of three volumes of pre-cooled (0-5 C.) acetonitrile containing 1% formic acid. Positive pressure was applied (4 bar, Waters Positive Pressure-96 Processor) and eluate was lyophilized. Lyophilizate was dissolved in 100 l of 10 mM ammonium formiate pH 4.0/acetonitrile 7/3 (v/v). Solution was centrifuged and supernatant was assayed for total treprostinil content by UPLC-MS/MS.

[0859] UPLC-MS/MS method for determination of treprostinil content:

[0860] The quantification of plasma treprostinil concentrations were carried out using a Waters Acquity UPLC coupled to a Thermo LTQ Orbitrap Discovery mass spectrometer via an ESI probe and with Waters BEH C18 (502.1 mm I.D., 1.7 m particle size) as analytical column (mobile phase A: 10 mM ammonium formate pH 4.6, mobile phase B: methanol, T=22 C.). The gradient system comprised a linear gradient from 0.1% B to 95% B in 4 min, an isocratic washing phase with 95% B (0.5 min), and a reconditioning phase (2.4 min) with a flow rate of 0.25 mL/min. Detection of the ions was performed in the selected reaction monitoring (SRM, negative ionization) mode, monitoring the transition pairs at the m/z 389.2 precursor ions to the m/z 331.2 product ions for treprostinil and m/z 269.1 precursor ions to the m/z 170.0 product ions for the internal standard (IS) tolbutamide.

[0861] The calibration curve was acquired by plotting the extracted peak area ratio area.sub.treprostinil/area.sub.tolbutamide against the nominal trepostinil concentrations of calibration standards. The results were fitted to linear regression using standard software.

[0862] The extracted peak area ratio area.sub.treprostinil/area.sub.tolbutamide of the quantification experiments at different time points were used to calculate the treprostinil content according to the calibration curve.

[0863] Treprostinil release at time points was expressed as % treprostinil release compared to total treprostinil content (see FIG. 1). By using a first order kinetics fit a half life of 4.5 d for the release kinetics of treprostinil from 25 in buffered rat plasma at 37 C. was obtained, which is in good agreement to release kinetics in pH 7.4 buffer at 37 C. (Example 15).

Example 31

PK of PEG Treprostinil Conjugate 25 in Monkeys

[0864] 25 (3 mg/mL in 10 mM pH 7.0 phosphate, 46 g/L mannitol) was given at a dose level of 0.5 mg/kg as a single dose by sc and iv injection in three male cynomolgus monkeys each. Blood samples were collected at given time points over two weeks. The plasma was assayed for PEG content and total treprostinil content (sum of released and carrier bound treprostinil). Due to the fast elimination of free treprostinil compared to carrier bound treprostinil, treprostinil plasma levels reflect the presence of treprostinil conjugate rather than free treprostinil levels.

[0865] For the analysis of total treprostinil content, 100 L plasma samples and treprostinil standards in cynomolgus monkey plasma were spiked with 20 L internal standard (2.8 g/mL tolbutamide in methanol/water 1/1 (v/v)) and 50 l of 0.5 M LiOH were added. The mixture was incubated in a shaker at RT for 2.5 h. After addition of 25 L 1 M HCl the mixture was transferred to an Ostro 96 well plate (Waters GmbH, Eschborn, Germany). Plasma proteins were precipitated by addition of three volumes of pre-cooled (0-5 C.) acetonitrile containing

[0866] 1% formic acid. Positive pressure was applied (4 bar, Waters Positive Pressure-96 Processor) and the eluate was lyophilized. The lyophilizate was dissolved in 100 l of 10 mM ammonium formiate pH 4.0/acetonitrile 7/3 (v/v). The solution was centrifuged and the supernatant was assayed for total treprostinil content by UPLC-MS/MS.

[0867] UPLC-MS/MS method for determination of treprostinil content:

[0868] The quantification of plasma treprostinil concentrations were carried out using a Waters Acquity UPLC coupled to a Thermo LTQ Orbitrap Discovery mass spectrometer via an ESI probe and with a Waters BEH C18 (502.1 mm I.D., 1.7 m particle size) as analytical column (mobile phase A: 10 mM ammonium formate pH 4.6, mobile phase B: methanol, T=22 C.). The gradient system comprised a linear gradient from 0.1% B to 95% B in 4 min, an isocratic washing phase with 95% B (0.5 min), and a reconditioning phase (2.4 min) with a flow rate of 0.25 mL/min. Detection of the ions was performed in the selected reaction monitoring (SRM, negative ionization) mode, monitoring the transition pairs at the m/z 389.2 precursor ions to the m/z 331.2 product ions for treprostinil and m/z 269.1 precursor ions to the m/z 170.0 product ions for the internal standard (IS) tolbutamide.

[0869] The calibration curve was acquired by plotting the extracted peak area ratio area.sub.treprostinil/area.sub.tolbutamide against the nominal trepostinil concentrations of calibration standards which were prepared in cynomolgus monkey plasma. The results were fitted to a linear regression using standard software.

[0870] The extracted peak area ratio area.sub.treprostinil/area.sub.tolbutamide of the quantification experiments at different time points were used to calculate the treprostinil content according to the calibration curve.

[0871] For analysis of total PEG content, plasma samples underwent basic preincubation in order to generate a uniform PEG material from 25. This was based on the fact that after injection of 25 different treprostinil carrier species are generated due to the sequential release of 4 treprostinils from carrier molecule over time.

[0872] 50 l plasma samples and 25 PEG treprostinil conjugate standards in cynomolgus monkey plasma were diluted with 50 L of 200 mM HEPES solution (pH 7.5) and 50 l of 0.5 M LiOH were added. Mixture was incubated in a shaker for 2 h at room temperature. After addition of 50 L 1 M HCl the mixture was assayed using the high sensitivity PEG ELISA kit P-0003 from Life Diagnostics Inc. West Chester, Pa., USA, according to the manufacturer's instructions.

[0873] The calibration curve was acquired by plotting the absorption values at 450 nm against the nominal PEG concentrations of calibration standards. The results were fitted to a sigmoidal curve using standard software.

[0874] The absorption values at 450 nm of the quantification experiments at different time points were used to calculate the PEG content according to the calibration curve.

[0875] Result: Total treprostinil content analysis after a single dose sc injection of 25 reveals a prolonged duration of circulation of treprostinil conjugate for more than two weeks in monkeys. (FIG. 2)

[0876] Single dose iv injection of 25 and plasma analysis for total treprostinil content revealed similar duration of circulation (FIG. 3). An apparent first order total treprostinil elimination half life time of 2.9 days (rate constant k.sub.apparent: 0.239 d.sup.1) was obtained by using standard software.

[0877] In contrast plasma analysis for PEG carrier content revealed a much slower elimination (FIG. 3). By fitting as a first order kinetics using standard software an PEG carrier elimination half life time of 6.6 d (rate constant k.sub.PEGelim: 0.105 d.sup.1) was obtained.

[0878] Equal elimination rate constant of different treprostinil carrier species e.g. generated by sequential linker hydrolysis/release of one to four treprostinils from PEG carrier are assumed.

[0879] The apparent faster elimination half life time of total treprostinil compared to PEG carrier is based on the combination of elimination of the PEG carrier and treprostinil release by linker hydrolysis. From the determined rate constant values of k.sub.apparent (0.239 d.sup.1) and k.sub.PEGelim (0.105 d.sup.1) a first order treprostinil release by linker cleavage rate constant k.sub.linker can be calculated:

exp(k.sub.apparent t)=exp(k.sub.PEGelim t)*exp(k.sub.linker t)=exp(t[k.sub.PEGelim+k.sub.linker])

[0880] After logmarithizing and rearrangement k.sub.linker can be calculated according to:

k.sub.linker=k.sub.apparentk.sub.PEGelim; k.sub.linker=0.239 d.sup.10.105 d.sup.1=0.134 d.sup.1

[0881] By help of the equation t.sub.half life time=ln(2)/k the half life time of treprostinil release by linker hydrolysis was calculated as 5.2 d, which is in good agreement with the 5 d linker treprostinil release half life time determined in vitro.

Example 32

PK of PEG Treprostinil Conjugate 25 and Free Treprostinil in Rats

[0882] 25 (3 mg/mL buffer (10 mM pH 7.0 phosphate, 46 g/L mannitol)) was injected at a dose level of 5.5 mg/kg as a single dose in male Wistar rats each. Three animals received sc injection and three animals received iv injection. Blood samples were collected at given time points over two weeks. Blood samples (250 L) were given directly into collection tubes containing 50 L acidic citrate buffer (0.5 M sodium citrate, pH 4.0). The plasma was assayed for free treprostinil content and total treprostinil content (sum of free and carrier bound treprostinil).

[0883] For the analysis of free treprostinil, 50 L plasma were thawed on ice and mixed with 5 l acidic citrate buffer and 10 L internal standard (0.28 g/mL tolbutamide in methanol/water 1/1 (v/v)). Samples were transferred to Ostro 96 well plates (Waters GmbH, Eschborn, Germany), and plasma proteins were precipitated by rapid addition of 400 L pre-cooled (0-5 C.) acetonitrile containing 1% formic acid. Positive pressure was applied (4 bar, Waters Positive Pressure-96 Processor) and the eluates were collected. Subsequently, the well plates were rinsed two times with 100 L ice-cold acetonitrile containing 1 vol. % formic acid. The eluates were transferred into 2 mL vials, placed into an Eppendorf Thermomixer (at 10 C.) and eluates were concentrated under a soft stream of nitrogen over 45 min to a final volume of 60-80 L. 30 l solvent mixture (10 mM aqueous ammonium formiate adjusted to pH 4.0 with formic acid/acetonitrile 7/3 (v/v)) were added to each vial and the solutions were analyzed by UHPLC-MS/MS.

[0884] For preparation of calibration standards, blank plasma samples were spiked with treprostinil and treated likewise.

[0885] UHPLC-MS/MS method for determination of free treprostinil content:

[0886] The quantification of plasma treprostinil concentrations were carried out using an Agilent 1290 UHPLC coupled to an Agilent Triplequad 6460 system (MassHunter Xcalibur software) in the ES-mode. As analytical column a Waters BEH C18 was used (502.1 mm I.D., 1.7 m particle size. Mobile phase A: 10 mM ammonium formate pH 5.7, mobile phase B: methanol. The gradient system comprised a linear gradient from 35% B to 99% B in 8 min, an isocratic washing phase with 99% B (0.9 min), and a reconditioning phase (3 min) with a flow rate of 0.200 mL/min (T=40 C.).

[0887] Detection of the ions was performed in the SRM mode, monitoring the transition pairs at the m/z 389.1 precursor ions to the m/z 331.1 product ions for treprostinil and m/z 269.0 precursor ions to the m/z 169.9 product ions for the internal standard (IS) tolbutamide. The calibration curve was acquired by plotting the extracted peak area ratio area treprostinil/area tolbutamide against the nominal trepostinil concentrations of calibration standards. The results were fitted to a linear regression using standard software.

[0888] The extracted peak area ratio area treprostinil/area tolbutamide of the quantification experiments at different time points were used to calculate the treprostinil content according to the calibration curve.

[0889] Total treprostinil plasma content was determined as given in Example 31.

[0890] Result: Free and total treprostinil content analysis after a single dose sc injection of 25 reveals a prolonged circulation of treprostinil conjugate and a burstless release of free treprostinil for more than four days in rats after iv (FIG. 4) or sc injection (FIG. 5).

Example 33

[0891] Isolation of Intermediate 6a by Enantioseparation of Racemic Mixture 6a/6b

[0892] Racemic mixture 6a/6b (107 g) was separated on a Chiralpak IA column (25076 mm, 20 m, flow rate 270 mL/min) using acetonitrile/acetic acid 1000/1 (v/v) as eluent. Combined eluates of second eluting enantiomer (6a) were mixed with 5 vol % water and evaporated under reduced pressure. The residue was taken up in DCM (500 mL) and extracted with 0.1 M HCl (500 mL, 2) and brine (500 mL). The organic phase was dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure.

[0893] Yield: 28.9 g (27%).

[0894] MS: m/z 552.2=[M+Na].sup.+ (MW calculated=.sup.5290.7.sup.5 g/mol).

[0895] Enantiomeric ratio of 6a/6b as determined by Chiralpak IC column (4.5250 mm, 5 m, eluent acetonitrile/acetic acid 1000/1 (v/v), flow rate 1 mL/min, 230 nm): 97.5/2.5

Example 34

Improved Synthesis of Treprostinil Linker Thiol 24a

[0896] ##STR00134##

[0897] Dmob protected treprostinil 8 (200 mg, 0.370 mmol), carboxylic acid 23 (294 mg, 0.555 mmol), EDC.HCl (248 mg, 1.295 mmol) and DMAP (158 mg, 1.295 mmol) were dissolved in DCM (2.9 mL, anhydrous, mol. sieve). The mixture was stirred at RT for 1 d. Ethyl acetate was added and the organic layer was washed with 0.1 N aqueous HCl (3) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and the solvent was removed under reduced pressure.

[0898] The residue was dissolved in HFIP (8 mL). After addition of TFA (200 L) and TES (200 L) the mixture was stirred for 30 min at RT. The solution was extracted with heptane (16 mL, 6) and diluted with DCM (16 mL). Solution was extracted with water (16 mL, 3). Combined water phases were back extracted with DCM (8 mL). The combined DCM phases were evaporated under reduced pressure.

[0899] UPLC analysis revealed a 9/1 ratio of regioisomers 24a and 24b (column: Kinetex 1002.1 mm, 1.7 m XB-C18 silica, pore size 100 , Phenomonex Ltd, Aschaffenburg, Germany; flow rate 0.25 mL/min; solvent A: water+0.05% TFA (v/v), solvent B: acetonitrile+0.04% TFA; gradient: 30-58% B (10 min), 58% B isocratic (10 min), 58-80% B (5 min), 80-99% (5 min), wavelength 280 nm).

[0900] The residue was taken up in acetonitrile/water and 24a was isolated by RP-HPLC (solvent A: H.sub.2O+0.01% HCl, solvent B: MeCN+0.01% HCl, gradient: 57-62% B over 15 min). Mixed fractions were subjected to repurification. Fractions containing pure 24a were combined and lyophilized.

[0901] Yield 24a: 98 mg (39%)

[0902] MS: m/z 660.3=[M+H].sup.+ (MW calculated=659.9 g/mol).

Abbreviations

[0903] AcOH acetic acid [0904] AIB 2-Aminoisobutyric acid [0905] BnBr benzylbromide [0906] Boc tert-Butoxycarbonyl- [0907] BSA N,O-Bis-(trimethylsilyl)-acetamide [0908] COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate

Abbreviations

[0909] d day [0910] DIPEA diisopropylethylamine [0911] DCM dichloromethane [0912] DMAP 4-(Dimethylamino)pyridine [0913] DMF N,N-Dimethylformamide [0914] Dmob 2,4-dimethoxybenzyl [0915] DMSO dimethyl sulfoxide [0916] Dpr 2,3-Diaminopropionic acid [0917] EDC N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide [0918] EDTA ethylenediamine tetraacetic acid disodium salt dihydrate [0919] EtOAc ethyl acetate [0920] eq equivalent [0921] h Hour [0922] HFIP 1,1,1,3,3,3-Hexafluoroisopropanol [0923] HPLC high performance liquid chromatography [0924] LC/MS mass spectrometry-coupled liquid chromatography [0925] Mal maleimido [0926] MeOH methanol [0927] MeCN acetonitrile [0928] min Minute [0929] Mmt 4-Methoxytriphenylmethyl [0930] mol. Molecular [0931] m/z Mass/charge [0932] NaOH Sodium hydroxide [0933] NHS N-hydroxysuccinimide [0934] PEG Polyethylene glycol [0935] Pfp Pentafluorophenyl [0936] PyBOB Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [0937] PP polypropylene

Abbreviations

[0938] RT room temperature [0939] RP reversed phase [0940] sat. saturated [0941] soln. solution [0942] T temperature [0943] T3P propyl phosphonic anhydride [0944] TCP 2-Chlorotrityl chloride resin [0945] TES Triethylsilane [0946] Trt Trityl [0947] Tmob 2,4,6-trimethoxybenzyl [0948] TMS trimethylsilyl [0949] TransCon transiently conjugated [0950] THF tetrahydrofuran [0951] TFA trifluoroacetic acid [0952] UPLC ultra performance liquid chromatography [0953] UV ultra violet

[0954] While this invention has been described in conjunction with the specific embodiments outlined above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the preferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the inventions as defined in the following claims.