CNP PRODRUGS WITH LARGE CARRIER MOIETIES

Abstract

The present invention relates to a CNP prodrug or a pharmaceutically acceptable salt thereof comprising a CNP moiety -D; and a carrier moiety —Z that is conjugated through a moiety -L.sup.2- to a reversible prodrug linker moiety -L.sup.1-, which reversible prodrug linker moiety -L.sup.1- is covalently and reversibly conjugated to -D; wherein -L.sup.2- is a chemical bond or a spacer; and —Z is a polymer having a molecular weight of at least 10 kDa. It further relates to pharmaceutical compositions comprising the CNP prodrug or a pharmaceutically acceptable salt thereof, their use as a medicament and to methods of treatment.

Claims

1. A CNP prodrug or a pharmaceutically acceptable salt thereof comprising a CNP moiety -D; and a carrier moiety —Z that is conjugated through a moiety -L.sup.2- to a reversible prodrug linker moiety -L.sub.1-, which reversible prodrug linker moiety -L.sub.1- is covalently and reversibly conjugated to -D; wherein -L.sup.2- is a chemical bond or a spacer; and —Z is a polymer having a molecular weight of at least 10 kDa.

2. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1, wherein —Z is a linear, branched, multi-arm or dendritic polymeric moiety.

3. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1, wherein —Z is a branched polymer.

4. The CNP prodrug or a pharmaceutically acceptable salt thereof of claims 1, wherein —Z is a branched polymer having one, two, three, four, five or six branching points.

5. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 4, wherein each branching points is independently selected from the group consisting of —N<, —CH< and >C<.

6. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1, wherein —Z has a molecular weight ranging from and including 10 kDa to 80 kDa.

7. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1, wherein —Z comprises a moiety ##STR00190##

8. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1, wherein —Z comprises a moiety of formula (a) ##STR00191## wherein the dashed line indicates attachment to -L.sup.2- or to the remainder of —Z; BP.sup.a is a branching point selected from the group consisting of —N<, —CR< and >C<; —R is selected from the group consisting of —H and C.sub.1-6 alkyl; a is 0 if BP.sup.a is —N< or —CR< and a is 1 if BP.sup.a is >C<; —S.sup.a—, —S.sup.a′—, —S.sup.a″— and —S.sup.a′″— are independently of each other a chemical bond or are selected from the group consisting of C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein 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.1, which are the same or different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.2)—, —S(O).sub.2N(R.sup.2)—, —S(O)N(R.sup.2)—, —S(O).sub.2—, —S(O)—, —N(R.sup.2)S(O).sub.2N(R.sup.2a)—, —S—, —N(R.sup.2)—, —OC(OR.sup.2)(R.sup.2a)—, —N(R.sup.2)C(O)N(R.sup.2a)—, and —OC(O)N(R.sup.2)—; each -T- is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each -T- is independently optionally substituted with one or more —R.sup.1, which are the same or different; each —R.sup.1 is independently selected from the group consisting of halogen, —CN, oxo (═O), —COOR.sup.3, —OR.sup.3, —C(O)R.sup.3, —C(O)N(R.sup.3R.sup.3a), —S(O).sub.2N(R.sup.3R.sup.3a, —S(O)N(R.sup.3R.sup.3a), —S(O).sub.2R.sup.3, —S(O)R.sup.3, —N(R.sup.3)S(O).sub.2N(R.sup.3aR.sup.3b), —SR.sup.3, —N(R.sup.3R.sup.3a), —NO.sub.2, —OC(O)R.sup.3, —N(R.sup.3)C(O)R.sup.3a, —N(R.sup.3)S(O).sub.2R.sup.3a, —N(R.sup.3)S(O)R.sup.3a, —N(R.sup.3)C(O)OR.sup.3a, —N(R.sup.3)C(O)N(R.sup.3aR.sup.3b), —OC(O)N(R.sup.3R.sup.3a), 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; each —R.sup.2, —R.sup.2a, —R.sup.3, —R.sup.3a and —R.sup.3b is independently selected from the group consisting of —H, and C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and —P.sup.a′, —P.sup.a″ and —P.sup.a′″ are independently a polymeric moiety.

9. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1, wherein —Z is of formula (d)
—Z.sup.b—Z.sup.a  (d), wherein the dashed line indicates attachment to -L.sup.2-; —Z.sup.b— is selected from the group consisting of C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein 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.1, which are the same or different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.2)—, —S(O).sub.2N(R.sup.2)—, —S(O)N(R.sup.2)—, —S(O).sub.2—, —S(O)—, —N(R.sup.2)S(O).sub.2N(R.sup.2a)—, —S—, —N(R.sup.2)—, —OC(OR.sup.2)(R.sup.2a)—, —N(R.sup.2)C(O)N(R.sup.2a)—, and —OC(O)N(R.sup.2)—; each -T- is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each -T- is independently optionally substituted with one or more —R.sup.1, which are the same or different; each —R.sup.1 is independently selected from the group consisting of halogen, —CN, oxo (═O), —COOR.sup.3, —OR.sup.3, —C(O)R.sup.3, —C(O)N(R.sup.3R.sup.3a), —S(O).sub.2N(R.sup.3R.sup.3a), —S(O)N(R.sup.3R.sup.3a), —S(O).sub.2R.sup.3, —S(O)R.sup.3, —N(R.sup.3)S(O).sub.2N(R.sup.3aR.sup.3b), —SR.sup.3, —N(R.sup.3R.sup.3a), —NO.sub.2, —OC(O)R.sup.3, —N(R.sup.3)C(O)R.sup.3a, —N(R.sup.3)S(O).sub.2R.sup.3a, —N(R.sup.3)S(O)R.sup.3a, —N(R.sup.3)C(O)OR.sup.3a, —N(R.sup.3)C(O)N(R.sup.3aR.sup.3b), —OC(O)N(R.sup.3R.sup.3a), 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; each —R.sup.2, —R.sup.2a, —R.sup.3, —R.sup.3a and —R.sup.3b is independently selected from the group consisting of —H, and C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and —Z.sup.a is ##STR00192## wherein BP.sup.a, —S.sup.a, —S.sup.a′—, —S.sup.a″—, —S.sup.a′″—, —P.sup.a′, —P.sup.a″ and —P.sup.a′″ and a are used as defined in claim 5.

10. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1, wherein -D has the sequence of SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:30.

11. The CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1, wherein -L.sub.1- is conjugated to the side chain of an amino acid residue of the ring moiety of -D or to the backbone of the ring moiety of -D.

12. A pharmaceutical composition comprising at least one CNP prodrug or a pharmaceutically acceptable salt thereof of claim 1 and at least one excipient.

13-17. (canceled)

18. A method of treating, controlling, delaying or preventing in a mammalian patient in need of the treatment of one or more diseases which can be treated with CNP, comprising the step of administering to said patient in need thereof a therapeutically effective amount of the CNP prodrug or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, wherein the one or more diseases which can be treated with CNP is selected from the group consisting of achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, camptomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia punctata, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, spondyloepimetaphyseal dysplasia, neurofibromatosis, Legius syndrome, LEOPARD syndrome, Noonan syndrome, hereditary gingival fibromatosis, neurofibromatosis type 1, Legius syndrome, cardiofaciocutaneous syndrome, Costello syndrome, SHOX deficiency, idiopathic short stature, growth hormone deficiency, osteoarthritis, cleidocranial dysostosis, craniosynostosis, dactyly, brachydactyly, camptodactyly, polydactyly, syndactyly, dyssegmental dysplasia, enchondromatosis, fibrous dysplasia, hereditary multiple exostoses, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Marfan syndrome, McCune-Albright syndrome, osteopetrosis and osteopoikilosis.

20. The method of claim 18, wherein the one or more diseases which can be treated with CNP is achondroplasia.

Description

BRIEF DESCRIPTION OF THE FIGURE

[1400] FIG. 1: Structure of CNP according to SEQ ID NO:1.

EXAMPLES

[1401] Materials and Methods

[1402] CNP SEQ ID No:1 was obtained from Bachem AG, Bubendorf, Switzerland (CNP-22, human, catalogue no. H-1296). CNP-34 SEQ ID No:40 and CNP-38 SEQ ID No:24 were obtained from CASLO ApS, Kongens Lyngby, Denmark.

[1403] Side chain protected CNP-38 on CTC resin having Boc protected N-terminus and ivDde protected side chain of Lys26 (synthesized by Fmoc-strategy) was obtained from CASLO ApS, Kongens Lyngby, Denmark.

[1404] Side chain protected CNP-34 on TCP Tentagel resin having Boc protected N-terminus and ivDde protected side chain of either Lys12, Lys16 or Lys22 (synthesized by Fmoc-strategy) was obtained from Peptide Specialty Laboratories GmbH, Heidelberg, Germany. Side chain protected CNP-38 on TCP tentagel resin having free N-terminus (synthesized by Fmoc-strategy) was obtained from Peptide Specialty Laboratories GmbH, Heidelberg, Germany.

[1405] Methoxy PEG amine 5 kDa was obtained from Rapp Polymere GmbH, Tuebingen, Germany. All other PEGs used in this work were acquired from NOF Europe N.V., Grobbendonk, Belgium.

[1406] FmocN-Me-Asp(OtBu)-OH was obtained from Bachem AG, Bubendorf, Switzerland. S-Trityl-6-mercaptohexanoic acid was purchased from Polypeptide, Strasbourg, France. HATU was obtained from Merck Biosciences GmbH, Schwalbach/Ts, Germany.

[1407] 2,4-Dimethylbenzyl alcohol was obtained from abcr GmbH, Karlsruhe, Germany.

[1408] Fmoc-N-Me-Asp(OBn)-OH was obtained from Peptide International Inc., Louisville, Ky., USA.

[1409] Neutral Endopeptidase (NEP) was obtained from Enzo Life Sciences GmbH, Lorrach,

[1410] Germany.

[1411] All other chemicals and reagents were purchased from Sigma Aldrich GmbH, Taufkirchen, Germany.

[1412] Syringes equipped with polyethylene frits (MultiSynTech GmbH, Witten, Germany) were used as reaction vessels or for washing steps for peptide resins.

[1413] General Procedure for the Removal of ivDde Protecting Group from Side Chain Protected CNPs on Resin

[1414] The resin was pre-swollen in DMF for 30 min and the solvent was discarded. The ivDde group was removed by incubating the resin with DMF/hydrazine hydrate 4/1 (v/v, 2.5 mL/g resin) for 8×15 min. For each step fresh DMF/hydrazine hydrate solution was used. Finally, the resin was washed with DMF (10×), DCM (10×) and dried in vacuo.

[1415] RP-HPLC Purification:

[1416] For preparative RP-HPLC a Waters 600 controller and a 2487 Dual Absorbance Detector was used, equipped with the following columns: Waters XBridgem BEH300 Prep C18 5 μm, 150×10 mm, flow rate 6 mL/min, or Waters XBridgem BEH300 Prep C18 10 μm, 150×30 mm, flow rate 40 mL/min. Linear gradients of solvent system A (water containing 0.1% TFA v/v or 0.01% conc. HCl v/v) and solvent system B (acetonitrile containing 0.1% TFA v/v or 0.01% conc. HCl v/v) were used.

[1417] HPLC fractions containing product were pooled and lyophilized if not stated otherwise.

[1418] Flash Chromatography

[1419] Flash chromatography purifications were performed on an Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges and n-heptane and ethyl acetate as eluents. Products were detected at 254 nm.

[1420] Analytical Methods

[1421] Analytical ultra-performance LC (UPLC)-MS was performed on a Waters Acquity system equipped with a Waters BEH.sub.300 C.sub.18 column (2.1×50 mm, 1.7 μm particle size, flow: 0.25 mL/min; solvent A: water containing 0.04% TFA (v/v), solvent B: acetonitrile containing 0.05% TFA (v/v)) coupled to a LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific or coupled to a Waters Micromass ZQ.

[1422] Size exclusion chromatography (SEC) was performed using an Amersham Bioscience AEKTAbasic system equipped with a Superdex 200 5/150 GL column (Amersham Bioscience/GE Healthcare) equipped with a 0.45 μm inlet filter, if not stated otherwise. 20 mM sodium phosphate, 140 mM NaCl, pH 7.4, was used as mobile phase.

[1423] Due to the reversible nature of the attachment of -L.sup.1- to -D measurements for NEP-stability and receptor affinity were made using stable analogs of the CNP prodrugs of the present invention, i.e. they were made using similar structures to those of the CNP prodrugs of the present invention which instead of a reversible attachment of —Z to -D have a stable attachment.

[1424] This was necessary, because the CNP prodrugs of the present invention would release CNP in the course of the experiment and said released CNP would have influenced the result.

[1425] Quantification of Plasma Total CNP-38 Concentrations

[1426] Plasma total CNP-38 concentrations (conjugated and released CNP-38) were determined by quantification of the N-terminal signature peptide (sequence: LQEHPNAR; residues 1-8 of SEQ ID NO:24) and C-terminal signature peptide (sequence: IGSMSGLGC; residues 30-38 of SEQ ID NO:24) after tryptic digestion.

[1427] LC-MS analysis was carried out by using an Agilent 1290 UPLC coupled to an Agilent 6550 iFunnel Q-TOF mass spectrometer via an ESI probe. Chromatography was performed on a Waters Acquity BEH300 C18 analytical column (50×2.1 mm I.D., 1.7 μm particle size) with pre-filter at a flow rate of 0.25 mL/min (T=25° C.). Water (UPLC grade) containing 0.2% formic acid (v/v) was used as mobile phase A and acetonitrile (UPLC grade) with 0.2% formic acid as mobile phase B. The gradient system comprised a short isocratic step at the initial parameters of 0.1% B for 3.0 min followed by a linear increase from 0.1% B to 16% B in 17 min. Mass analysis was performed in the single ion monitoring (SIM) mode, monitoring the ions m/z 482.75 [M+2H].sup.2+ (N-terminal) and m/z 824.36 [M+H].sup.1+ (C-terminal). As internal standard deuterated CNP-38 peptide was used.

[1428] Calibration standards of CNP-38 conjugate in blank plasma were prepared as follows: The thawed Li-heparin cynomolgous plasma was first homogenized, then centrifuged for 5 minutes. The CNP-38 conjugate formulation was diluted to a working solution of 10 μg/mL (conjugate CNP-38 eq.) in DMSO and spiked into blank plasma at concentrations between 9.3 ng/100 μL (conjugate CNP-38 eq.) and 139.5 ng/100 μL (conjugate CNP-38 eq.). These solutions were used for the generation of a calibration curve. Calibration curves were weighted 1/x.sup.2 for both signature peptides (N- and C-Terminal). For quality control, three quality control samples were prepared accordingly with contents of 116.2 ng/100 μL (high QC, conjugate CNP-38 eq.), 69.75 ng/100 μL (mid QC, conjugate CNP-38 eq.) and 23.25 ng/100 μL (low QC, conjugate CNP-38 eq.).

[1429] For sample preparation, protein precipitation was carried out by addition of 300 μL of precooled (0° C.) methanol to 100 μL of the plasma sample. 200 μL of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 35° C.). 100 μL of reconstitution solvent (Thermo digestion buffer, order number 60109-101, Thermo Fisher Scientific GmbH, Dreieich, Germany) were used to dissolve the residue. 20 μg of trypsin (order number V5111, Promega GmbH, Mannheim, Germany) were dissolved in 20 μL of 10 mM acetic acid. 2 μL of the trypsin solution were added to each cavity.

[1430] After 4 hours incubation at 37° C. (water bath), 5 μL of a 0.5 M TCEP solution were added to each cavity and incubated again for 5 min at 96° C. After the samples had cooled to room temperature, 3 μL acetonitrile were added. The eluates were transferred into vials. 10 μL were injected into the UPLC-MS system.

Example 1

[1431] Synthesis of Linker Reagent 1f

[1432] Linker reagent 1f was synthesized according to the following scheme:

##STR00161##

[1433] To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48 mmol) and NaCNBH.sub.3 (819 mg, 12.63 mmol) in MeOH (20 mL) was added 2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) portion wise. The mixture was stirred at rt for 90 min, acidified with 3 M HCl (4 mL) and stirred further 15 min. The reaction mixture was added to saturated NaHCO.sub.3 solution (200 mL) and extracted 5× with CH.sub.2Cl.sub.2. The combined organic phases were dried over Na.sub.2SO.sub.4 and the solvents were evaporated under reduced pressure. The resulting N-methyl-N-Boc-N′-Tmob-ethylenediamine 1a was dried in vacuo and used in the next reaction step without further purification.

[1434] Yield: 3.76 g (11.48 mmol, 89% purity, 1a: double Tmob protected product=8:1)

[1435] MS: m/z 355.22=[M+H].sup.+, (calculated monoisotopic mass=354.21).

[1436] To a solution of 1a (2 g, 5.65 mmol) in CH.sub.2Cl.sub.2 (24 mL) COMU (4.84 g, 11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol) and 2,4,6-collidine (2.65 mL, 20.34 mmol) were added. The reaction mixture was stirred for 3 h at rt, diluted with CH.sub.2Cl.sub.2 (250 mL) and washed 3× with 0.1 M H.sub.2SO.sub.4 (100 mL) and 3× with brine (100 mL). The aqueous phases were re-extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtrated and the residue concentrated to a volume of 24 mL. 1b was purified using flash chromatography.

[1437] Yield: 5.31 g (148%, 6.66 mmol)

[1438] MS: m/z 796.38=[M+H].sup.+, (calculated monoisotopic mass=795.37).

[1439] To a solution of 1b (5.31 g, max. 4.52 mmol) in THF (60 mL) DBU (1.8 mL, 3% v/v) was added. The solution was stirred for 12 min at rt, diluted with CH.sub.2Cl.sub.2 (400 mL) and washed 3× with 0.1 M H.sub.2SO.sub.4 (150 mL) and 3× with brine (150 mL). The aqueous phases were re-extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and filtrated. 1c was isolated upon evaporation of the solvent and used in the next reaction without further purification.

[1440] MS: m/z 574.31=[M+H].sup.+, (calculated monoisotopic mass=573.30).

[1441] 1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile (26 mL) and COMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid (2.12 g, 5.42 mmol) and 2,4,6-collidine (2.35 mL, 18.08 mmol) were added. The reaction mixture was stirred for 4 h at rt, diluted with CH.sub.2Cl.sub.2 (400 mL) and washed 3× with 0.1 M H.sub.2SO.sub.4 (100 mL) and 3× with brine (100 mL). The aqueous phases were re-extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtrated and 1d was isolated upon evaporation of the solvent. Product 1d was purified using flash chromatography.

[1442] Yield: 2.63 g (62%, 94% purity)

[1443] MS: m/z 856.41=[M+H].sup.+, (calculated monoisotopic mass=855.41).

[1444] To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H.sub.2O (11 mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture was stirred for 70 min at rt. The mixture was diluted with CH.sub.2Cl.sub.2 (200 mL) and washed 3× with 0.1 M H.sub.2SO.sub.4 (50 mL) and 3× with brine (50 mL). The aqueous phases were re-extracted with CH.sub.2Cl.sub.2 (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtrated and 1e was isolated upon evaporation of the solvent. 1e was purified using flash chromatography.

[1445] Yield: 2.1 g (88%)

[1446] MS: m/z 878.4=[M+Na].sup.+, (calculated monoisotopic mass=855.40).

[1447] To a solution of 1e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) were added DCC (123 mg, 0.59 mmol), and a catalytic amount of DMAP. After 5 min N-hydroxy-succinimide (114 mg, 0.99 mmol) was added and the reaction mixture was stirred at rt for 1 h. The reaction mixture was filtered, the solvent was removed in vacuo and the residue was taken up in 90% acetonitrile plus 0.1% TFA (3.4 mL). The crude mixture was purified by RP-HPLC.

[1448] Product fractions were neutralized with 0.5 M pH 7.4 phosphate buffer and concentrated. The remaining aqueous phase was extracted with DCM and 1f was isolated upon evaporation of the solvent.

[1449] Yield: 154 mg (81%)

[1450] MS: m/z 953.4=[M+H].sup.+, (calculated monoisotopic mass=952.43).

Example 2

[1451] Synthesis of N.sup.εK4/εK10-CNP mono-linker thiol 2, N.sup.εK4-CNP mono-linker thiol 2c and N.sup.εK10-CNP mono-linker thiol 2d

##STR00162##

[1452] N.sup.εK4/εK10-CNP mono-linker thiol (mixture of regioisomers with linker conjugated at side chain amino group of Lys4 or Lys10) 2 is prepared by dissolving CNP-22 (5.2 μmol) in 0.6 mL DMSO. 0.15 mL 0.375 M borate buffer, adjusted to pH 8.5 with tetrabutyl-ammoniumhydroxide hydrate, 60 μL DIPEA and 1f (6.1 mg, 7.1 μmol) in 0.34 mL of DMSO are added and the mixture is stirred for 30 min at rt. Reaction mixture is diluted with 2 mL acetonitrile/water 1/1 (v/v) and 200 μL AcOH and the protected N.sup.εK4/εK10-CNP mono-linker conjugate is isolated from the reaction mixture by RP-HPLC.

[1453] Optimized RP-HPLC gradients can be used for isolation of N.sup.εK4-CNP mono-linker thiol 2a and N.sup.εK10-CNP mono-linker thiol 2b.

[1454] Removal of protecting groups is affected by treatment of lyophilized product fractions with 0.6 mL of 90/10/2/2 (v/v/v/v) HFIP/TFA/TES/water for 1 h at rt. The deprotected N.sup.εK4/εK10-CNP mono-linker thiol 2 is purified by RP-HPLC. Identity and purity of the product is determined by ESI-LCMS.

[1455] Deprotected N.sup.εK4-CNP mono-linker thiol 2c and N.sup.εK10-CNP mono-linker thiol 2d can be obtained likewise from 2a and 2b, respectively.

Example 3

[1456] Synthesis of N.sup.αG1-CNP mono-linker thiol 3

##STR00163##

[1457] N.sup.αG1-CNP mono-linker thiol 3 is prepared by dissolving CNP-22 (5.2 μmol) in 0.6 mL DMSO. 0.25 mL 0.5 M phosphate buffer pH 7.4 and if (6.1 mg, 7.1 μmol) in 0.34 mL of DMSO are added and the mixture is stirred for several hours at rt. Reaction mixture is diluted with 2 mL acetonitrile/water 1/1 (v/v) and 200 μL AcOH and the protected N.sup.αG1-CNP mono-linker thiol is isolated from the reaction mixture by RP-HPLC.

[1458] Removal of protecting groups is affected by treatment of lyophilized product fractions with 0.6 mL of 90/10/2/2 (v/v/v/v) HFIP/TFA/TES/water for 1 h at rt. The deprotected N.sup.αG1-CNP mono-linker thiol 3 is purified by RP-HPLC. Identity and purity of the product is determined by ESI-LCMS.

Example 4

[1459] PEGylation of CNP Mono-Linker Thiols 2c, 2d and 3

##STR00164##

[1460] 1 μmol CNP mono-linker thiol 2c is dissolved in 0.5 mL acetonitrile/0.2 M succinate buffer pH 3.8 1/1 (v/v) 1.2 μmol of linear 40 kDa PEG-maleimide is added and the mixture is stirred at rt. The reaction is quenched by addition of 20 μL AcOH and CNP conjugate 4 is purified by preparative RP-HPLC.

[1461] CNP conjugates 5 and 6 are prepared likewise from 1 μmol CNP mono-linker thiols 2d and 3.

[1462] CNP content is determined by quantitative amino acid analysis after total hydrolysis under acidic conditions.

Example 5

[1463] Release Kinetics In Vitro

[1464] CNP conjugates 4, 5 and 6 are dissolved in 60 mM sodium phosphate, 3 mM EDTA, 0.01% Tween-20, pH 7.4 at a concentration of approximately 2 mg/mL and filtered sterile. Mixtures are incubated at 37° C. At time points aliquots are withdrawn and analysed by RP-HPLC and ESI-MS. UV-signals correlating to liberated CNP are integrated and plotted against incubation time.

[1465] Curve-fitting software is applied to estimate the corresponding halftime of release.

Example 6

[1466] Pharmacokinetics and cGMP Production in Rats

[1467] Equimolar doses of CNP-22, CNP conjugates 4, 5 or 6 are injected iv and sc in normal rats. Plasma CNP and cGMP levels over time are determined as described in the literature (U.S. Pat. No. 8,377,884 B2).

Example 7

[1468] Synthesis of Dmb Protected 6-Mercaptohexanoic Acid 7

[1469] Compound 7 was synthesized according to the following scheme:

##STR00165##

[1470] To a solution of 6-mercaptohexanoic acid (7.10 g, 47.90 mmol) in trifluoroacetic acid (20 mL), 2,4-dimethylbenzyl alcohol (13.5 g, 95.80 mmol) was added. The mixture was stirred at RT for 60 min and then the trifluoroacetic acid was removed in vacuo. The residue was dissolved in a mixture of 95.8 mL LiOH (3 M) and THF (81 mL) and stirred at rt for 60 min. The solvent was removed in vacuo and the aqueous residue was extracted 3× with EtOAc (200 mL). The combined organic phases were dried over MgSO.sub.4, and the solvent was removed in vacuo. 7 was purified by RP-HPLC.

[1471] Yield: 2.27 g (8.52 mmol, 18%)

[1472] MS: m/z 267.01=[M+H].sup.+, (calculated monoisotopic mass=266.13).

Example 8

[1473] Synthesis of Linker Reagent 8c

[1474] Linker reagent 8c was synthesized according to the following scheme:

##STR00166##

[1475] To a solution of 1b (21.6 g, 27.18 mmol) in isopropanol (401 mL) were added water (130 mL) and LiOH (3.90 g, 163.06 mmol). The reaction mixture was stirred for 3 h at rt, then it was diluted with toluene (300 mL) and washed 3× with 0.1 M HCl (200 mL). The combined aqueous phases were washed 3× with toluene (100 mL). The aqueous phase was basified with 4 M NaOH (4 mL) to a pH of 8.5 and extracted 8× with CH.sub.2Cl.sub.2 (200 mL). The combined CH.sub.2Cl.sub.2 phases were washed with brine (50 mL), dried over Na.sub.2SO.sub.4. 8b was isolated upon evaporation of the solvent and used in the next reaction without further purification.

[1476] Yield: 11.89 g (24.59 mmol, 90%)

[1477] MS: m/z 484.16=[M+H].sup.+, (calculated monoisotopic mass=483.26).

[1478] To a solution of 7 (293 mg, 1.10 mmol) and PyBOP (572 mg, 1.10 mmol) in THF (10 mL) was added DIEA (0.52 mL, 3.00 mmol) under a N.sub.2-atmosphere. The reaction mixture was stirred for 60 min at rt. A solution of 8b (484 mg, 1.00 mmol) in THF (2 mL) was added and the reaction was stirred for a further 60 min. The reaction was quenched with 2 M citric acid solution (10 mL) and the THF was removed in vacuo. The resulting aqueous phase was then extracted 2× with EtOAc (15 mL) and the combined organic layers were washed with water (10 mL) and brine (10 mL), and dried over MgSO.sub.4. The solvent was removed in vacuo and 8c was purified by RP HPLC.

[1479] Yield: 330 mg (0.451 mmol, 45%)

[1480] MS: m/z 732.34=[M+H].sup.+, (calculated monoisotopic mass=731.38).

Example 9

[1481] Synthesis of Linker Reagent 9

[1482] Linker reagent 9 was synthesized according to the following scheme:

##STR00167##

[1483] To a solution of 8b (2.00 g, 4.14 mmol) and Fmoc-C.sub.1 (1.07 g, 4.14 mmol) in dioxane (20 mL) was added 1 M Na.sub.2CO.sub.3 solution (20 mL). The reaction mixture was stirred for 40 min at rt. Water (100 mL) and diethyl ether (100 mL) were added and the aqueous phase was extracted 2× with diethyl ether (100 mL). The aqueous phase was acidified with conc. HCl until pH 1 and again extracted 3× with diethyl ether. The combined organic phases were dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo. 9 was used in the next step without further purification.

[1484] Yield: 2.63 g (3.73 mmol, 90%)

[1485] MS: m/z 728.32=[M+Na].sup.+, (calculated monoisotopic mass=705.33).

Example 10

[1486] Synthesis of Reversible Lys26 CNP-38 PEG2×20 kDa Conjugate 10f

[1487] Conjugate 10f was synthesized according to the following scheme:

##STR00168## ##STR00169##

[1488] 2.00 g (0.21 mmol) of side chain protected CNP-38 on CTC resin having Boc protected N-terminus and ivDde protected side chain of Lys26 was ivDde deprotected according to the procedure given in Materials and Methods to obtain 10a. A solution of linker reagent 8c (336 mg, 0.46 mmol), PyBOP (239 mg, 0.46 mmol) and DIEA (182 μL, 1.04 mmol) in DMF (5 mL) was incubated for 10 min at rt, then added to the resin 10a. The suspension was shaken for 2 h at rt. The resin was washed 10× with DMF (10 mL) and 10× with DCM (10 mL) and dried in vacuo for 15 min. Cleavage of the peptide from resin and removal of protecting groups was achieved by treatment of the resin with 15 mL pre-cooled (−18° C.) cleavage cocktail 68.5/10/10/5/3.5/1 (v/w/v/v/v/v) TFA/DTT/thioanisole/phenol/water/TIPS. The mixture was allowed to warm to rt and was agitated for 60 min. The resin was filtered off and crude 10c was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The combined HPLC fractions were used directly in the next step.

[1489] MS: m/z 1124.60=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1124.59).

[1490] To the combined HPLC fractions of 10c (250 mL) 40 mL of 0.5 M citric acid buffer (pH=5.00) and 7 mL of a 0.01 M solution of 2,2′-dithiobis(pyridine-N-oxide) solution in 1/1 (v/v) acetonitrile/water were added. After incubation for 5 min at rt the reaction was complete. The mixture was diluted with 500 mL water containing 0.1% TFA (v/v) and acidified with AcOH (20 mL) to a pH of approx. 2. 10d was purified by RP-HPLC.

[1491] Yield: 101 mg (17.3 μmol, 9%) CNP-38-linker-Dmb * 10 TFA

[1492] MS: m/z 1124.10=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1124.09).

[1493] Cleavage of the Dmb protecting group was achieved by adding 30 mL pre-cooled (−18° C.) cleavage cocktail 100/5/3/2/1 (v/v/w/v/v) TFA/MSA/DTT/water/thioanisole to 10d (101 mg, 17.3 μmol) and stirring for 3 h at 0° C. Crude 10e was precipitated in pre-cooled (−18° C.) diethyl ether. The precipitate was dissolved in water containing 0.1% TFA (v/v) and incubated for 10 min in order to hydrolyze any TFA esters. 10e was purified by RP-HPLC.

[1494] Product fractions were combined and freeze dried.

[1495] Yield: 46 mg (8.34 μmol, 48%) CNP-38-linker-thiol * 10 TFA

[1496] MS: m/z 1094.58=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1094.57).

[1497] To a solution of 10e (46 mg, 8.43 μmol) in 1.15 mL water containing 0.1% TFA (v/v) was added a solution of PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 870 mg, 21.75 μmol) in 4.35 mL water containing 0.1% TFA (v/v), followed by 0.5 M lactic acid buffer (1.07 mL, pH=4.20). The mixture was stirred at rt for 4 h. Conjugate 10f was purified by RP-HPLC.

[1498] Yield: 233 mg (5.21 μmol, 62%) conjugate 10f * 10 HCl

Example 11

Synthesis of Reversible Lys26 CNP-38 PEG4×10 kDa Conjugate Conjugate 11i

[1499] Conjugate 11i was synthesized according to the following scheme:

##STR00170## ##STR00171##

[1500] To a solution of 9 (353 mg, 0.50 mmol) and PyBOP (260 mg, 0.50 mmol) in DMF (9 mL) was added DIEA (105 μL, 0.60 mmol). This mixture was drawn onto Lys26-side-chain deprotected CNP-38 resin 10a (2.00 g, 0.21 mmol) and the suspension was shaken for 2 h at RT in order to afford resin 11a. The resin was washed 10× with DMF (7 mL). Cleavage of the Fmoc protecting group in 11a was carried out with a solution of HOBt (0.68 g, 5.03 mmol) and piperazine (3.00 g, 34.83 mmol) in DMF (47 mL). Therefore, the resin was incubated 5× with 10 mL of the cleavage mixture for 15 min at rt each time. Then, the resin was washed 7× with DMF (7 mL).

[1501] A solution of Fmoc-Lys(Fmoc)-OH (449 mg, 0.76 mmol), COMU (325 mg, 0.76 mmol) and DIEA (165 μL, 0.95 mmol) in DMF (9 mL) was prepared and drawn onto the resin. The mixture was shaken for 2 h at rt. The procedure was repeated twice, each for 1 h with freshly prepared coupling mixture. The resin was washed 10× with DMF (7 mL) and the remaining free amino groups were capped with 8 mL 1/1/2 (v/v/v) Ac.sub.2O/pyridine/DMF.

[1502] Cleavage of the Fmoc protecting groups in 11c was carried out with a solution of HOBt (0.68 g, 5.03 mmol), piperazine (3.00 g, 34.83 mmol) in DMF (47 mL). Therefore, the resin was incubated 5× with 10 mL of the cleavage mixture for 15 min at rt each time. The resin was washed 7× with DMF (7 mL) To a solution of 7 (266 mg, 1.00 mmol) and PyBOP (520 mg, 1.00 mmol) in DMF (9 mL) was added DIEA (209 μL, 1.20 mmol). This mixture was drawn onto the resin and was shaken for 2 h at rt. The resin was washed 7× with DMF (7 mL) affording resin 11e. Cleavage of the peptide from resin and removal of protecting groups was achieved by treatment of the resin with 15 mL pre-cooled (−18° C.) cleavage cocktail 68.5/10/10/5/3.5/1 (v/w/v/v/v/v) TFA/DTT/thioanisole/phenol/water/TIPS. The mixture was allowed to warm to rt and was agitated for 3 h at. The resin was filtered off and crude 11f was precipitated in pre-cooled (−18° C.) diethyl ether and purified by RP-HPLC. The combined HPLC fractions were used directly in the next step.

[1503] MS: m/z 1218.66=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1218.65).

[1504] To the combined HPLC product fractions of 11f (1 L) 160 mL of 0.5 M citric acid buffer (pH=5.00) and 100 mL of a 50 mM 2,2′-dithiobis(pyridine-N-oxide) solution in 9/1 (v/v) acetonitrile/water were added. The mixture was stirred for 4 h at rt and then diluted with 1 L of water containing 0.1% TFA (v/v). 11 g was purified by RP-HPLC. The product fractions were combined and lyophilized.

[1505] Yield: 64.3 mg (10.7 μmol, 6%) CNP-38-linker-DMB * 10 TFA

[1506] MS: m/z 1218.15=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1218.14).

[1507] Cleavage of the Dmb protecting group was achieved by adding 45 mL of pre-cooled (−18° C.) cleavage cocktail 100/5/3/2/1 (v/v/w/v/v) TFA/MSA/DTT/water/thioanisole to 11g (61.8 mg, 10.3 μmol), and then stirring for 4 h at 0° C. Crude 11h was precipitated in pre-cooled (−18° C.) ether. The precipitate was dissolved in a solution of 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) and incubated for 4 h at rt in order to hydrolyze any TFA esters. 11h was purified by RP-HPLC.

[1508] Yield: 38.4 mg (6.65 μmol, 65%) CNP-38-linker-thiol * 10 TFA

[1509] MS: m/z 1159.11=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1159.10).

[1510] To a solution of 11h (34.6 mg, 5.99 μmol) in 1 mL water containing 0.1% TFA (v/v) was added a solution of PEG 2×10 kDa maleimide (Sunbright GL2-200MA, 1.12 g, 56.03 μmol) in 6.1 mL water containing 0.1% TFA (v/v), followed by 0.5 M lactic acid buffer (1.46 mL, pH=4.00). The mixture was stirred at rt for 4 h. Conjugate 11i was purified by RP-HPLC.

[1511] Yield: 227 mg (4.96 μmol, 83%) conjugate 11i * 10 HCl

Example 12

Synthesis of Permanent Lys26 CNP-38 PEG4×10 kDa Conjugate 12g

[1512] Conjugate 12g was synthesized according to the following scheme:

##STR00172## ##STR00173##

[1513] To a solution of Fmoc-Lys(Fmoc)-OH (365 mg, 0.62 mmol) and PyBOP (322 mg, 0.62 mmol) in DMF (4.6 mL) was added DIEA (0.11 mL, 0.62 mmol). The mixture was drawn onto resin 10a (2.0 g, 0.21 mmol). The suspension was shaken for 2 h at rt. The resin was washed 10× with DMF (7 mL). Cleavage of the Fmoc protecting groups in 12a was carried out with a solution of HOBt (1.35 g, 9.99 mmol), piperazine (6.00 g, 69.66 mmol) in DMF (94 mL). Therefore, the resin was incubated 5× with the cleavage mixture for 15 min at rt each time, affording resin 12b. Then the resin was washed 7× with DMF (7 mL).

[1514] To a solution of 7 (283 mg, 1.06 mmol) and PyBOP (552 mg, 1.06 mmol) in DMF (6.5 mL), DIEA (185 μL, 1.06 mmol) was added and the mixture was drawn onto resin 12b (2.07 g, 0.10 mmol/g, 0.21 mmol). The mixture was shaken for 2 h at rt. Then, the resin was washed x each with DMF (7 mL) and CH.sub.2Cl.sub.2 (7 mL) and dried in vacuo.

[1515] Cleavage of the peptide from resin and removal of protecting groups was achieved by treatment of the resin with 15 mL pre-cooled (−18° C.) cleavage cocktail 68.5/10/10/5/3.5/1 (v/w/v/v/v/v) TFA/DTT/thioanisole/phenol/water/TIPS. The mixture was allowed to warm to rt and was agitated for 2.5 h. The resin was filtered off and crude 12d was precipitated in pre-cooled diethyl ether (−18° C.) and purified by RP-HPLC. The combined HPLC fractions were used directly in the next step.

[1516] MS: m/z 1172.37=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1172.37).

[1517] To the combined HPLC product fractions of 12d (390 mL) 58.5 mL of 0.5 M citric acid buffer (pH=5.00) and 8.9 mL of a 10 mM 2,2′-dithiobis(pyridine-N-oxide) solution in 1/1 (v/v) acetonitrile/water were added. The mixture was stirred for 10 min at rt then diluted with 400 mL of water containing 0.1% TFA (v/v). 12e was purified by RP-HPLC.

[1518] Yield: 100 mg (17.5 μmol, 8% over 6 steps) CNP-38-linker-Dmb * 9 TFA

[1519] MS: m/z 1171.87=[M+4H]4+, (calculated monoisotopic mass for [M+4H]4+=1171.86).

[1520] Cleavage of the Dmb protecting group was achieved by adding 65 mL pre-cooled (−18° C.) cleavage cocktail 100/5/3/2/1 (v/v/w/v/v) TFA/MSA/DTT/water/thioanisole to 12e (100 mg, 17.5 μmol) and stirring for 3.5 h at 0° C. Crude 12f was precipitated in pre-cooled (−18° C.) diethyl ether. The precipitate was dissolved in water containing 0.1% TFA (v/v) and incubated for 2 h at rt in order to hydrolyze any TFA esters. 12f was purified by RP-HPLC.

[1521] Yield: 43.4 mg (7.92 μmol, 45%) CNP-38-linker-thiol * 9TFA

[1522] MS: m/z 1112.83=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1112.82).

[1523] To a solution of 12f (39.6 mg, 7.22 μmol) in 1 mL water containing 0.1% TFA (v/v) was added a solution of PEG 2×10 kDa maleimide (Sunbright GL2-200MA, 1.22 g, 59.94 μmol) in 6.16 mL water containing 0.1% TFA (v/v), followed by 0.5 M lactic acid buffer (1.41 mL, pH=4.20). The mixture was stirred at rt for 4 h. Conjugate 12g was purified by RP-HPLC.

[1524] Yield: 204 mg (4.48 μmol, 57%) conjugate 12g * 9 HCl

Example 13

Synthesis of PEG5 kDa Thiol 13c

[1525] PEG5 kDa thiol 13c was synthesized according to the following scheme:

##STR00174##

[1526] To a solution of 13b (58.6 mg, 0.15 mmol), HOBt (22.9 mg, 0.15 mmol) and EDC hydrochloride (28.8 mg, 0.15 mmol) in DCM (1.00 mL) 2,4,6-collidine (121 mg, 1.00 mmol) was added. Then, a solution of methoxy PEG amine 5 kDa 13a (500 mg, 0.10 mmol) in DCM (4.00 mL) was added and the mixture was stirred for 16 h at rt. The solvent was evaporated and the mixture was dissolved in ACN/water and purified by RP-HPLC. The amount of solvent was reduced in vacuo and the aqueous residue was extracted with DCM (1×100 mL, 2×50 mL). The combined organic layers were reduced in vacuo to 20 mL. TFA (1.6 mL) and TES (3.5 mL) were added and the mixture was stirred at rt for 4.5 h. 13c was precipitated in diethyl ether, stored over night at −20° C., filtered and dried in vacuo.

[1527] Yield: 372 mg (72 μmol, 72%)

Example 14

Synthesis of Permanent N-Terminal CNP-34 PEG 5 kDa Conjugate 14e

[1528] Conjugate 14e was synthesized according to the following scheme:

##STR00175##

[1529] Side chain protected CNP-34 on TCP tentagel resin having free N-terminus 14a (0.78 g, 70 μmol) was pre-swollen in DMF for 30 min. A solution of maleimido hexanoic acid (85.3 mg, 0.40 mmol), DIC (50.9 mg, 0.40 mmol) and Oxyma (57.4 mL, 0.40 mmol) in DMF (6 mL) was drawn onto the resin and the mixture was shaken for 30 min at rt. The coupling then was repeated once with freshly prepared coupling solution. The resin was washed 10× each with DMF and CH.sub.2Cl.sub.2 and dried in vacuo affording 14b.

[1530] Cleavage of the peptide from resin and removal of protecting groups was achieved by treatment of the resin with 6 μmL cleavage cocktail 100/3/2/1 (v/v/v/v) TFA/TES/water/thioanisole for 1.5 h at rt. The crude peptide was precipitated in pre-cooled (−18° C.) diethyl ether.

[1531] MS: m/z 937.77=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=937.74).

[1532] The precipitate was dissolved in 15 mL TFA. A solution of diphenylsulfoxide (68.06 mg, 0.34 mmol) and anisole (0.18 mL, 1.68 mmol) in 5 mL TFA was added. Trichloromethylsilane (0.47 mL, 4.17 mmol) was added and the mixture was stirred for 15 min at rt. Ammonium fluoride (0.38 g, 10.3 mmol) was added and the solution was agitated for a further 2 min. The crude material was precipitated in pre-cooled (−18° C.) diethyl ether and purified by RP-HPLC affording 14d.

[1533] Yield: 8.30 mg (1.78 μmol, 82% purity, 1.4% over 3 steps) CNP-34-Malhx * 8 TFA

[1534] MS: m/z 937.26=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=937.23).

[1535] To a solution of 14d (7.34 mg, 1.57 μmol) in 200 μL 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) was added a solution of 13c (20 mg, 3.90 μmol) in 200 μL water containing 0.1% TFA (v/v), followed by 200 μL 0.5 M acetate buffer (pH=5.00). The mixture was incubated at rt for 30 min. Conjugate 14e was purified by RP-HPLC.

[1536] Yield: 9.92 mg (1.01 μmol, 57%) conjugate 14e * 8 TFA

Example 15

[1537] Synthesis of Permanent N-Terminal CNP-38 PEG 5 kDa Conjugate 15e Conjugate 15e was synthesized according to the following scheme:

##STR00176##

[1538] Compound 15d was synthesized as described for 14d, except that side chain protected CNP-38 on TCP tentagel resin having free N-terminus 15a (1.34 g, 0.12 mmol) was used as starting material.

[1539] Yield: 15.6 mg (2.94 μmol, 6.6%) CNP-38-Malhx * 9 TFA

[1540] MS: m/z 1064.05=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1064.04).

[1541] Conjugate 15e was synthesized as described for 14e, except that 15d (8.34 g, 1.58 mmol) was used as starting material.

[1542] Yield: 9.47 mg (0.91 μmol, 31%) conjugate 15e * 9 TFA

Example 16

[1543] Synthesis of Permanent Lys12 CNP-34 PEG 5 kDa Conjugate 16e

[1544] Conjugate 16e was synthesized according to the following scheme:

##STR00177##

[1545] 1.0 g (0.10 mmol) of side chain protected CNP-34 on TCP tentagel resin having Boc protected N-terminus and ivDde protected side chain of Lys12 was ivDde deprotected according to the procedure given in Materials and Methods to obtain 16a.

[1546] Compound 16d was synthesized as described for 14d, except that resin 16a (1.00 g, 0.10 mmol) was used as starting material.

[1547] Yield: 17.0 mg (3.65 μmol, 3.7%) CNP-34-Lys12-Malhx * 8 TFA

[1548] MS: m/z 937.25=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=937.23).

[1549] Conjugate 16e was synthesized as described for 14e, except that 16d (17 mg, 3.65 μmol) was used as starting material.

[1550] Yield: 12.2 mg (1.25 μmol, 34%) conjugate 16e * 8 TFA

Example 17

[1551] Synthesis of Permanent Lys16 CNP-34 PEG 5 kDa Conjugate 17e

[1552] Conjugate 17e was synthesized according to the following scheme:

##STR00178##

[1553] 0.78 g (0.07 mmol) of side chain protected CNP-34 on TCP tentagel resin having Boc protected N-terminus and ivDde protected side chain of Lys16 was ivDde deprotected according to the procedure given in Materials and Methods to obtain 17a.

[1554] Compound 17d was synthesized as described for 14d, except that resin 17a (0.78 g, 0.13 mmol) was used as starting material.

[1555] Yield: 5.39 mg (1.16 μmol, 1.7%) CNP-34-Lys16-Malhx * 8 TFA

[1556] MS: m/z 937.26=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=937.23).

[1557] Conjugate 17e was synthesized as described for 14e, except that 17d (5.39 mg, 1.16 μmol) was used as starting material.

[1558] Yield: 10.7 mg (1.09 μmol, 94%) conjugate 17e * 8 TFA

Example 18

[1559] Synthesis of Permanent Lys22 CNP-34 PEG 5 kDa Conjugate 18e

[1560] Conjugate 18e was synthesized according to the following scheme:

##STR00179##

[1561] 1.07 g (0.11 mmol) of side chain protected CNP-34 on TCP tentagel resin having Boc protected N-terminus and ivDde protected side chain of Lys22 was ivDde deprotected according to the procedure given in Materials and Methods to obtain 18a.

[1562] Compound 18d was synthesized as described for 14d, except that resin 18a (1.07 g, 0.11 mmol) was used as starting material.

[1563] Yield: 5.20 mg (1.12 μmol, 1.0%) CNP-34-Lys22-Malhx * 8 TFA

[1564] MS: m/z 937.26=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=937.23).

[1565] Conjugate 18e was synthesized as described for 14e, except that 18d (5.2 mg, 1.12 μmol) was used as starting material.

[1566] Yield: 4.20 mg (0.43 μmol, 38%) conjugate 18e * 8 TFA

Example 19

[1567] Synthesis of Permanent Lys26 CNP-38 PEG 5 kDa Conjugate 19e

[1568] Conjugate 19e was synthesized according to the following scheme:

##STR00180##

[1569] (0.865 g, 0.10 mmol) of side chain protected CNP-38 on TCP tentagel resin having Boc protected N-terminus and ivDde protected side chain of Lys26 was ivDde deprotected according to the procedure given in Materials and Methods to obtain 19a.

[1570] Compound 19d was synthesized as described for 14d, except that resin 19a (0.865 g, 0.10 mmol) was used as starting material.

[1571] Yield: 10.3 mg (1.95 μmol, 2.0%) CNP-38-Lys26-Malhx * 9 TFA

[1572] MS: m/z 1064.05=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1064.04).

[1573] Conjugate 19e was synthesized as described for 14e, except that 19d (4.70 mg, 1.10 μmol) was used as starting material.

[1574] Yield: 3.20 mg (0.31 μmol, 28%) conjugate 19e * 9 TFA

Example 20

[1575] Release Kinetics In Vitro

[1576] CNP conjugates 10f and 11i were dissolved in a PBS buffer containing 3 mM EDTA and 10 mM methionine, pH 7.4 at a concentration of approximately 1 mg conjugate/mL. The solutions was filtered sterile and were incubated at 37° C. At time points aliquots were withdrawn and analysed by RP-HPLC and ESI-MS. UV-signals correlating to liberated CNP were integrated and plotted against incubation time.

[1577] Curve-fitting software was applied to estimate the corresponding halftime of release.

[1578] Results:

[1579] For conjugate 10f a release half life time of 8.5 d (1 d) was obtained.

[1580] For conjugate 11i a release half life time of 9.5 d (1.5 d) was obtained.

Example 21

[1581] Digest of CNP Variants by Neutral Endopeptidase In Vitro

[1582] In order to determine the in vitro stability of various CNP variants including different peptide chain lengths and PEGylations using different PEGylation sites and PEG molecules in the presence of Neutral Endopeptidase (NEP), a NEP digest assay was established. This assay monitored the decrease of the non-digested CNP variant (normalized with the internal standard PFP) over time in reference to the to-time point.

[1583] In detail, recombinant human NEP (2.5 μg/mL final concentration) and the standard pentafluorophenol (PFP; 40 μg/mL final concentration) were added to the CNP variant (100 μg CNP equivalents/mL) in digest buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaCl). The solution was incubated at 37° C. and 500 rpm for up to 4 days. Samples were taken at different time intervals. The reaction was stopped by a combined reduction and heat denaturation adding TCEP (tris(2-carboxyethyl)phosphine; 25 mm final concentration) and incubating the mixture at 95° C., 500 rpm for 5 minutes. The resulting reaction products were assigned using HPLC-MS. The half life of each CNP variant was calculated via the ratio change in the HPLC-UV peak areas of CNP and PFP over time. To compensate for variations in the C protease activity, a CNP-38 or CNP-34 digest was carried out in every batch measurement as reference.

[1584] Table 1 lists the half-lives, based on the in vitro NEP cleavage assay, of various CNP variants of different lengths and having various PEG molecules attached to different side chains.

TABLE-US-00010 Compound CNP-variant PEGylation half life norm. [h] CNP-22.sup.1 CNP-22 — 0.32 CNP-34.sup.1 CNP-34 — 4.15 14e.sup.1 CNP-34 5 kDa PEG, Almost no proteolysis N-Terminus after 4 days. 17e.sup.1 CNP 34 5 kDa PEG, Lys16 54.23 18e.sup.1 CNP-34 5 kDa PEG, Lys22 38.87 16e.sup.1 CNP-34 5 kDa PEG, Lys12 No evaluation possible. CNP-38.sup.2 CNP-38 — 12.10 19e.sup.2 CNP-38 5 kDa PEG, Lys26 62.76 15e.sup.2 CNP-38 5 kDa PEG, Almost no proteolysis N-Terminus after 4 days. 12g.sup.2 CNP-38 4 × 10 kDa Almost no proteolysis PEG, -Lys26 after 4 days. .sup.1Due to variations in NEP catalytic activity between experiments, a mean was formed of all CNP-34 half life measurements (4.15 h) and the CNP-34 conjugates' half life measurements were normalized to this mean using a coefficient to calculate the adjusted t.sub.1/2. .sup.2Due to variations in NEP catalytic activity between experiments, a mean was formed of all CNP-38 half life measurements (12.10 h) and the CNP-38 conjugates' half life measurements were normalized to this mean using a coefficient to calculate the adjusted t.sub.1/2.

[1585] The rank order of resistance towards NEP is as follows: The longer CNP-variant (CNP-38) is more stable than the shorter CNP variant (CNP-34), which in turn is more stable than the shorter CNP-22. The order of the PEG-attachment sites is as follows: N-terminal >next-to-ring >ring. Therefore, an N-terminal PEG attachment confers the highest stability towards the proteolytic digest with NEP for the tested conjugates. The stability of CNP-38 PEGylated at Lys26 can be increased with increasing PEG size.

Example 22

[1586] Functional cGMP stimulation in NIH-3T3 cells with CNP variants

[1587] Functional activity of CNP variants were determined in a cell-based assay with NIH-3T3 cells (Murine Embryo Fibroblast cell line). These cells express endogenously NPR-B on the cell surface. Stimulation of NPR-B with CNP leads to intracellular production of the second messenger cGMP which is detected with a commercially available cGMP assay. NIH-3T3 cells were routinely cultured in DMEM F-12 medium with 5% FBS and 5 mM glutamine at 37° C. and 5% CO.sub.2. For each assay, 50,000 cells were resuspended in stimulation buffer (Dulbecco's PBS with IBMX) and incubated with the CNP variants in different concentrations. CNP (dilutions were made in PBS with 0.2% BSA). After incubation of 30 min at 37° C. and 5% CO.sub.2, the cells were lyzed and cGMP levels were determined with a commercially available cGMP TR-FRET assay (Cisbio, cGMP kit, Cat. No. 62GM2PEB). PEGylated CNP variants were always characterized in comparison with the non-PEGylated version in the same experiment batch. If possible, evaluation of the residual activity was done via the EC50- parameter of the resulting dose-response curve (restricted model with common slope).

TABLE-US-00011 TABLE 2 Residual NPR-B activity of PEGylated CNP variants in a cell-based assay as determined against the non-PEGylated CNP variant EC.sub.50 compound/ Compound CNP Variant PEGylation EC.sub.50 CNP-38 15e CNP-38 5 kDa PEG, N-Terminus    >5 19e CNP-38 5 kDa PEG, Lys26  >100 12g CNP-38 4 × 10 kDa PEG, Lys26 >>100 11i CNP-38 4 × 10 kDa PEG, Lys26 >>100 10f CNP-38 2 × 20 kDa PEG, Lys26 >>100

[1588] Comparing the tested PEG attachment sites, the attachment at the Lys26 (ring-lysine) showed the highest functional activity reduction, whereas the N-terminal attachment showed relatively high residual functional activity values. Increasing the PEG size resulted in a better shielding of the CNP molecule and a lower residual functional activity.

Example 23

[1589] Growth Study in FVB Mice after 5 Weeks Treatment with CNP-38 by Daily Subcutaneous Bolus Injection or by Continuous Subcutaneous Infusion

[1590] This study was performed in order to test the effect of daily subcutaneous bolus injection vs. continuous subcutaneous infusion of CNP-38 on animal growth. 21- to 22-days-old wild-type FVB male mice (n=9/group) were given 50 nmol/kg/d CNP-38 or vehicle (30 mM acetate pH 4 containing 5% sucrose and 1% benzylic alcohol) either by daily subcutaneous bolus injection or by continuous subcutaneous infusion in the scapular region over 35 days. Continuous infusion was applied by Alzet osmotic pumps model 1002 for week 1-2, followed by model 1004 for week 3-5. CNP-38 concentrations in the pumps were adjusted for the mean animal weight at study day 7 (pump model 1002) or study day 25 (pump model 1004). Growth was determined at d 35 by total body length measurement and X-ray measurements of the right femur and tibia.

[1591] Results of animals treated by daily subcutaneous bolus injection: At d 35, total body length of CNP-38 treated animals was 110.2%, right femur length was 105.6% and right tibia length was 104.0% compared to vehicle treated animals.

[1592] Results of animals treated by continuous subcutaneous infusion: At d 35, total body length of CNP-38 treated animals was 121.7%, right femur length was 107.5% and right tibia length was 112.2% compared to vehicle treated animals.

[1593] It was concluded that continuous subcutaneous infusion or related slow release formulations of CNP-38 (e.g. a slow releasing CNP-38 prodrug) are more effective than daily subcutaneous bolus injection in eliciting growth in the appendicular and axial skeleton.

Example 24

[1594] Pharmacokinetic Study of Permanent Lys26 CNP-38 PEG4×10 kDa Conjugate 12g in Cynomolgus Monkeys

[1595] This study was performed in order to show the suitability of 12g as a model compound for a slow release CNP-38 prodrug in cynomolgus monkeys. Male cynomolgus monkeys (2-4 years old, 3.5-4.1 kg) received either a single intravenous (n=3 animals) or a single subcutaneous (n=2 animals) administration of 12g at a dose of 0.146 mg CNP-38 eq/kg. Blood samples were collected up to 168 h post dose, and plasma was generated. Plasma total CNP-38 concentrations were determined by quantification of the N-terminal signature peptide (sequence: LQEHPNAR; residues 1-8 of SEQ ID NO:24) and C-terminal signature peptide (sequence: IGSMSGLGC; residues 30-38 of SEQ ID NO:24) after tryptic digestion as described in Materials and Methods.

[1596] Results: Dose administrations were well tolerated with no visible signs of discomfort during administration and following administration. No dose site reactions were observed any time throughout the study. After intraveneous injection the CNP-38 t.sub.max was observed at 15 min (earliest time point analyzed), followed by a slow decay in CNP-38 content with a half life time of approx. 24 h. After subcutaneous injection the CNP-38 concentration peaked at a t.sub.max of 48 h. At 168 h the CNP-38 concentration was still as high as ca. 50% of c.sub.max. The bioavailability was ca. 50%.

[1597] Similar PK curves were obtained for the N- and the C-terminal signature peptide up to 168 h post dose, indicating the presence of intact CNP-38 in the conjugate.

[1598] The favourable long lasting PK over several days and the stability of CNP-38 in the conjugate indicates the suitability of the permanent model compound Lys26 CNP-38 PEG 4×10 kDa conjugate 12g as a slow releasing CNP-38 prodrug after subcutaneous injection. It can be concluded that similar conjugates having a transiently Lys26 linked CNP-38 (like e.g. 11i) are suitable CNP-38 prodrugs providing long lasting levels of released bioactive CNP-38 over several days.

Example 25

[1599] Pharmacokinetic Study of Transient Conjugates 10f and 11i in Cynomolgus Monkeys

[1600] This study was performed in order to show the suitability of 10f and 11i as slow release CNP-38 prodrugs in cynomolgus monkeys. Male cynomolgus monkeys (2-4 years old, 3-5 kg) received either a single subcutaneous administration (n=3 animals) of compound 10f or a single subcutaneous (n=3 animals) administration of 11i at a dose of 0.146 mg CNP-38 eq/kg. Blood samples were collected up to 168 h post dose, and plasma was generated. Plasma levels of total CNP-38 content were analyzed as described in example 24. In order to analyze the plasma content of free CNP-38, the blood samples were acidified after withdrawal by adding 20 vol % of 0.5 M sodium citrate buffer pH 4 to stop further CNP-38 release from the conjugate. Free CNP-38 levels in plasma can e.g. be determined by ELISA using a CNP antibody that binds to the ring region of CNP, as described in the literature (U.S. Pat. No. 8,377,884 B2), or by LC-MS/MS.

[1601] Results: Dose administrations were well tolerated with no visible signs of discomfort during administration and following administration. No dose site reactions were observed any time throughout the study. After dose administration the total CNP-38 t.sub.max was observed at 12 h for compound 10f and 24 h for compound 11i. Total CNP-38 plasma levels were below LOQ (100 ng/mL, C-terminal peptide) after 120 h for compound 10f, while the plasma level was still as high as ca. 30% of c.sub.max. for compound 11i after 168 h (C-terminal peptide). For compound 11i similar terminal half life of 3-4 d was found for the C-terminal and the N-terminal peptide, indicating the presence of intact CNP-38 in the conjugate.

[1602] Conclusion: The favourable long lasting PK over several days and the stability of CNP-38 in the conjugate 11i indicates its suitability as CNP-38 prodrug for providing long lasting levels of released bioactive CNP-38 over several days.

Example 27

[1603] Synthesis of Fluorescein Labelled CNP-38 27d and NPR-C Affinity Assay

[1604] Compound 27d was synthesized according to the following scheme:

##STR00181##

[1605] Side chain protected CNP-38 on TCP tentagel resin having free N-terminus 27a (0.50 g, 35.4 μmol) was pre-swollen in DMF for 30 min. A solution of 5(6)-carboxyfluorescein-N-succinimidyl ester (41.9 mg, 88.5 μmol) and DIEA (30.9 μL, 177 μmol) in DMF (1.6 mL) was drawn onto the resin and the mixture was shaken over night at rt. The resin was washed 10× each with DMF and CH.sub.2Cl.sub.2 and dried in vacuo affording 27b.

[1606] Cleavage of the peptide from resin and removal of protecting groups was achieved by treatment of the resin with 7 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole for 1 h at rt. The resin was filtered off and crude 27c was precipitated in pre-cooled (−18° C.) diethyl ether and purified by RP-HPLC affording 27c. The combined HPLC fractions were used directly in the next step.

[1607] MS: m/z 1105.80=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1105.81).

[1608] To the combined HPLC product fractions of 27c (115 mL), 30 mL of 0.5 M citric acid buffer (pH=5.00) and 8 mL of a 10 mM 2,2′-dithiobis(pyridine-N-oxide) solution in 1/1 (v/v) acetonitrile/water were added. The mixture was stirred for 60 min at rt and then diluted with 350 mL of water containing 0.1% TFA (v/v). 27d was purified by RP-HPLC.

[1609] Yield: 16.1 mg (2.90 μmol, 8.2% over 3 steps) labelled CNP-38 * 10 TFA

[1610] MS: m/z 1105.30=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1105.30).

[1611] For the NPR-C affinity assay, a NPR-C expressing Hek293 cell line was developed. The coding region of the NPR-C sequence (BC131540) was cloned into a lentiviral vector under CMV promoter for constitutive receptor expression. A bicistronic element located on the vector for puromycin resistence was used as eukaryotic selection marker. After transduction, stably growing cell pools were subjected to qRT-PCR for confirmation of receptor mRNA-expression compared to parental Hek293 cells. An NPR-C-expressing cell pool was expanded and frozen as master cell bank for CNP sample testing.

[1612] For the assay, growing cells were trypsinized from the cell flask bottom, counted, and seeded in a 96-well plate (1.5×10.sup.5/well) and centrifuged. Supernatants were discarded. CNP standard and sample were serially diluted over 9 steps in PBS 0.2% BSA and transferred to the micro plate in duplicates and mixed with cells. After 30 min incubation at room temperature, fluorescein-labelled CNP 27d was added to each well with a constant concentration and cells were incubated for additional 45 min at room temperature. Subsequently, cells were analyzed by flow cytometry using mean fluorescence intensity of the FITC channel (FL1, Beckman Coulter FC500MPL) as read out.

[1613] Standard curve and sample curve were generated in an analysis software (PLA 2.0) using a 4PL fit for potency and/or IC.sub.50 calculation.

TABLE-US-00012 TABLE 3 Residual NPR-C affinity of PEGylated CNP-38 variants in a cell-based assay versus CNP-38 IC.sub.50 of PEGylated CNP- Compound PEGylation 38/IC.sub.50 CNP-38 15e 5 kDa PEG, N-Terminus 0.53 19e 5 kDa PEG, Lys26 1.1 10f 2 × 20 kDa PEG, Lys26 12 (reversible conjugate, first carrier branching point close to CNP moiety) 12g 4 × 10 kDa PEG, Lys26 143 (permanent conjugate, first carrier branching point close to the CNP moiety) 11i 4 × 10 kDa PEG, Lys26 91 (reversible conjugate, first carrier branching point close to the CNP moiety) 31d 4-arm PEG 40 kDa, Lys26 1.7 (reversible conjugate, first carrier branching point not close to the CNP moiety)

Example 29

[1614] Synthesis of Asn-Linker Reagent 29b

[1615] Asn-linker reagent 29b was synthesized according to the following scheme:

##STR00182##

[1616] To a solution of 1b (12.85 g, 16.14 mmol) in isopropanol (238 mL), H.sub.2O (77.5 mL) and LiOH (2.32 g, 96.87 mmol) were added. The reaction mixture was stirred for 4 h at rt.

[1617] Afterwards, the reaction mixture was diluted with toluene (300 mL). The phases were separated and the organic phase was washed 3× with 0.1 M HCl (200 mL). The phases were separated again. The aqueous phase was extracted 3× with toluene (100 mL). The product was found in the acidic aqueous phase and the pH value of this phase was adjusted to pH 8.5 by the addition of 4 N NaOH. Then, the aqueous phase was extracted 3× with CH.sub.2Cl.sub.2 (200 mL). The organic phase was washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and filtrated. 29a was isolated upon evaporation of the solvent and used in the next reaction without further purification.

[1618] Yield: 6.46 g (13.36 mmol, 83%)

[1619] MS: m/z 484.06=[M+H].sup.+, (calculated monoisotopic mass=483.26).

[1620] To a solution of 6-maleimidohexanoic acid (1.73 g, 8.19 mmol) in THF (70 mL), PyBOP (4.26 g, 8.19 mmol) and DIEA (3.89 mL, 22.33 mmol) were added. Then, the reaction mixture was stirred for 2 h at rt. Afterwards, 29a (3.60 g, 7.44 mmol) was dissolved in THF (10 mL) and added to the reaction mixture. The reaction was stirred at rt overnight. Then, methyl-tert-butylether (300 mL) was added. The organic phase was washed 2× with 0.1 M HCl solution (200 mL). The combined aqueous phases were extracted 2× with methyl-tert-butylether (200 mL). The combined organic phases were washed with brine (150 mL), dried over Na.sub.2SO.sub.4 and filtrated. The solvent was evaporated in vacuo. 29b was purified using flash chromatography.

[1621] Yield: 3.34 g (4.94 mmol, 66%)

[1622] MS: m/z 677.34=[M+H].sup.+, (calculated monoisotopic mass=676.33).

Example 30

[1623] Synthesis of 4-Arm-Thiol-PEG 40 kDa 30c

[1624] 4-arm-thiol PEG 30c was synthesized according to the following scheme:

##STR00183##

[1625] To a solution of 6-tritylmercapto-hexanoic acid (111.72 mg, 286.02 μmol), HOBt (43.81 mg, 286.06 μmol) and EDC*HCl (54.84 mg, 286.06 μmol) in CH.sub.2Cl.sub.2 (5 mL) was added 2,4,6-collidine (251 μL, 1.91 mmol). Then, this solution was added to a solution of 4-arm amino PEG 40 kDa (NOF, Sunbright PTE-400PA, 1.30 g, 31.78 μmol) in CH.sub.2Cl.sub.2 (10 mL). The reaction mixture was stirred over night at rt. Afterwards, the solvent was evaporated (water bath 30° C.). 30b was purified by RP-HPLC.

[1626] Yield: 650.5 mg (48%).

[1627] Cleavage of the Trt protecting group was achieved by adding the cleavage cocktail (DTT 500 mg/TFA 500 μL/water 500 μL, TES 2.5 mL/HFIP 5.0 mL/CH.sub.2Cl.sub.2 25.0 mL) to 30b (500 mg, 11.79 μmol) and incubating for 30 min at rt. 30c was obtained after precipitation in pre-cooled (−18° C.) diethyl ether.

[1628] Yield: 401.3 mg (82%; 93.3% purity).

Example 31

[1629] Synthesis of Conjugate 31d

[1630] Conjugate 31d was synthesized according to the following scheme:

##STR00184## ##STR00185##

[1631] A solution of linker reagent 29b (3.82 g, 5.64 mmol), OxymaPure (802 mg, 5.64 mmol) and DIC (868 μL, 5.64 mmol) in DMF (42.5 mL) was added to the resin 10a (18 g, 1.85 mmol). The suspension was shaken for 100 min at rt to afford resin 31a. The resin was washed 10× with DMF (10 mL) and 10× with DCM (10 mL) and dried in vacuo for 15 min. Cleavage of the peptide from resin and removal of protecting groups was achieved by treatment of the resin with 135 mL cleavage cocktail 100/3/2/1 (v/v/v/v) TFA/TES/water/thioanisol. The mixture was agitated for 60 min at rt. Crude 31b was precipitated in pre-cooled diethyl ether (−18° C.).

[1632] The precipitate was dissolved in TFA (423 mL). To this solution, a solution of diphenyl sulfoxide (1.87 g, 9.25 mmol) and anisole (5.05 mL, 46.25 mmol) in TFA (40 mL) was added. Afterwards, trichloromethylsilane (13.3 mL, 114.7 mmol) was added and the reaction mixture was stirred for 15 min at rt. Then, ammonium fluoride (10.96 g, 296 mmol) was added and the solution was stirred for 2 min in a water bath at rt. Crude 31c was precipitated in pre-cooled diethyl ether (−18° C.) and purified by RP-HPLC.

[1633] Yield: 187 mg (34.2 μmol, 16%) CNP-38-linker * 9 TFA

[1634] MS: m/z 1110.33=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1110.33).

[1635] To a solution of 31c (88.0 mg, 16.1 μmol) in 4.40 mL MeCN/H.sub.2O (1:1) containing 0.1% TFA (v/v) was added a solution of 4-arm-thiol-PEG 40 kDa 30c (107.35 mg, 2.59 μmol) in 1.45 mL water containing 0.1% TFA and 1 mM EDTA, followed by 0.5 M phosphate buffer containing 3 mM EDTA (1.46 mL, pH 6.0). The mixture was incubated for 2 h at rt. Conjugate 31d was purified by RP-HPLC.

[1636] Yield: 129 mg (2.09 μmol, 80%) conjugate 16d * 36 TFA

Example 32

[1637] Alternative synthesis of Dmb protected 6-mercaptohexanoic acid 7 Dmb-protected mercapto hexanoic acid 7 was synthesized according to the following scheme:

##STR00186##

[1638] To a solution of 6-bromohexanoic acid (100 g, 0.513 mol) in EtOH (1.0 L) was added thiourea (47 g, 0.615 mol) in one portion at 20° C. Then, the suspension was heated up to 78° C. (a clear solution was formed) and stirred for 12 h. A solution of NaOH (62 g, 1.54 mol) in H.sub.2O (1.0 L) was added dropwise with a constant pressure funnel. Afterwards, refluxing was continued for additional 2 h. The reaction mixture was poured into H.sub.2O (1 L) and extracted with EtOAc (1 L). The aqueous phase was acidified with con. HCl towards pH=2 and then extracted 3× with EtOAc (500 mL). The combined organic phases were washed with brine (400 mL). Afterwards, the combined organic phases were dried over Na.sub.2SO.sub.4, filtrated and the solvent was evaporated under reduced pressure at 45° C. The 6-mercaptohexanoic acid was used in the next reaction without further purification.

[1639] Yield: 62 g (crude)

[1640] .sup.1H-NMR (400 MHz, CDCl.sub.3):

[1641] δ=2.50-2.55 (q, J=7.2 Hz, 2H), 2.36 (t, J=7.6 Hz, 2H), 1.66-1.61 (m, 4H), 1.41-1.49 (m, 2H), 1.34 (t, J=7.6 Hz, 1H) ppm.

[1642] 6-mercaptohexanoic acid (27.0 g, 0.182 mol) was charged in a 1 L three-necked bottom flask with anhydrous THF (540 mL). The solution was degassed by freeze-pump-thaw technique and then cooled to 0° C. with an external ice bath. NaH (18.2, 455.4 mmol, 4.16 mL, 60% purity) was added with spoon horns over 30 min at 0° C. Then, 2,6-dimethylbenzylchloride (28.2 g, 0.182 mol) was added in one portion. The reaction mixture was warmed up to 20° C. and stirred for 12 h. The reaction mixture was poured into H.sub.2O (540 mL) and extracted 2× with MTBE (540 mL). Afterwards, the aqueous phase was acidified with conc. HCl towards pH=2 and then extracted 3× with MTBE (500 mL). The combined organic phases were washed with brine (500 mL), dried over Na.sub.2SO.sub.4 and filtrated. 7 was isolated upon evaporation of the solvent under reduced pressure at 45° C. as a yellow oil.

[1643] Yield: 41.5 g (0.16 mol, 85%)

[1644] .sup.1H-NMR (400 MHz, DMSO-d.sub.6):

[1645] δ=11.99 (s, 1H), 7.05-7.07 (d, J=6.8 Hz, 1H), 6.97 (s, 1H), 6.91-6.92 (d, J=6.8 Hz, 1H), 3.66 (s, 2H), 2.38-2.39 (m, 2H), 2.29 (s, 3H), 2.23 (s, 3H), 2.16-2.19 (m, 2H), 1.40-1.55 (m, 4H), 1.22-1.38 (m, 2H) ppm MS (neg. mode): m/z 265.0=[M−H].sup.−, (calculated monoisotopic mass=265.13).

Example 33

[1646] Synthesis of Linker Reagent 33c

[1647] Linker reagent 33c was synthesized according to the following scheme:

##STR00187##

[1648] Four reactions were carried out in parallel. To a solution of compound 1b (60 g, 75 mmol) in CH.sub.2Cl.sub.2 (300 mL) was added piperidine (58 g, 0.68 mol, 67 mL). The reaction mixture was stirred at rt for 4 h. The four reactions which were performed in parallel were combined for work-up. The reaction mixture was diluted with H.sub.2O (500 mL) and adjusted with a 0.5 N HCl solution towards pH=3˜4. The organic phase was separated and the aqueous phase was extracted with CH.sub.2Cl.sub.2 (800 mL). The combined organic phases were washed with brine (400 mL) and 5% saturated NaHCO.sub.3 solution (400 mL) in turn. Then, the combined organic phases were dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated in vacuo. 33a was purified by chromatography on silica (100-200 mesh) with DCM/MeOH (20/1 to 4/1).

[1649] Yield: 150 g (87%)

[1650] .sup.1H-NMR (400 MHz, DMSO-d.sub.6):

[1651] δ=7.34-7.38 (m, 4H), 6.25-7.29 (m, 2H), 5.08-5.19 (m, 2H), 4.60-4.68 (m, 1H), 4.32-4.40 (m, 2H), 3.73-3.79 (m, 9H), 3.10-3.27 (m, 3H), 2.65-3.05 (m, 8H), 1.36 (s, 9H) ppm.

[1652] Two reactions were carried out in parallel. To a solution of Fmoc-L-Lys(Fmoc)-OH (79 g, 0.13 mol), 33a (70 g, 0.12 mol), 4-ethyl-morpholine (70 g, 0.61 mol, 77 mL) in MeCN (850 mL) was added dropwise T.sub.3P (50% in EtOAc; 140 g, 0.22 mol) over a period of 30 min. After addition, the reaction mixture was stirred at rt for 18 h. The two reactions which were performed in parallel were combined for work-up. The reaction mixture was diluted with H.sub.2O/CH.sub.2Cl.sub.2 (1:1, 2 L) and then adjusted with 0.5 N HCl solution towards pH=3˜4. The organic phase was separated and the aqueous phase was extracted with CH.sub.2Cl.sub.2 (1 L). The combined organic phases were washed with brine (800 mL) and 5% NaHCO.sub.3 solution (800 mL) in turn. Then, the combined organic phases were dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated in vacuo. 33b was purified by chromatography on silica (100-200 mesh) with petroleum ether/ethyl acetate (5/1 to 1/1).

[1653] Yield: 160 g (57%)

[1654] .sup.1H-NMR (400 MHz, DMSO-d.sub.6):

[1655] δ=7.80-7.90 (m, 4H), 7.61-7.68 (m, 5H), 7.20-7.40 (m, 14H), 6.14-6.28 (m, 3H), 5.01-5.07 (m, 2H), 4.15-4.36 (m, 8H), 3.71-3.77 (m, 9H), 2.80-3.53 (m, 9H), 2.66-2.75 (m, 4H), 2.36-2.39 (m, 1H), 1.52-1.55 (m, 2H), 0.88-1.19 (m, 13H) ppm.

[1656] Two reactions were carried out in parallel. To a solution of 33b (60 g, 52 mmol) in MeOH (1.2 L) was added 10% Pd/C (18 g) in a 2 L hydrogenated bottle. The reaction mixture was degassed and purged 3× with H.sub.2 and then stirred at 25° C. under H.sub.2-atmosphere (45 psi) for 2.5 h. The two reactions which were performed in parallel were combined for work-up. The reaction mixture was filtered by diatomite and the filtrate was concentrated in vacuo to give crude 33c. 33c was purified by chromatography on silica (100-200 mesh) with DCM/MeOH (200/1 to 100/3).

[1657] Yield: 70 g (63%)

[1658] .sup.1H-NMR (400 MHz, DMSO-d.sub.6):

[1659] δ=12.15 (s, 1H), 7.87-7.89 (m, 4H), 7.50-7.70 (m, 5H), 7.31-7.40 (m, 9H), 6.20-6.23 (m, 2H), 4.13-4.36 (m, 10H), 3.70-3.77 (m, 9H), 2.62-3.10 (m, 12H), 2.30-2.34 (m, 1H), 2.14-2.18 (m, 1H), 1.50-1.58 (m, 2H), 1.25-1.34 (m, 13H) ppm

[1660] MS: m/z 1056.4=[M+H].sup.+, (calculated monoisotopic mass=1056.50).

Example 34

[1661] Alternative Synthesis of 11c

[1662] Compound 11c was synthesized according to the following scheme:

##STR00188##

[1663] A solution of linker reagent 33c (3.21 g, 3.04 mmol), PyBOP (1.58 g, 3.04 mmol) and DIPEA (848 mL, 4.86 mmol) in DMF (24.0 mL) was incubated for 5 min at rt, then added to the resin 10a (12 g, 1.21 mmol). The suspension was shaken for 2.5 h at rt. The resin was washed 10× with DMF (10 mL) and 10× with DCM (10 mL) and dried in vacuo for 60 min.

Example 35

[1664] CNP-38 and Conjugate 11i: Evaluation of Cardiovascular Effects in the Conscious Mouse (Subcutaneous Administration)

[1665] The purpose of this study was to evaluate the haemodynamic side effects of 11i at dose level equivalent to a CNP-38 dose level eliciting haemodynamic side effects (decrease in blood pressure) in the telemetered mouse.

[1666] Male Crl:CD1(ICR) mice (age range 8-13 weeks and body weight range 27.3-35.6 g at start of dosing) were surgically implanted with a TA11PA-C10 telemetry transmitter (Data Sciences International (DSI)) in the carotid artery. The body of the transmitter was placed subcutaneously in the lateral flank of the mouse. The mice were dosed subcutaneously in a latin square crossover design with at least 72 hours between dosing occasions. Mice were dosed with 1) vehicle (10 mM succinate, 46.0 g/L mannitol, pH 4.00), 2) CNP-38 (800 μg CNP-38/kg, 10 mM succinate, 46.0 g/L mannitol, pH 4.00) or 3) 11i (800 μg CNP-38 eq/kg, 10 mM succinate, 46.0 g/L mannitol, pH 4.00). At least four mice were included at each dose level. Blood pressure (systolic (SAP), diastolic (DAP) and mean (MAP) and heart rate (HR, derived from blood pressure), were recorded using a digital data capture system linked with a DSITM Ponemah data acquisition and analysis system. The capture system allowed recording of the cardiovascular parameters whilst the mice were in individual cages. On the day of each test session the animals were weighed and a predose recording was performed for at least 60 min prior to dosing. Each mouse was returned to the home cage and the cardiovascular parameters were recorded for approximately 48 hours postdose. Blood pressure and HR were reported at the following time points: -30, -20, -10, 5, 15 and 30 min postdose and 1, 2, 6, 12, 18, 24, 30, 36, 42 and 48 hours postdose. Each time point was presented as the average value of five minute's recording prior to the time point. The monitoring period was selected to cover exposure to the test items both prior to and after T.sub.max.

[1667] Results: Compared to predose values, vehicle dosed animals had increased MAP at the 5, 15, and 30 min post dose sampling time point. This was considered a normal physiological response due to handling and dosing. The same physiological increase in MAP was seen for animals dosed with 11i at the 5, 15, and 30 min post dose sampling time point predose. In 3 of 4 animals dosed with CNP-38 the physiological increase in MAP was not evident. On the contrary, 3 of 4 CNP-38 dosed animals showed a significant decrease in MAP at the 5, 15, and 30 min post dose sampling time point. During the remaining ten time points there were no difference in MAP between animals dosed with vehicle, CNP-38 and 11i.

[1668] MAP (mmHg) predose to 30 min post dose (mean+SD)

TABLE-US-00013 Vehicle (n = 10) 11i (n = 4) CNP-38 (n = 4) predose 101.9 ± 10.0 106.4 ± 10.7 106.8 ± 13.4  5 min post dose 125.9 ± 7.3  122.8 ± 5.9  102.0 ± 7.5   15 min post dose 126.3 ± 6.9  121.5 ± 7.5  89.5 ± 29.4 30 min post dose 114.4 ± 15.3 111.5 ± 13.7 99.5 ± 25.2

[1669] Similar trends were seen for SAP and DAP for all dose levels. HR was not impacted by treatment with CNP-38 or 11i.

[1670] In conclusion, subcutaneous administration of 11i did not decrease blood pressure as seen for an equivalent dosage CNP-38.

Example 36

[1671] Pharmacokinetic Profile of CNP-38 after Subcutaneous Single-Dose Administration to Cynomolgus Monkeys

[1672] This study was performed in order to test the pharmacokinetics of CNP-38 after subcutaneous (s.c.) administration in cynomolgus monkeys. Three male monkeys (2-4 years old, 3-5 kg) received a single s.c. injection at a dose of 40 μg/kg of CNP-38. Blood samples were collected at 5, 10, 15, 30, 45 min and 1, 2, 4, 8 hours upon dose.

[1673] Method: Plasma levels of CNP were analysed using a commercially available competitive radioimmuno-assay (RK-012-03, Phoenix Pharmaceuticals, CA). The assay was applied essentially as described by the manufacturer. The assay is based on competitive binding between 1251-labelled CNP (supplied in the kit) and unlabeled CNP (from study sample or calibrants) to an anti-CNP antibody. When the concentration of CNP in the sample increases, the amount of 125I-labelled CNP that is able to bind to the antibody decreases. By measuring the amount of 125I-labelled CNP bound as a function of the concentration of peptide, it is possible to construct a calibration curve from which the concentration of peptide in the sample can be determined.

[1674] A few changes to the supplied assay protocol were made. These changes included using in-house CNP calibrant and QC samples to secure consistency between assay runs. In order to shorten the duration of the assay, the initial incubation of samples with antibodies was performed at room temperature for 5 hours (instead of 16-24 hours at 4° C.). Due to matrix effects in monkey plasma, the minimal required dilution was set at 1:10, yielding an assay range of 150-1080 μg/mL CNP.

[1675] Results: Administration of CNP-38 to cynomolgus monkeys was well tolerated. After s.c. injection, the CNP-38 median T.sub.max was observed at 10 min, with a mean half-life time of approximately 7 min.

TABLE-US-00014 PK Parameter Result T.sub.max (median)  10 min C.sub.max (mean) 7.9 ng/mL AUC.sub.tlast (mean) 2.5 h*ng/mL Half-life (mean) 6.6 min

Example 37

[1676] Pharmacokinetic Profile of Conjugate 11i after Subcutaneous Single-Dose Administration to Cynomolgus Monkeys

[1677] This study was performed in order to investigate pharmacokinetics of 11i after s.c. administration in cynomolgus monkeys. Four male animals (2-4 years old, 3-5 kg) received a single s.c. injection of 11i at a dose of 40 μg CNP-38 eq/kg. Blood samples were collected up to 168 h post dose and plasma was generated (LiHeparin). Total CNP-38 concentrations were determined by LC-MS/MS

[1678] Method: The term “total CNP-38” refers to a combination of both free CNP-38 and CNP-38 bound in the CNP-38 conjugate. Plasma total CNP-38 concentrations were determined by quantification of the C-terminal signature peptide (sequence: IGSMSGLGC; residues 30-38 of SEQ ID NO:24) after tryptic digestion and disulfide bridge reduction.

[1679] LC-MS analysis was carried out by using an Agilent 1290 UPLC coupled to an Agilent 6460 Triple Quad mass spectrometer via an ESI probe. Chromatography was performed on a Waters Acquity BEH C18 analytical column (50×1.0 mm I.D., 1.7 μm particle size, 130 Å) with pre-filter at a flow rate of 0.5 mL/min (T=45° C.). Water (Ultrapure ≤500 ppt sodium grade) containing 0.1% formic acid (v/v) was used as mobile phase A and acetonitrile (ULC/MS grade) with 0.1% formic acid as mobile phase B. The gradient system comprised a short isocratic step at the initial parameters of 0.1% B for 0.5 min followed by a linear increase from 0.1% B to 30% B in 1.5 min. Mass analysis was performed in the multiple reaction monitoring (MRM) mode, monitoring the reactions of the ionsation m/z 824.5 [M+H].sup.1+ to 515.2. As internal standard deuterated CNP-38 conjugate was used.

[1680] Calibration standards of CNP-38 conjugate in blank plasma were prepared as follows: The thawed Li-heparin cynomolgus plasma was first homogenized, then centrifuged for 5 minutes. The CNP-38 conjugate formulation was diluted to eight different calibration working solutions containing between 0.103 and 51.28 μg/mL (CNP-38 eq.) in 50% methanol/50% water/0.1% formic acid (v/v/v). The working solutions were spiked into blank plasma at concentrations between 10.3 ng/mL (CNP-38 eq.) and 5128 ng/mL (CNP-38 eq.). The standards were used for the generation of a calibration curve. A calibration curve was generated based on analyte to internal standard peak area ratios using weighted (1/x.sup.2) linear regression and the sample concentrations were determined by back-calculation against the calibration curve.

[1681] For sample preparation, protein precipitation was carried out by addition of 200 μL of precooled (0° C.) acetonitrile to 50 μL of the plasma sample and 10 μL of internal standard solution (2.8 μg/mL CNP-38 eq. in 50% methanol/50% water/0.1% formic acid (v/v/v)). 200 μL of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 35° C.). For reconstitution solvent 100 μg Trypsin (order number V5111, Promega GmbH, Mannheim, Germany) were dissolved in 100 μL 10 mM acetic acid. 2.5 mL Tris buffer and 500 μL methanol were added. 50 μL of the resulting reconstitution solvent were added to each cavity of the-well plate.

[1682] After 3 hours incubation at 37° C. (Eppendorf ThermoMixer with ThermoTop), 5 μL of a 0.5 M TCEP solution were added to each cavity and incubated again for 30 min at 37° C. After the samples had cooled to room temperature, 2 μL 60% formic acid in water were added. 10 μL were injected into the UHPLC-MS system. Results: Administration of 11i to cynomolgus monkeys was well tolerated. After s.c. injection the 11i median T.sub.max was 36 h, and with a mean half-life time of 107 h.

TABLE-US-00015 PK Parameter Result T.sub.max (median)  36 hours C.sub.max (mean) 316 ng/mL AUC.sub.tlast (mean) 38.051 h*ng/mL Half-life (mean) 107 hours

Example 38

[1683] Functional cGMP Stimulation in NIH-3T3 Cells with Released CNP

[1684] 11i was incubated under physiological conditions (1 mg CNP-38 eq/mL), as described in Example 20. After 7 d, released CNP-38 was isolated by RP-HPLC and analyzed for bioactivity as described in Example 22.

TABLE-US-00016 CNP EC.sub.50 compound/ Compound Variant PEGylation EC.sub.50 CNP-38 Released CNP-38 CNP-38 — 1

Example 39

[1685] Alternative Synthesis of 11h

##STR00189##

[1686] Alternative synthesis of compound 11h: 39a is synthesized by solid phase synthesis as described in Material and Methods. Protecting group PG1 for the ring lysin side chain and protecting groups PG2 for the cysteine side chains is Mint. Mild resin cleavage and disulfide formation by iodine treatment affords compound 39c. After coupling of linker molecule 39d and global deprotection, 11h is purified by RP-HPLC.

Abbreviations

[1687] ACH achondroplasia [1688] ACN acetonitrile [1689] AcOH acetic acid [1690] AUC.sub.tlast Area Under the Curve to the last quantifiable time point [1691] Bn benzyl [1692] Boc tert-butyloxycarbonyl [1693] BSA bovine serum albumin [1694] cGMP cyclic guanosine monophosphate [1695] Cmax Maximum concentration [1696] CMV cytomegalovirus [1697] CNP C-type natriuretic peptide [1698] COMU (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate [1699] conc. Concentrated [1700] d day [1701] CTC Chlorotritylchloride polystyrol [1702] DAP Diastolic arterial pressure [1703] DBU 1,3-diazabicyclo[5.4.0]undecene [1704] DCC N,N′-dicyclohexylcarbodiimide [1705] DCM dichloromethane [1706] DIC N,N′-diisopropylcarbodiimide [1707] DIEA N,N-diisopropylethylamine [1708] DIPEA N,N-diisopropylethylamine [1709] DMAP dimethylamino-pyridine [1710] DMEM Dulbecco's modified Eagle's medium [1711] Dmb 2,4-dimethylbenzyl [1712] DMEM Dulbecco's modified eagle medium [1713] DMF N,N-dimethylformamide [1714] DMSO dimethylsulfoxide [1715] DTT dithiothreitol [1716] EC50 half maximal effective concentration [1717] EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [1718] EDTA ethylenediaminetetraacetic acid [1719] ELISA enzyme-linked immunosorbent assay [1720] eq stoichiometric equivalent [1721] ESI-MS electrospray ionization mass spectrometry [1722] Et ethyl [1723] EtOAc ethyl acetate [1724] EtOH ethanol [1725] FBS fetal bovine serum [1726] FGFR3 fibroblast-growth-factor-receptor 3 [1727] FITC fluorescein isothiocyanate [1728] Fmoc 9-fluorenylmethyloxycarbonyl [1729] h hour [1730] HATU O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate [1731] HCH hypochondroplasia [1732] HFIP hexafluoroisopropanol [1733] HPLC high performance liquid chromatography [1734] HOBt N-hydroxybenzotriazole [1735] HR Heart rate [1736] IBMX 3-isobutyl-1-methylxanthine [1737] iPrOH 2-propanol [1738] iv intravenous [1739] ivDde 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl [1740] LC liquid chromatography [1741] LTQ linear trap quadrupole [1742] Mal 3-maleimido propyl [1743] MAP Mean arterial pressure [1744] Me methyl [1745] MeOH methanol [1746] min minutes [1747] Mint monomethoxytrityl [1748] MS mass spectrum/mass spectrometry [1749] MSA methanesulfonic acid [1750] MTBE methyl-tert-butylether [1751] Mtt methyltrityl [1752] MW molecular weight [1753] m/z mass-to-charge ratio [1754] NEP neutral endopeptidase [1755] NHS N-hydroxy succinimide [1756] NPR natriuretic peptide receptor [1757] OtBu tert-butyloxy [1758] PBS phosphate buffered saline [1759] PEG poly(ethylene glycol) [1760] PFP pentafluorophenol [1761] pH potentia Hydrogenii [1762] Pr propyl [1763] PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [1764] Q-TOF quadrupole time-of-flight [1765] qRT-PCR quantitative real-time polymerase chain reaction [1766] RP-HPLC reversed-phase high performance liquid chromatography [1767] rpm rounds per minute [1768] rt room temperature [1769] SIM single ion monitoring [1770] SAP Systolic arterial pressure [1771] SEC size exclusion chromatography [1772] sc subcutaneous [1773] Su succinimidyl [1774] T.sub.3P 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide [1775] TCEP tris(2-carboxyethyl)phosphine [1776] TCP tritylchloride polystyro [1777] TD thanatophoric dysplasia [1778] TES triethylsilane [1779] TFA trifluoroacetic acid [1780] THF tetrahydrofuran [1781] TIPS triisoproylsilane [1782] T.sub.max Time of maximum concentration [1783] TMEDA N,N,N′N′-tetramethylethylene diamine [1784] Tmob 2,4,6-trimethoxybenzyl [1785] TR-FRET time-resolved fluorescence energy transfer [1786] Trt triphenylmethyl, trityl [1787] UPLC ultra performance liquid chromatography [1788] UV ultraviolet [1789] vs. versus [1790] ZQ single quadrupole