HK1197731A

HK1197731A - Carrier-linked treprostinil prodrugs

Info

Publication number: HK1197731A
Application number: HK14111396.1A
Authority: HK
Inventors: Ulrich Hersel; Harald Rau; Torben Lessmann; Nicola BISEK; Guillaume Maitro; Kennett Sprogøe; Thomas Wegge; Oliver Keil; Joachim Zettler
Original assignee: Ascendis Pharma A/S
Priority date: 2011-08-12
Filing date: 2012-08-10
Publication date: 2015-02-13

Description

Carrier-linked treprostinil prodrugs

Pulmonary Arterial Hypertension (PAH) is an increase in blood pressure in the pulmonary arteries, pulmonary veins, or pulmonary capillaries, leading to shortness of breath, dizziness, fainting, and other symptoms, all of which worsen with exertion. PAH can be a serious disease, markedly reduced exercise endurance and heart failure. It is a rare disease that occurs in about 2-3 per million people per year and in about 15 per million when prevalent. The median survival range for patients with untreated PAH is 2-3 years from the time of diagnosis, the cause of death is usually right ventricular failure.

Pulmonary hypertension involves vasoconstriction or tightening of blood vessels connecting the lungs and the lungs. Over time, fibrosis causes the affected blood vessels to become stiffer and thicker, which further increases the blood pressure in the lungs and affects their blood flow. In addition, increased cardiac workload leads to right ventricular hypertrophy, which ultimately leads to right heart failure. As blood flow through the lungs decreases, the left heart receives less blood and the oxygen supply is lower than desired, particularly during physical activity.

Many active agents are introduced for the treatment of PAH, with prostacyclin generally being considered the most effective. One prostacyclin is epoprostenol, which is a synthetic prostacyclin and is commercially available as(GlaxoSmithKline). It is administered to patients by continuous infusion and requires a semi-permanent central venous catheter, which may lead to sepsis and thrombosis.Unstable and therefore must be placed on ice at the time of application. Because its half-life is only 3 to 5 minutes, the infusion requires continuous night and day time, and any interruption can be fatal. Thus usingTreatment of PAH is a significant burden on the patient.

Another prostacyclin, ofIloprost (Ilomedin) marketed (Bayer) is the only inhaled form of prostacyclin approved for use in the united states and europe, until the inhaled form of treprostinil was approved by the FDA in 2009 at 7 months, with a commercial trade name of(UnitedTherapeutics)。

Inhaled prostacyclin suffers from the following disadvantages: failure to provide fully effective plasma levels of the drug throughout the dosing period makes inhalation therapy rarely required in severe patients.

Therefore, there is a need to develop other prostanoids, such as those described in US4306075A and EP159784B 1. One such prostaglandin is treprostinil, trade name(Unitedpherapeutics). Treprostinil has a half-life of 4 hours, but it is still administered by continuous subcutaneous infusion or continuous intravenous infusion via an infusion pump and must be worn by the patient at any time.

Subcutaneous infusion of treprostinil is usually painful to some extent and the patient cannot tolerate the pain, so the mode of administration becomes an intravenous infusion. However, intravenous Remodulin has been reported to increase the risk of sepsis.

Since subcutaneous infusion is accompanied by pain, there is a need to develop a prostacyclin that can be administered by subcutaneous administration, but with reduced pain levels. This can be achieved by administering a carrier-linked prodrug of prostacyclin, where the absorption of the prodrug is very fast and the release of prostacyclin from the prodrug is very slow, thus minimizing subcutaneous exposure to free prostacyclin molecules.

Prostacyclin is the standard treatment for PAH, especially in more severe patients. While inhaled treprostinil is more convenient and does not have the intense pain often associated with subcutaneous infusion treprostinil, inhalation is considered less effective and therefore is rarely prescribed.

Treprostinil has the following structure:

accordingly, there is a need to provide more effective and/or more comfortable treprostinil treatment for patients.

This object is achieved with a carrier-linked treprostinil prodrug of formula (I):

wherein each T is independently selected from structures (i) to (v):

wherein the dotted line indicates a connection to X⁰；

y is an integer in the range of 1 to 64, preferably in the range of 1 to 16, more preferably y is selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, even more preferably y is 4,6 or 8, most preferably 8,

each X⁰Independently is (X)^0A)_m1-(X^0B)_m2；

m1, m2 are independently 0 or 1;

X^0Ais T⁰；

X^0BIs branched or unbranched or cyclic C_1-15Alkylene which is unsubstituted or substituted by one or more R³Substituted, said R³Are the same or different; more preferably, X is^0BIs a branched or unbranched cyclic C_1-15Alkylene which is unsubstituted or substituted by one or more R³Substituted, said R³Are the same or different;

R³is halogen; c_1-6An alkyl group; CN; c (O) R⁴；C(O)OR⁴；OR⁴；C(O)R⁴；C(O)N(R⁴R^4a)；S(O)₂N(R⁴R^4a)；S(O)N(R⁴R^4a)；S(O)₂R⁴；S(O)R⁴；N(R⁴)S(O)₂N(R^4aR^4b)；SR⁴；N(R⁴R^4a)；NO₂；OC(O)R⁴；N(R⁴)C(O)R^4a；N(R⁴)SO₂R^4a；N(R⁴)S(O)R^4a；N(R⁴)C(O)N(R^4aR^4b)；N(R⁴)C(O)OR^4a；OC(O)N(R⁴R^4a) (ii) a Or T⁰；

R⁴、R^4a、R^4bIndependently selected from H; t is⁰；C_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl, wherein C_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl is optionally substituted by one or more R⁵Substituted, said R⁵Are the same or different;

R⁵is halogen; CN; c (O) R⁶；C(O)OR⁶；OR⁶；C(O)R⁶；C(O)N(R⁶R^6a)；S(O)₂N(R⁶R^6a)；S(O)N(R⁶R^6a)；S(O)₂R⁶；S(O)R⁶；N(R⁶)S(O)₂N(R^6aR^6b)；SR⁶；N(R⁶R^6a)；NO₂；OC(O)R⁶；N(R⁶)C(O)R^6a；N(R⁶)SO₂R^6a；N(R⁶)S(O)R^6a；N(R⁶)C(O)N(R^6aR^6b)；N(R⁶)C(O)OR^6a；OC(O)N(R⁶R^6a)；

R⁶、R^6a、R^6bIndependently selected from H; c_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl, wherein C_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl is optionally substituted with one or more halo, the halo being the same or different;

T⁰is phenyl; a naphthyl group; azulene radical; an indenyl group; indanyl; c_3-7A cyclic hydrocarbon group; a 3-to 7-membered heterocyclic group; or 8 to 11 membered heterobicyclic group, wherein T⁰Optionally substituted by one or more R⁷Substituted, said R⁷Are the same or different;

R⁷is halogen; CN; COOR⁸；OR⁸；C(O)R⁸；C(O)N(R⁸R^8a)；S(O)₂N(R⁸R^8a)；S(O)N(R⁸R^8a)；S(O)₂R⁸；S(O)R⁸；N(R⁸)S(O)₂N(R^8aR^8b)；SR⁸；N(R⁸R^8a)；NO₂；OC(O)R⁸；N(R⁸)C(O)R^8a；N(R⁸)S(O)₂R^8a；N(R⁸)S(O)R^8a；N(R⁸)C(O)OR^8a；N(R⁸)C(O)N(R^8aR^8b)；OC(O)N(R⁸R^8a) (ii) a Oxo (= O), wherein the ring is at least partially saturated; c_1-6An alkyl group; c_2-6An alkenyl group; or C_2-6Alkynyl, wherein C_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl is optionally substituted by one or more R⁹Substituted, said R⁹Are the same or different;

R⁸、R^8a、R^8bindependently selected from H; c_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl, wherein C_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl is optionally substituted by one or more R¹⁰Substituted, said R¹⁰Are the same or different;

R⁹、R¹⁰independently selected from halogen; CN; c (O) R¹¹；C(O)OR¹¹；OR¹¹；C(O)R¹¹；C(O)N(R¹¹R^11a)；S(O)₂N(R¹¹R^11a)；S(O)N(R¹¹R^11a)；S(O)₂R¹¹；S(O)R¹¹；N(R¹¹)S(O)₂N(R^11aR^11b)；SR¹¹；N(R¹¹R^11a)；NO₂；OC(O)R¹¹；N(R¹¹)C(O)R^11a；N(R¹¹)SO₂R^11a；N(R¹¹)S(O)R^11a；N(R¹¹)C(O)N(R^11aR^11b)；N(R¹¹)C(O)OR^11a(ii) a And OC (O) N (R)¹¹R^11a)；

R¹¹、R^11a、R^11bIndependently selected from H; c_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl, wherein C_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl being optionally substituted by one or more halogensAnd (ii) the halogens are the same or different;

Z¹is a carrier comprising a covalently bound polymer, preferably a pharmaceutically acceptable polymer,

wherein the carrier is covalently linked to the moiety X⁰Provided that one of m1, m2 is 1, and wherein if m1, m2=0, the carrier is covalently linked to T,

or a pharmaceutically acceptable salt thereof.

It has surprisingly been found that such carrier-linked treprostinil prodrugs can be used to obtain dosage forms of treprostinil that at least partially overcome the above disadvantages.

Within the present invention, the terms are used with the following meanings.

The terms "drug", "bioactive molecule", "bioactive moiety", "bioactive agent", "active substance" and the like refer to any substance that affects any physical or biochemical property of a biological organism, including but not limited to viruses, bacteria, fungi, plants, animals and humans. In particular, the term as used herein includes any substance used to diagnose, cure, alleviate, treat or prevent a disease of an organism, particularly a human or other animal, or otherwise enhance physical or mental well-being of an organism, particularly a human or animal. Treprostinil is understood as a bioactive molecule.

"biologically active moiety D" means the portion of the drug linker conjugate that is obtained after cleavage of the known biologically active drug D-OH or D-H. Thus, the "biologically active moiety treprostinil" or "treprostinil moiety" refers to the portion of the carrier-linked treprostinil prodrug that is obtained after cleavage of the known biologically active drug treprostinil.

By "free form" of a drug, such as treprostinil, is meant an unmodified, pharmaceutically active form of the drug, e.g., following release of a carrier-linked prodrug.

Targeting moieties refer to moieties that, when present in a molecule, such as a prodrug, result in the preferred localization of such larger molecules within a particular targeted region of the organism to which they are administered. Such specifically targeted regions may be organs, certain cell types or subcellular compartments. By "preferably localized" is meant that at least 5%, 10%, preferably at least 20% and more preferably at least 30% of the biologically active moiety administered to the patient reaches the specific target area.

Targeting moieties can be classified into 3 classes according to size:

small molecule targeting moieties such as C-glucuronide, cobalamin, vitamins such as folic acid (folate) and analogues and derivatives, carbohydrates, bisphosphonates, N-acetylgalactosamine;

peptides, e.g. bombesin, somatostatin, LHRH, EGF, VEGF, hCG, fragments of Luteinizing Hormone (LH), octreotide, vapreotide, lanreotide, the RC-3940 series, dabigatran, leuprolide acetate, norrexed, cetrorelix, peptides or pseudopeptides containing or derived from the NGR or RGD motif such as CNGRC (linear), GNGRG (cyclic), ACDC RGD CFCG (cyclic), CDCRGDCFC, CNGRC (cyclic), CRGDCGG, CNGRC, or other peptides such as ATWLPPR, Thrombin Sensitive Protein (TSP) -1 mimetics, (RGD pseudopeptide), CTTHWGFTLC, CGNKRTRGC, neuropeptide P substances, SSP, Sar9, Met (O2)11P substance analogues, cholecystokinin (CCK), corticotropin-releasing hormone/factor (CRH/CRF), dermorphin, FGF-2 or basic fibroblast growth factor, glactin, melanoidin, neurotensin, Cyclic RGDfK and cyclic RGDyV; preferably bombesin, somatostatin, LHRH, EGF, VEGF, hCG, fragments of Luteinizing Hormone (LH), octreotide, vapreotide, lanreotide, the RC-3940 series, dabigatran, leuprolide acetate, nordread, cetrorelix, peptides or pseudopeptides containing or derived from the NGR or RGD motif such as CNGRC (linear), GNGRG (cyclic), ACDC RGD CFCG (cyclic), CDCRGDCFC, CNGRC (cyclic), CRGDCGG, CNGRC, or other peptides such as ATWLPPR, Thrombin-sensitive protein (TSP) -1 mimetics, (RGD pseudopeptides), CTTHWGFTLC, CGNKRTRGC, neuropeptide substance P, SSP, Sar9, Met (O2)11P substance analogs, cholecystokinin (CCK), corticotropin releasing hormone/factor (CRH/CRF), dermorphin, FGF-2 or basic fibroblast growth factor, galanin, melanoidin, neurotensin.

And protein or macromolecular targeting moieties such as IL-2, GM-CSF, TNF-a, transferrin, immunoglobulins, acetylated-LDL, lactoferrin (Lf) (also known as lactoferrin) and lactoferricin (Lcin), Gambogic Acid (GA).

In principle, any ligand of a cell surface receptor may be advantageously used as targeting moiety. For example, the ATWLPPR peptide is a potent antagonist of VEGF; thrombin-sensitive protein-1 (TSP-1) induces endothelial cell apoptosis, RGD-motif mimetics block integrin receptors, NGR-containing peptides inhibit aminopeptidase N, and cyclic peptides containing HWGF sequences selectively inhibit MMP-2 and MMP-9. The LyP-1 peptide specifically binds to tumor lymphatic vessels. Exemplary additional ligands include peptide ligands identified by library screening, tumor cell specific peptides, tumor cell specific aptamers, tumor cell specific carbohydrates, tumor cell specific monoclonal or polyclonal antibodies, Fab or scFv (i.e., single chain variable region) fragments of antibodies such as the Fab fragment of antibodies targeting EphA2 or other proteins specifically expressed or uniquely accessible on metastatic cancer cells, small organic molecules derived from combinatorial libraries, growth factors such as EGF, FGF, insulin and insulin-like growth factor, and homologous polypeptides, somatostatin and analogs thereof, transferrin, lipoprotein complexes, bile salts, selectins, steroid hormones, Arg-Gly-Asp containing peptides, retinoids, various galectins, delta-opioid receptor ligands, cholecystokinin A receptor ligands, ligands specific ligands for angiotensin AT1 or AT2 receptors, and the like, Peroxisome proliferator activated receptor lambda ligands, beta-lactam antibiotics such as penicillins, small organic molecules including antimicrobials, and other molecules that specifically bind to receptors preferentially expressed on the surface of tumor cells or on infected organisms, ligands that are designed to conform to the binding pocket of a particular receptor based on the crystal structure of the receptor or other cell surface protein, antimicrobials and other drugs, tumor antigens, or other molecules preferentially expressed on the surface of tumor cells, or fragments of any of these molecules. Examples of tumor-specific antigens that can be used as targeting moieties include extracellular epitopes of Ephrin family members of proteins (e.g., EphA 2). EphA2 expression was restricted to cell-cell junctions in normal cells, but EpbA2 was distributed throughout the cell surface of metastatic cells. Thus, EpbA2 on metastatic cells readily binds, e.g., to a Fab fragment of an antibody linked to an immunogen, whereas proteins do not readily bind to a Fab fragment on normal cells, resulting in targeting moieties specific for metastatic cancer cells.

Further examples of such targeting moieties are: FSH-33, somatostatin 1, liver cancer targeting peptides, peptide GFE, anti-EGFR antibodies and/or antibody fragments, in particular cetuximab, CendR, iRGD peptide (RGD-CendR hybrid peptide), small molecules, antibodies and/or antibody fragments that bind a cancer-specific epitope such as CEA, the gastrin releasing peptide receptor, the somatostatin receptor, the galanin receptor, the follicle stimulating hormone receptor, the p32 protein, the fibroblast growth factor receptor, HepG2, the epidermal growth factor receptor, integrin α v β 6, the transmembrane protein-1 receptor and the VEGF receptor.

The phrases "associated form", "linked" or "moiety" refer to a partial substructure of a larger molecule. The phrases "associated with" and "linked" are used to simplify and refer to a nomenclature or list of parts of a reagent, starting material, or hypothetical starting material well known in the art, while "associated with" or "linked" refer to, for example, one or more hydrogen groups (-H) present in the reagent or starting material, or one or more activating or protecting groups not present in the part of a larger molecule.

Such drugs may be combined with a carrier in order to enhance the physicochemical or pharmacokinetic properties of the drug in vivo. If a drug such as treprostinil is transiently conjugated to a carrier and/or linker, as in the present invention, such a system is often referred to as a "carrier-linked prodrug". Carrier-linked prodrugs are those prodrugs comprising a given active substance temporarily linked to a temporary carrier group which gives improved physicochemical or pharmacokinetic properties and which can be easily removed in vivo by hydrolysis, according to the definition provided by IUPAC (as given under http:// www.chem.qmul.ac.uk/IUPAC/medchem/ah. html, accepted 3/7/2011).

The term "promoity" denotes the portion of a prodrug which is not a drug and thus denotes a linker and a carrier and/or any optional spacer moiety.

The term "reversible prodrug linker" or "transient prodrug linker" denotes a reversible linker comprising, in particular a linker consisting of, a reversible linker that is non-hydrolytically degradable (i.e. cleavable) under physiological conditions (aqueous buffer, ph7.4, 37 ℃) and has a half-life ranging, for example, from 1 hour to 3 months. On the other hand, stable or permanent connections are usually non-cleavable permanent bonds, meaning that they have a half-life of at least 6 months under physiological conditions (aqueous buffer, pH7.4, 37 ℃).

By "traceless prodrug linker" is meant a prodrug linker from which the drug is released in its free form, meaning that the drug does not contain any trace of the front moiety when released from the front moiety.

The term "polymer" describes a molecule comprising, in particular consisting of, repeating structural units linked by chemical bonds in a linear, cyclic, branched, cross-linked or dendritic manner or a combination thereof, which may be of synthetic or biological origin or a combination of both. Typically, the polymer has a molecular weight of at least 500 Da. It will be understood that when the polymer is a polypeptide, the individual amino acids of the polypeptide may be the same or may be different.

The term "polymerized" refers to a moiety comprising one or more polymers.

The term "poly (ethylene glycol) -based polymeric chain" or "PEG-based polymeric chainBy "is meant a polymer comprising at least 20% by weight of ethylene glycol moieties, more preferably at least 50% by weight, even more preferably at least 80% by weight of ethylene glycol moieties, the chains of which are optionally end-capped and/or which optionally further comprise one or more functional groups, such as amino groups. It is to be understood that the PEG-based polymeric chain may be terminated or interrupted by alkyl or aryl groups, and optionally substituted with heteroatoms and/or functional groups. For PEG-based polymeric chains, suitable end capping or terminating groups are, for example, CH₃-、CH₃-O-and CH₃-CH₂-. Thus, a PEG-based polymer is a polymer comprising at least 20 wt.% of ethylene glycol moieties, more preferably at least 50 wt.%, even more preferably at least 80 wt.% of ethylene glycol moieties.

The term "hydrogel" may be defined as a three-dimensional, hydrophilic or amphiphilic polymeric network capable of absorbing large amounts of water, which in an aqueous medium results in swelling of the hydrogel. The network consists of homopolymers or copolymers and is insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. Crosslinking provides network structure and physical integrity.

The terms "spacer", "spacer group", "spacer molecule" and "spacer moiety" are used interchangeably and denote any moiety suitable for linking two moieties, e.g. C_1-50Alkyl radical, C_2-50Alkenyl or C_2-50Alkynyl, a fragment of which is optionally interrupted by one or more groups selected from-NH-, -N (C)_1-4Alkyl) -, -O-, -S-, -C (O) NH-, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O)₂-、-O-C(O)NH-、-O-(CO)N(C_1-4Alkyl) -, 4-to 7-membered heterocyclyl, phenyl or naphthyl. Preferably, the term denotes C_1-50Alkyl radical, C_2-50Alkenyl or C_2-50Alkynyl, a fragment of which is optionally interrupted by one or more groups selected from-NH-, -N (C)_1-4Alkyl) -, -O-, -S-, -C (O) NH-, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O)₂-, 4-to 7-membered heterocyclyl, phenyl or naphthyl.

The term "terminal" denotes the last carbon atom or heteroatom of a linear or branched chain of carbon atoms and/or heteroatoms, i.e. "terminal" denotes a carbon atom or heteroatom to which exactly another carbon atom or heteroatom is attached.

"terminal/terminally" or "terminally linked" refers to a moiety that is linked to an end or to the end of another moiety.

"pharmaceutical composition" or "composition" means a composition comprising one or more drugs or prodrugs, and optionally one or more excipients, and any product resulting, directly or indirectly, from combination, complexation or aggregation of any two or more excipients and/or drugs or prodrugs, or from one or more excipients and/or drugs and/or prodrugs, or from other types of reactions or interactions of one or more excipients and/or drugs and/or prodrugs. Accordingly, the pharmaceutical compositions of the present invention include any composition obtainable by mixing a carrier-linked treprostinil prodrug of the present invention and a pharmaceutically acceptable excipient.

The term "excipient" means a diluent, adjuvant, or carrier with which the carrier-linked treprostinil prodrug is administered. Such pharmaceutical excipients may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred excipients when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferred as liquid vehicles for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, pH buffering agents and the like, for example acetates, succinates, tris (hydroxymethyl) aminomethane (tris), carbonates, phosphates, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), MES (2- (N-morpholino) ethanesulfonic acid), or may contain detergents, for example tweens, poloxamines, CHAPS, polyethylene glycol phenyl ethers or amino acids, for example glycine, lysine or histidine. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The compositions may be formulated as suppositories with conventional binders and excipients such as triglycerides. Oral formulations may include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical excipients are described in e.w. martin, "Remington's Pharmaceutical Sciences". These compositions will comprise a therapeutically effective amount of treprostinil in the form of at least one carrier-linked treprostinil prodrug of the invention, preferably in purified form, together with a suitable amount of excipients, to provide a form suitable for administration to a patient. The formulation should be suitable for the mode of administration.

The term "pharmaceutically acceptable" means approved by a regulatory agency, such as EMEA (europe) and/or FDA (us) and/or any other national regulatory agency, for use in animals, preferably humans.

By "dry composition" is meant that the pharmaceutical composition comprising the carrier-linked treprostinil prodrug of the invention is provided in a dry form in a container. Suitable drying methods are spray drying and freeze drying (freeze drying). The dry composition of such carrier-linked treprostinil prodrugs contains a residual water content of at most 10%, preferably less than 5% and more preferably less than 2% (determined according to Karl Fischer method). The preferred method of drying is lyophilization. By "lyophilized composition" is meant a pharmaceutical composition comprising a carrier-linked treprostinil prodrug that is first frozen and subsequently water is removed by reduced pressure. This technique does not exclude other drying steps, which may be carried out during the preparation process before the composition is filled into the final container.

"lyophilization" (freeze-drying) is a dehydration process characterized by freezing a composition and then reducing the ambient pressure, optionally with heating, such that the frozen water in the composition sublimes directly from the solid phase to the gaseous state. Typically, the sublimated water is collected by desublimation.

The term "functional group" denotes a particular group of atoms within a molecule that can undergo a characteristic chemical reaction. Examples of functional groups are hydroxyl, carbonyl, aldehyde, carboxyl, ester, ketal, hemiketal, acetal, hemiacetal, primary/secondary/tertiary amine, cyanate, disulfide, thiol, sulfonyl, phosphate.

If a functional group is coupled to another functional group, the resulting chemical structure is referred to as "attached". For example, the reaction of an amine functional group with a carboxyl functional group produces an amide linkage. Further examples of linkages are esters, ethers, ketals, acetals, primary/secondary/tertiary amines, amides, sulfides and disulfides.

"alkyl" means a straight chain (linear, unbranched) or branched carbon chain (unsubstituted alkyl). Optionally, one or more hydrogen atoms of an alkyl carbon may be substituted with a substituent as shown herein, which is referred to as "substituted alkyl". Generally, the preferred alkyl group is C_1-6An alkyl group.

“C_1-4Alkyl "denotes an alkyl chain having 1 to 4 carbon atoms (unsubstituted C_1-4Alkyl), for example if present at the molecular end: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or when two parts of the molecule are linked by an alkyl group (also referred to as C)_1-4Alkylene) such as-CH₂-、-CH₂-CH₂-、-CH(CH₃)-、-CH₂-CH₂-CH₂-、-CH(C₂H₅)-、-C(CH₃)₂-. Optionally, C_1-4One or more hydrogen atoms of the alkyl carbon may be substituted with substituents as shown herein. Thus, "C_1-50Alkyl "means an alkyl chain having 1 to 50 carbon atoms.

“C_1-6Alkyl "denotes an alkyl chain having 1 to 6 carbon atoms, for example if present at the end of the molecule: c_1-4Alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, or when two parts of the molecule are linked by an alkyl group (also known as C)_1-6Alkylene) such as-CH₂-、-CH₂-CH₂-、-CH(CH₃)-、-C(CH₂)-、-CH₂-CH₂-CH₂-、-CH(C₂H₅)-、-C(CH₃)₂-。C_1-6One or more hydrogen atoms of the alkyl carbon may be substituted with substituents as shown herein. The term C is defined accordingly_1-15Alkyl or C_1-15An alkylene group.

“C_2-6Alkenyl "means an alkenyl chain having 2 to 6 carbon atoms, for example if present at the molecular end: -CH = CH₂、-CH=CH-CH₃、-CH₂-CH=CH₂、-CH=CH-CH₂-CH₃、-CH=CH-CH=CH₂Or when two parts of the molecule are linked through an alkenyl group, for example-CH = CH-. C_2-6One or more hydrogen atoms of an alkenyl carbon may be substituted with a substituent as shown herein.

The term C is defined accordingly_2-4An alkenyl group.

“C_2-6Alkynyl "means an alkynyl chain having 2 to 6 carbon atoms, for example if present at a molecular terminus: -C ≡ CH, -CH₂-C≡CH、CH₂-CH₂-C≡CH、CH₂-C≡C-CH₃Or when two moieties of the molecule are linked by an alkynyl group, for example-C.ident.C-. C_2-6One or more hydrogen atoms of an alkynyl carbon may be substituted with a substituent as shown herein. The term C is defined accordingly_2-4Alkynyl.

“C_2-50Alkenyl "denotes a branched or unbranched alkenyl chain having 2 to 50 carbon atoms (unsubstituted C)_2-50Alkenyl), for example if present at the molecular end: -CH = CH₂、-CH=CH-CH₃、-CH₂-CH=CH₂、-CH=CH-CH₂-CH₃、-CH=CH-CH=CH₂Or when two parts of the molecule are linked through an alkenyl group, for example-CH = CH-. Optionally, C_2-50One or more hydrogen atoms of an alkenyl carbon may be substituted with further specified substituents. Thus, the term "alkenyl" denotes a carbon chain having at least one carbon-carbon double bond. Optionally, one or more triple bonds are present. The term "C" is defined accordingly_2-15Alkenyl ".

“C_2-50Alkynyl "denotes a branched or unbranched alkynyl chain having 2 to 50 carbon atoms (unsubstituted C)_2-50Alkynyl), for example if present at the molecular end: -C ≡ CH, -CH₂-C≡CH、CH₂-CH₂-C≡CH、CH₂-C≡C-CH₃Or when two moieties of the molecule are linked by an alkynyl group, for example-C.ident.C-. Optionally, C_2-50One or more hydrogen atoms of an alkynyl carbon may be substituted with further specified substituents. Thus, the term "alkynyl" denotes a carbon chain having at least one carbon triple bond. One or more double bonds may optionally be present. The term "C" is defined accordingly_2-15Alkynyl ".

“C_3-7Cycloalkyl "or" C_3-7Cycloalkyl ring "denotes a cyclic alkyl chain having 3 to 7 carbon atoms, which may have an at least partially saturated carbon-carbon double bond (unsubstituted C)_3-7Cycloalkyl), such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Optionally, one or more hydrogen atoms of a cyclic hydrocarbyl carbon may be substituted with a substituent as shown herein. The term "C_3-7Cycloalkyl "or" C_3-7Cyclic hydrocarbyl rings "also include bridged bicyclic rings, such as norbornane (norbornyl) or norbornene (norbornenyl). Thus, "C_3-5The cycloalkyl group "means a cycloalkyl group having 3 to 5 carbon atoms. Thus, "C_3-10The cycloalkyl group "means a cycloalkyl group having 3 to 10 carbon atoms. The term "C" is defined accordingly_5-6A cyclic hydrocarbon group ".

"halogen" means fluorine, chlorine, bromine or iodine. It is generally preferred that the halogen is fluorine or chlorine.

"4-to 7-membered heterocyclyl" or "4-to 7-membered heterocycle" means a ring having 4, 5, 6 or 7 ring atoms which may contain the maximum number of double bonds (aromatic or non-aromatic rings, which are fully, partially or unsaturated), wherein at least one ring atom has up to 4 ring atoms selected from sulfur (including-S (O) -, -S (O))₂-), oxygen and nitrogen (including heteroatoms = n (o) -, and wherein the ring is attached to the remainder of the molecule through a carbon or nitrogen atom (an unsubstituted 4-to 7-membered heterocyclyl group). For completeness, in some embodiments of the invention it means that the 4 to 7 membered heterocyclyl group must meet other requirements. Examples of 4-to 7-membered heterocycles are azetidine, propylene oxide, cyclopropane sulfide (thietane), furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, pyridine,Azole,Oxazoline, heteroOxazole, isoOxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, and,Oxazolidine, isozymeOxazolidines, thiazolidines, isothiazolidines, thiadiazolidines, sulfolanes, pyrans, dihydropyrans, tetrahydropyrans, imidazolidines, pyridines, pyridazines, pyrazines, pyrimidines, piperazines, piperidines, morpholines, tetrazoles, triazoles, triazolanes, tetrazoles, diazepanes, aza-cycloheptanesOr homopiperazine. Optionally, one or more hydrogen atoms of the 4-to 7-membered heterocyclic group may be replaced by a substituent. The terms "3-to 7-membered heterocyclyl" and "5-or 6-membered heterocyclyl" are defined accordingly.

"8 to 11 membered heterobicyclic group" or "8 to 11 membered heterobicyclic" means a heterocyclic ring system of two rings having 8 to 11 ring atoms, wherein at least one ring atom is shared by the two rings, and which may contain the maximum number of double bonds (aromatic or non-aromatic rings, which are full, partial or unsaturated), wherein at least one ring atom has up to 6 ring atoms selected from sulfur (including-s (o) -, -s (o))₂-), oxygen and nitrogen (including heteroatoms = n (o) -, and wherein the ring is attached to the remainder of the molecule through a carbon or nitrogen atom (an unsubstituted 8-to 11-membered heterobicyclic group). Examples of 8-to 11-membered heterobicyclics are indole, indoline, benzofuran, benzothiophene, benzoAzole, benzisohOxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepinePurine or pteridine. The term 8-to 11-membered heterobicyclic also includes two ring spiro structures, e.g., 1, 4-dioxa-8-azaspiro [4.5 ]]Decane or bridged heterocycles, e.g. 8-aza-bicyclo [3.2.1]Octane. The term "9 to 11 membered heterobicyclic group" or "9 to 11 membered heterobicyclic" is defined accordingly.

The term "aliphatic" means fully saturated.

The term "interrupted" denotes a different carbon-terminal insertion group (e.g., -O-or-NH-) between two carbon atoms, e.g., a linker or spacer, or between a different carbon atom and a hydrogen atom.

In general, the term "substituted" preferably denotes substituents, which are identical or different and which are independently selected from halogen, CN, COOR^b9、OR^b9、C(O)R^b9、C(O)N(R^b9R^b9a)、S(O)₂N(R^b9R^b9a)、S(O)N(R^b9R^b9a)、S(O)₂R^b9、S(O)R^b9、N(R^b9)S(O)₂N(R^b9aR^b9b)、SR^b9、N(R^b9R^b9a)、NO₂、OC(O)R^b9、N(R^b9)C(O)R^b9a、N(R^b9)S(O)₂R^b9a、N(R^b9)S(O)R^b9a、N(R^b9)C(O)OR^b9a、N(R^b9)C(O)N(R^b9aR^b9b)、OC(O)N(R^b9R^b9a)、T^b、C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50An alkynyl group,

wherein T is^b、C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl is optionally substituted by one or more R^b10Substituted, said R^b10Are the same or different, and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl group is optionally interrupted by one or more groups selected from: t is^b；-C(O)O-；-O-；-C(O)-；-C(O)N(R^b11)-；-S(O)₂N(R^b11)-；-S(O)N(R^b11)-；-S(O)₂-；-S(O)-；-N(R^b11)S(O)₂N(R^b11a)-；-S-；-N(R^b11)-；-OC(O)R^b11；-N(R^b11)C(O)-；-N(R^b11)S(O)₂-；-N(R^b11)S(O)-；-N(R^b11)C(O)O-；-N(R^b11)C(O)N(R^b11a) -; and-OC (O) N (R)^b11R^b11a)；

R^b9、R^b9a、R^b9bIndependently selected from H; t is^b(ii) a And C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50An alkynyl group,

T^bSelected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C_3-10Cycloalkyl, 4-to 7-membered heterocyclyl and 9-to 11-membered heterobicyclic group, wherein T^bOptionally substituted by one or more R^b10Substituted, said R^b10Are the same or different and are each a different,

R^b10is halogen, CN, oxo (= O), COOR^b12、OR^b12、C(O)R^b12、C(O)N(R^b12R^b12a)、S(O)₂N(R^b12R^b12a)、S(O)N(R^b12R^b12a)、S(O)₂R^b12、S(O)R^b12、N(R^b12)S(O)₂N(R^b12aR^b12b)、SR^b12、N(R^b12R^b12a)、NO₂、OC(O)R^b12、N(R^b12)C(O)R^b12a、N(R^b12)S(O)₂R^b12a、N(R^b12)S(O)R^b12a、N(R^b12)C(O)OR^b12a、N(R^b12)C(O)N(R^b12aR^b12b)、OC(O)N(R^b12R^b12a) Or C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more halogens, which are the same or different,

R^b11、R^b11a、R^b12、R^b12a、R^b12bindependently selected from H; c_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more halogens, which may be the same or different.

The term "interrupted" means that there is an intervening group between two carbons, or a group inserted at the end of the carbon chain between a carbon and a hydrogen.

In general, the term "dashed line" used to indicate the attachment of one moiety to another moiety is different from the dashed line used to indicate stereochemistry. One skilled in the art can distinguish between the two.

Typically the term "comprising" or "includes" also includes "consisting of … ….

The term "water-soluble" in "water-soluble carrier" is a carrier that is soluble in water at room temperature. Typically, a solution of a water-soluble carrier transmits at least about 75%, more preferably at least about 95%, of the light, after filtration, through the same solution. On a weight basis, the water-soluble carrier or portion thereof is preferably at least about 35% by weight soluble in water, more preferably at least about 50% by weight soluble in water, more preferably about 70% by weight soluble in water, and more preferably about 85% by weight soluble in water. Most preferably, however, the water-soluble carrier or portion thereof is about 95% by weight soluble in water or completely soluble in water.

The carrier-linked treprostinil prodrugs of the present invention comprise a treprostinil moiety. Treprostinil in purified form or a pharmaceutically acceptable salt thereof is a drug known per se to those skilled in the art.

As used herein, the dosage of treprostinil compound alone is given in mg, and the concentration of treprostinil compound in the pharmaceutical composition is given in mg/mL. Since treprostinil compounds are carrier-linked prodrugs, the concentration is based on the amount of free treprostinil released by the prodrug. Aliquots of the compositions were subjected to treprostinil release conditions (aqueous buffer, pH7.4, 37 ℃, or accelerated conditions at elevated pH) by methods well known in the art until no significant increase in treprostinil concentration was observed and the total amount of treprostinil released was determined.

In the present invention, the carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof does not comprise treprostinil in free form or a pharmaceutically acceptable salt thereof, but is in bound form. Trionile is bound to the rest of the molecule via its functional groups, for example via hydroxyl or carboxyl groups, and as part of the moiety T it is linked to the moiety X⁰Or if m1 and m2 are both 0, to a moiety Z of formula (I)¹The above. This means that the carrier-linked treprostinil prodrug of the invention comprises treprostinil as the biologically active moiety. As the biologically active moiety is cleaved from the carrier-linked treprostinil prodrug when administered to a patient in need thereof, treprostinil is released in its free form or a pharmaceutically acceptable salt thereof. In other words, the carrier-linked treprostinil prodrug comprises one or more moieties T, each of which is bound by a moiety X⁰Substitution (provided that at least one of m1 and m2 is 1), which in turn is covalently bound to the support Z¹The above. The carrier comprises a covalently bound polymer, preferably a pharmaceutically acceptable polymer having a molecular weight of at least 500 daltons.

In another preferred embodiment, the molecular weight of the polymer, preferably the pharmaceutically acceptable polymer, is at most 160kDa, preferably at most about 100kDa, even more preferably at most about 50 kDa.

Preferably, the moiety X⁰(provided that at least one of m1 and m2 is 1) and moiety T are linked by a carbonate or ester linkage, most preferably moiety X⁰And moiety T are linked by an ester linkage.

Preferably, the moiety X⁰Is unsubstituted. More preferably, each portion X⁰Is unsubstituted.

In a preferred embodiment, m1 is 0 and m2 is 1.

In another preferred embodiment, both m1 and m2 are 0.

In another preferred embodiment, both m1 and m2 are 1.

Preferably, the substructure X⁰-Z¹Is C (R)¹R²)-CH₂-Z¹Wherein R is¹、R²Independently selected from H and C_1-4Alkyl group with the proviso that at least R¹、R²One is not H; or (CH)₂)_n-Z¹Wherein n is 2,3, 4, 5, 6, 7 or 8.

Preferably, the vector Z¹Is covalently linked to the moiety X through an amide group⁰The above.

Preferably, R³Is halogen; CN; c (O) R⁴；C(O)OR⁴；OR⁴；C(O)R⁴；C(O)N(R⁴R^4a)；S(O)₂N(R⁴R^4a)；S(O)N(R⁴R^4a)；S(O)₂R⁴；S(O)R⁴；N(R⁴)S(O)₂N(R^4aR^4b)；SR⁴；N(R⁴R^4a)；NO₂；OC(O)R⁴；N(R⁴)C(O)R^4a；N(R⁴)SO₂R^4a；N(R⁴)S(O)R^4a；N(R⁴)C(O)N(R^4aR^4b)；N(R⁴)C(O)OR^4a；OC(O)N(R⁴R^4a) (ii) a Or T⁰。

Preferably, R⁴、R^4a、R^4bIndependently selected from H; t is⁰；C_1-4An alkyl group; c_2-4An alkenyl group; and C_2-4Alkynyl, wherein C_1-4An alkyl group; c_2-4An alkenyl group; and C_2-4Alkynyl is optionally substituted by one or more R⁵Substituted, said R⁵Are the same or different.

More preferably, the carrier-linked treprostinil prodrug is of formula (II):

wherein each T is independently selected from structures (i) to (v) (preferably (iii)):

wherein the dotted line represents the attachment to the rest of the molecule;

y is an integer in the range of 1 to 64, preferably in the range of 1 to 16, more preferably y is selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, even more preferably y is 4,6, 8, 10 or 12; even more preferably y is 4,6 or 8; most preferably 4;

R^a1selected from unsubstituted alkyl groups; a substituted alkyl group; unsubstituted phenyl; substituted phenyl; an unsubstituted naphthyl group; substituted naphthyl; an unsubstituted indenyl group; a substituted indenyl group; unsubstituted indanyl; substituted indanyl; unsubstituted tetralinyl; substituted tetralinyl; unsubstituted C_3-10A cyclic hydrocarbon group; substituted C_3-10A cyclic hydrocarbon group; an unsubstituted 4-to 7-membered heterocyclyl; a substituted 4-to 7-membered heterocyclyl; unsubstituted 9-to 11-membered heterobicyclic group; and substituted 9-to 11-membered heterobicyclic groups;

R^a2selected from the group consisting of H, unsubstituted alkyl, and substituted alkyl;

R^a3and R^a4Independently selected from the group consisting of H, unsubstituted alkyl, and substituted alkyl;

n is 0 or 1;

optionally, R^a1And R^a3Taken together with the atoms to which they are attached to form ring a;

a is selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c_3-10A cyclic hydrocarbon group; a 4-to 7-membered aliphatic heterocyclic group; or a 9-to 11-membered aliphatic heterobicyclic group, wherein A is unsubstituted or substituted;

preferably, A is selected from C_3-10A cyclic hydrocarbon group; a 4-to 7-membered aliphatic heterocyclic group; and 9-to 11-membered aliphatic heterobicyclic groups, wherein a is unsubstituted or substituted;

q is a spacer moiety;

Z¹is a carrier comprising a covalently bound polymer, preferably a pharmaceutically acceptable polymer;

or a pharmaceutically acceptable salt thereof.

Preferably, R^a1Is C_1-6Alkyl or substituted C_1-6Alkyl, more preferably C_1-4Alkyl or substituted C_1-4An alkyl group.

More preferably, R^a1Selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and benzyl.

Preferably, R^a2Is H.

Preferably, R^a3Is H, C_1-6Alkyl or substituted C_1-6Alkyl, more preferably C_1-4Alkyl or substituted C_1-4An alkyl group. More preferably, R^a3Selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and benzyl.

More preferably, R^a3Is H.

Preferably, R^a4Selected from H, C_1-6Alkyl or substituted C_1-6Alkyl, more preferably C_1-4Alkyl or substituted C_1-4An alkyl group. More preferably, R^a4Selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and benzyl.

More preferably, R^a4Is H.

In another preferred embodiment, R^a1And R^a3Taken together with the atoms to which they are attached to form ring a; wherein A is selected from cyclopropane, cyclobutane, cyclopentane, cyclohexane and cycloheptane. Even more preferably cyclohexane.

Preferably, Q in formula (II) is selected from COOR^a9；OR^a9；C(O)R^a9；C(O)N(R^a9R^a9a)；S(O)₂N(R^a9R^a9a)；S(O)N(R^a9R^a9a)；S(O)₂R^a9；S(O)R^a9；N(R^a9)S(O)₂N(R^a9aR^a9b)；SR^a9；N(R^a9R^a9a)；OC(O)R^a9；N(R^a9)C(O)R^a9a；N(R^a9)S(O)₂R^a9a；N(R^a9)S(O)R^a9a；N(R^a9)C(O)OR^a9a；N(R^a9)C(O)N(R^a9aR^a9b)；OC(O)N(R^a9R^a9a)；W；C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50An alkynyl group,

w, C therein_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl is optionally substituted by one or more R^a10Substituted, said R^a10Are the same or different and are each a different,

and wherein C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50The alkynyl group is optionally interrupted by one or more groups selected from: -W-; -C (O) O-; -O-; -c (o) -; -C (O) N (R)^a11)-；-S(O)₂N(R^a11)-；-S(O)N(R^a11)-；-S(O)₂-；-S(O)-；-N(R^a11)S(O)₂N(R^a11a)-；-S-；-N(R^a11)-；-OC(O)R^a11；-N(R^a11)C(O)-；-N(R^a11)S(O)₂-；-N(R^a11)S(O)-；-N(R^a11)C(O)O-；-N(R^a11)C(O)N(R^a11a) -; and-OC (O) N (R)^a11R^a11a)；

R^a9、R^a9a、R^a9bIndependently selected from H; w; and C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50An alkynyl group,

and wherein C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50The alkynyl group is optionally interrupted by one or more groups selected from: w; -C (O) O-; -O-; -c (o) -; -C (O) N (R)^a11)-；-S(O)₂N(R^a11)-；-S(O)N(R^a11)-；-S(O)₂-；-S(O)-；-N(R^a11)S(O)₂N(R^a11a)-；-S-；-N(R^a11)-；-OC(O)R^a11；-N(R^a11)C(O)-；-N(R^a11)S(O)₂-；-N(R^a11)S(O)-；-N(R^a11)C(O)O-；-N(R^a11)C(O)N(R^a11a) -; and-OC (O) N (R)^a11R^a11a)；

W is selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c_3-10A cyclic hydrocarbon group; 4-to 7-membered heterocyclyl; or 9-to 11-membered heterobicyclic group wherein W is optionally substituted with one or more R^a10Substituted, said R^a10Are the same or different and are each a different,

R^a10is halogen; CN; oxo (= O); COOR^a12；OR^a12；C(O)R^a12；C(O)N(R^a12R^a12a)；S(O)₂N(R^a12R^a12a)；S(O)N(R^a12R^a12a)；S(O)₂R^a12；S(O)R^a12；N(R^a12)S(O)₂N(R^a12aR^a12b)；SR^a12；N(R^a12R^a12a)；NO₂；OC(O)R^a12；N(R^a12)C(O)R^a12a；N(R^a12)S(O)₂R^a12a；N(R^a12)S(O)R^a12a；N(R^a12)C(O)OR^a12a；N(R^a12)C(O)N(R^a12aR^a12b)；OC(O)N(R^a12R^a12a) (ii) a Or C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more halogens, which are the same or different;

R^a11、R^a11a、R^a12、R^a12a、R^a12bindependently selected from H; and C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more halogens, which may be the same or different.

More preferably, each-Q-is independently-Q^1a-Q¹-, wherein the asterisk indicates the link to Z¹And wherein

Q^1aIs a bond; -C (O) O-; -O-; -c (o) -; -C (O) N (R)^a9a)-；-S(O)₂N(R^a9a)-；-S(O)N(R^a9a)-；-S(O)₂-；-S(O)-；-N(R^a9a)S(O)₂N(R^a9b)-；-S-；-N(R^a9a)-；-OC(O)-；-N(R^a9a)C(O)-；-N(R^a9a)S(O)₂-；-N(R^a9a)S(O)-；-N(R^a9a)C(O)O-；-N(R^a9a)C(O)N(R^a9b)-；-OC(O)N(R^a9a) -; or-W-, preferably-C (O) N (R)^a9a) -or-N (R)^a9a)C(O)-；

Q¹Is selected from C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl optionally substituted by one or more R^a10Substituted, optionally interrupted by one or more groups selected from (with the proviso that Q is¹Is at least C₂)：C_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

Wherein each of said groups may be independently present one or more times; and C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl may optionally be attached to Z¹Is terminated by a group selected from: c_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

R^a12、R^a12aand R^a12bIndependently selected from H; and C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more halogens, which are the same or different;

R¹³and R^13aIndependently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group; preferably, R¹³And R^13aIndependently selected from C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6Alkynyl.

In formula (I), the moiety X⁰Is of formula (IIa):

wherein the dotted line with an asterisk indicates attachment to T, and the unlabeled dotted line indicates attachment to the remainder of the carrier-attached treprostinil prodrug;

and is

Q, R therein^a1、R^a2、R^a3And R^a4As defined in formula (II).

Even more preferably, the carrier-linked treprostinil prodrug has the structure of formula (II-a):

wherein each T is independently selected from structures (i) to (v):

wherein the dotted line represents the attachment to the rest of the molecule;

y is an integer in the range of 1 to 64, preferably in the range of 1 to 16, more preferably y is selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, even more preferably y is 4,6, 8, 10 or 12; most preferably 4;

R^a4selected from the group consisting of H, unsubstituted alkyl, and substituted alkyl;

q is a spacer moiety;

Z¹is a carrier comprising a covalently bound polymer, preferably a pharmaceutically acceptable polymer.

Preferably, R of formula (II-A)^a2Is H.

Preferably, R of formula (II-A)^a4Selected from H, C_1-6Alkyl or substituted C_1-6Alkyl, more preferably C_1-4Alkyl or substituted C_1-4An alkyl group. More preferably, R^a4Selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and benzyl.

More preferably, R of formula (II-A)^a4Is H.

Preferably, Q in formula (II-A) is selected from COOR^a9；OR^a9；C(O)R^a9；C(O)N(R^a9R^a9a)；S(O)₂N(R^a9R^a9a)；S(O)N(R^a9R^a9a)；S(O)₂R^a9；S(O)R^a9；N(R^a9)S(O)₂N(R^a9aR^a9b)；SR^a9；N(R^a9R^a9a)；OC(O)R^a9；N(R^a9)C(O)R^a9a；N(R^a9)S(O)₂R^a9a；N(R^a9)S(O)R^a9a；N(R^a9)C(O)OR^a9a；N(R^a9)C(O)N(R^a9aR^a9b)；OC(O)N(R^a9R^a9a)；W；C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50An alkynyl group,

and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl group is optionally interrupted by one or more groups selected from: -W-; -C (O) O-; -O-; -c (o) -; -C (O) N (R)^a11)-；-S(O)₂N(R^a11)-；-S(O)N(R^a11)-；-S(O)₂-；-S(O)-；-N(R^a11)S(O)₂N(R^a11a)-；-S-；-N(R^a11)-；-OC(O)R^a11；-N(R^a11)C(O)-；-N(R^a11)S(O)₂-；-N(R^a11)S(O)-；-N(R^a11)C(O)O-；-N(R^a11)C(O)N(R^a11a) -; and-OC (O) N (R)^a11R^a11a)；

and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl group is optionally interrupted by one or more groups selected from: w; -C (O) O-; -O-; -c (o) -; -C (O) N (R)^a11)-；-S(O)₂N(R^a11)-；-S(O)N(R^a11)-；-S(O)₂-；-S(O)-；-N(R^a11)S(O)₂N(R^a11a)-；-S-；-N(R^a11)-；-OC(O)R^a11；-N(R^a11)C(O)-；-N(R^a11)S(O)₂-；-N(R^a11)S(O)-；-N(R^a11)C(O)O-；-N(R^a11)C(O)N(R^a11a) -; and-OC (O) N (R)^a11R^a11a)；

More preferably, the carrier-linked treprostinil prodrug is of formula (IIaa):

wherein each T is independently selected from the structures (i) or (iii):

wherein the dotted line represents the attachment to the rest of the molecule;

R^a2selected from the group consisting of H, unsubstituted alkyl, and substituted alkyl; preferably, R^a2Selected from H and substituted or unsubstituted C_1-6An alkyl group;

R^a4selected from the group consisting of H, unsubstituted alkyl, and substituted alkyl; preferably, R^a4Selected from H and substituted or unsubstituted C_1-6An alkyl group;

ring A¹Is C_3-10A cyclic hydrocarbon group; a 4-to 7-membered aliphatic heterocyclic group; or a 9-to 11-membered aliphatic heterobicyclic group wherein A¹Is unsubstituted or substituted;

Q¹is selected from C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl optionally substituted by one or more R^a10Substituted, optionally interrupted by one or more groups selected from (with the proviso that Q¹Is at least C₂)：C_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

Wherein each of said groups may be independently present once orMultiple times; and C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl may optionally be attached to Z¹Is terminated by a group selected from: c_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

R^a12、R^a12aand R^a12bIndependently selected from H; and C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more halogens, which are the same or different,

R¹³and R^13aIndependently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group; preferably, R¹³And R^13aIndependently selected from C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

Z¹is comprised of a covalently bound polymer, preferablyA carrier for a pharmaceutically acceptable polymer.

It is understood that R of formula (IIaa)^a4And R^a2Are ortho-positioned.

Preferably, the carrier-linked treprostinil prodrug is of formula (IIab):

wherein, T, R^a2、R^a4、A¹、Q¹、Z¹And y is the use as defined in formula (IIaa).

It is understood that R of formula (IIab)^a4And R^a2Are ortho-positioned.

Preferably, A of formulae (IIaa) and (IIab)¹Selected from cyclopentane, cyclohexane or cycloheptane. More preferably, A¹Is cyclohexane.

Preferably, R of formula (IIaa) or (IIab)^a2And R^a4Are all H.

Preferably Q of formula (IIaa) or (IIab)¹Is selected from C_1-50Alkyl optionally substituted by one or more R^a10Substituted, said R^a10Are the same or different; and wherein C_1-50The alkyl group is optionally interrupted by one or more groups selected from (provided that Q is¹Is at least C₂)：C_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

Wherein each of said groups may be independently present one or more times; and C_1-50Alkyl may optionally be attached to Z¹Is terminated by a group selected from: c_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

And wherein R¹³And R^13aUse as defined in formula (IIaa);

preferably, y of formula (IIaa) or (IIab) is 4,6 or 8. More preferably y of formula (IIaa) or (IIab) is 4 or 8, most preferably y of formula (IIaa) or (IIab) is 4.

Even more preferably, the carrier-linked treprostinil prodrug is of formula (IIac) or (IIad):

t, Z therein¹And y is as defined for formula (IIaa),

x is selected from 2,3, 4, 5, 6, 7 or 8, more preferably x is selected from 3, 4, 5, 6, 7 or 8,

X₁is selected from C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl optionally substituted by one or more R^a10Is substituted, and C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50The alkynyl group is optionally interrupted by one or more groups selected from (provided that X is₁Is at least C₂)：C_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

and wherein R¹³And R^13aUse as defined in formula (IIaa).

Preferably, X of formulae (IIac) and (IIad)₁Is selected from C_1-15Alkyl radical, C_2-15Alkenyl and C_2-15Alkynyl optionally substituted or interrupted by one or more groups selected from: c_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

Wherein R is¹³And R^13aIndependently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6Alkynyl.

More preferably, y of formula (IIac) or (IIad) is 4,6 or 8, even more preferably y is 4 or 8, most preferably y is 4.

More preferably, x of formula (IIac) or (IIad) is 4, 5 or 6, most preferably x is 6.

Even more preferably, the carrier-linked treprostinil prodrug is of formula (IIb):

wherein

X₁and y is as defined in formulae (IIac) and (IIad), and

x is selected from 2,3, 4, 5, 6, 7 or 8.

More preferably, y of formula (IIb) is 4,6 or 8, even more preferably y is 4 or 8, and most preferably y is 4.

More preferably, x of formula (IIb) is 4, 5 or 6, even more preferably x is 5 or 6, and most preferably x is 6.

Most preferably, y in formula (IIb) is 4 and x in formula (IIb) is 6.

More preferably, the carrier-linked treprostinil prodrug of formula (IIb) has the structure of formula (IIba):

wherein Z¹Is the use as defined in formula (IIaa), and y is the use as defined in formulae (IIac) and (IIad).

Preferably, y of formula (IIba) is 4,6 or 8. More preferably, y of formula (IIba) is 4 or 8, most preferably y of formula (IIba) is 4.

More preferably, X of formula (IIac)₁Is that

Wherein q is selected from 1 to 4; preferably, q is 1.

More preferably, X of formula (IIad)¹Is that

Wherein q is selected from 1,2, 3 or 4, and is preferably 2.

Preferably, X of formula (I)⁰Selected from the following structures:

wherein the dotted line marked with an asterisk indicates a connection to T, an

The unlabeled dashed line represents the remainder of the treprostinil prodrug attached to the carrier.

Preferably, all moieties T of the carrier-linked treprostinil prodrug of formula (I), (II), (IIaa), (IIab), (IIac) and (IIad) have the same structure.

Preferably, all moieties T of formula (I) have a structure of formula (v) or formula (ii) or formula (iii), more preferably a structure of formula (v).

Preferably, all moieties T of formulae (II), (IIaa), (IIab), (IIac) and (IIad) have the same structure and are one of formulae (II), (iii) or (iv). More preferably, all moieties T of formulae (II), (IIaa), (IIab), (IIac) and (IIad) have the same structure and are of formulae (II) or (iv).

Preferred substructures of the formula (I) -X⁰-T is selected from the following structures:

wherein the dotted line indicates the connection to Z¹。

In another preferred embodiment, the carrier-linked treprostinil prodrug has the structure of formula (IId):

wherein the dotted line represents T connected to formula (I);

Y^vis-N (R)^1v)-；

X^vis-C (R)^4v)(R^4av)-；-N(R^4v)-；-O-；-C(R^4v)(R^4av)-C(R^5v)(R^5av)-；-C(R^4v)(R^4av)-N(R^6v)-；-N(R^6v)-C(R^4v)(R^4av)-；-C(R^4v)(R^4av)-O-；-O-C(R^4v)(R^4av)-；-C(O)-N(R^6v) -; or-N (R)^6v)-C(O)-；

X^1vIs that

X^2vis-C (R)^7v)(R^7av) -; or-C (R)^7v)(R^7av)-C(R^8v)(R^8av)-；

X^3vIs = O; = S; or = N-CN;

R^1v、R^1av、R^2v、R^2av、R^3v、R^3av、R^4v、R^4av、R^5v、R^5av、R^6v、R^7v、R^7av、R^8v、R^8avindependently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-20Heteroalkyl group and Y₁-T^v(ii) a And independently none, one or more pairings R^1av/R^4av、R^1av/R^5av、R^4av/R^5av、R^7av/R^8avAre absent and the corresponding carbon atoms to which they are attached form a cis double bond;

Y^1vis a chemical bond or C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6An alkynyl group;

T^vis selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c_3-10A cyclic hydrocarbon group; 4-to 7-membered heterocyclyl; or 9-to 11-membered heterobicyclic group, wherein T^vOptionally substituted by one or more R⁹Substituted, said R⁹Are the same or different;

R^9vis halogen; -CN; oxo (= O); -C (O) OH; -OH; -S (O)₂NH₂；-S(O)NH₂；-S(O)₂OH；-S(O)OH；-SH；-NH₂；-NO₂；C_1-6Alkyl or C_1-10A heterocyclic group;

optionally, one or more pairings R^1v/R^1av、R^1v/R^4v、R^1v/R^6v、R^1v/R^5v、R^2v/R^2av、R^2v/R^3v、R^4v/R^4av、R^4v/R^5v、R^5v/R^5av、R^7v/R^7av、R^7v/R^8v、R^8v/R^8avTaken together with the atoms to which they are attached to form a ring T;

optionally, R^3v/R^3avTaken together with the nitrogen atom to which they are attached to form a 4-to 7-membered heterocyclic ring;

and wherein R^1v、R^1av、R^2v、R^2av、R^3v、R^3av、R^4v、R^4av、R^5v、R^5av、R^6v、R^7v、R^7av、R^8v、R^8avA is Z of formula (I)⁰And (4) substitution.

The carrier Z of the formulae (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) and (IIba)¹Comprising a covalently bound polymer, preferably a pharmaceutically acceptable polymer.

Preferred polymers are selected from the group consisting of 2-methacryloyl-oxyethylphosphoryl choline, hydrogels, PEG-based hydrogels, poly (acrylic acid), poly (acrylate), poly (acrylamide), poly (alkyloxy) polymers, poly (amide), poly (amidoamine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), polybutylene terephthalate, poly (caprolactone), poly (carbonate), poly (cyanoacrylate), poly (dimethylacrylamide), poly (ester), poly (ethylene glycol), poly (ethylene oxide), poly (ethylphosphate), poly (ethylacetate), poly (amide), poly (amino acid), poly (anhydride), poly (asparagine)Oxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethylOxazoline), poly (hydroxy methacrylate), poly (hydroxy propyl methacrylamide), poly (hydroxy propyl methacrylate)Oxazoline), poly (iminocarbonate), poly (lactic acid-glycolic acid copolymer), poly (methacrylamide), poly (methacrylate), poly (methyl methacrylate)Oxazolines, poly (organophosphazenes), poly (orthoesters), poly(s)Oxazoline), poly (propylene glycol), poly (siloxane), poly (urethane), poly (vinyl alcohol), poly (vinylamine), poly (vinyl methyl ether), poly (vinyl pyrrolidone), siloxane, cellulose, formyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate based polymers, xylan and copolymers thereof.

Preferably, the carrier Z of the formulae (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) and (IIba)¹Comprising poly (A), (B), (C), (Oxazoline) or PEG-based polymers. More preferably, the vector Z¹Comprising a PEG-based polymer.

In one embodiment, the carrier Z of the formulae (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) and (IIba)¹May be a hydrogel (as an alternative to polymers) as is known in the artAlternative). Suitable hydrogels are described in WO-A2006/003014 or EP-A1625856. If the vector Z is¹Is a hydrogel, preferably it is a PEG-based hydrogel as disclosed in WO-a2011/012715, which is incorporated herein by reference.

Preferably, the carrier Z of the formulae (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) and (IIba)¹Is a water soluble carrier.

In one embodiment, the carrier Z of the formulae (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) and (IIba)¹Having the structure of formula (III):

wherein the dotted lines respectively indicate the connection to X⁰(if the carrier-linked treprostinil prodrug is of formula (I)), attached to Q (if the carrier-linked treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-attached treprostinil prodrug is of formula (IIaa) or (IIab)), attached to X₁(if the carrier-linked treprostinil prodrug is of formulae (IIac), (IIad) and (IIb)) or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)), and

wherein m, n and p of each formula (III) are independently integers in the range of 5 to 500,

and wherein q of formula (III) ranges from 2 to 32.

In one embodiment, the vector Z of formulae (I), (II), (IIaa), (IIab), (IIac), (IIad) and (IIb)¹A structure having formula (IIIa):

wherein the dotted lines respectively indicate the connection to X⁰(if carrier-linked TreponinilThe prodrug is of formula (I)), attached to Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X₁(if the carrier-linked treprostinil prodrug is of formulae (IIac), (IIad) and (IIb)), and

wherein m, n and p of each formula (IIIa) are independently integers in the range of 5 to 500,

and wherein q of formula (IIIa) ranges from 2 to 32.

Preferably, q in formulae (III) and (IIIa) is an integer ranging from 2 to 14, and more preferably q in formulae (III) and (IIIa) is 6.

Preferably, each of m, n and p in formulae (III) and (IIIa) independently ranges from 10 to 250, more preferably from 50 to 150. Preferably, m, n and p in formulae (III) and (IIIa) are the same.

In an alternative embodiment, the carrier Z of the formulae (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) and (IIba)¹Having the structure of formula (IV):

wherein the dotted lines respectively indicate the connection to X⁰(if the carrier-linked treprostinil prodrug is of formula (I)), attached to Q (if the carrier-linked treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X₁(if the carrier-linked treprostinil prodrug is of formulae (IIac), (IIad) and (IIb)) or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)), with the proviso that one of m1, m2 is 1, and wherein the carrier is covalently linked to T if m1, m2=0, and

wherein m, n and p of each formula (IV) are independently integers ranging from 5 to 500, and wherein q of formula (IV) ranges from 0 to 14.

Preferably, q in formula (IV) is an integer ranging from 2 to 6, and more preferably q of formula (IV) is 2.

Preferably, m, n and p of each formula (IV) are independently in the range of 10 to 250, more preferably 50 to 150. Preferably, m, n and p in formula (IV) are the same.

In another preferred embodiment, Z of formulae (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb), and (IIba)¹Having the structure of formula (V):

Hyp¹ _mx-POL^x-Hyp² (V)，

wherein

POL^xIs a polymeric moiety having a molecular weight in the range of 0.5kDa to 160kDa,

Hyp¹and Hyp²Independently a highly branched portion, and

mx is 0 or 1.

Polymeric moiety POL^xHas a molecular weight of 0.5kDa to 160kDa, preferably 2kDa to 80kDa, and more preferably 5kDa to 40 kDa.

POL^xMay be selected from the following polymers: for example, polypeptides, 2-methacryloyl-oxyethylphosphoryl choline, water-soluble hydrogels, water-soluble PEG-based hydrogels, water-soluble hyaluronic acid-based hydrogels, poly (acrylic acid), poly (acrylate), poly (acrylamide), poly (alkoxy) polymers, poly (amide), poly (amidoamine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), polybutylene terephthalate, poly (caprolactone), poly (carbonate), poly (cyanoacrylate), poly (dimethylacrylamide), poly (ester), poly (ethylene glycol), poly (ethylene oxide), poly (ethylphosphate), poly (ethylhexylate), poly (ethylene oxide), poly (ethylphosphate), poly (isopropylester), polyOxazoline), poly (glycolic acid)) Poly (hydroxyethyl acrylate), poly (hydroxyethyl acrylate)Oxazoline), poly (hydroxy methacrylate), poly (hydroxypropyl methacrylamide), poly (hydroxypropyl methacrylate)Oxazoline), poly (iminocarbonate), poly (lactic acid-glycolic acid copolymer), poly (methacrylamide), poly (methacrylate), poly (methyl methacrylate)Oxazolines, poly (organophosphazenes), poly (orthoesters), poly(s)Oxazoline), poly (propylene glycol), poly (siloxane), poly (urethane), poly (vinyl alcohol), poly (vinylamine), poly (vinyl methyl ether), poly (vinyl pyrrolidone), siloxane, cellulose, formyl cellulose, hydroxypropyl methyl cellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate based polymers, xylan and copolymers thereof.

The polymeric moiety POL of formula (V)^xMay comprise linear or branched polymers. Preferably, POL of formula (V)^xComprising, in particular consisting of, a linear polymer.

In a preferred embodiment, the POL of formula (V)^xComprising a PEG-based polymer or poly(s) ((s))Oxazoline) polymers, more preferably linear PEG-based polymers, in particular from PEG-based polymers or poly(s)Oxazoline), more preferably linear PEG based polymers. Even more preferably, POL of formula (V)^xConsisting of a PEG-based linear polymer.

If m in formula (V) is 0, POL of formula (V) is preferred^xComprising the formula X1- (OCH)₂CH₂)_p-O-(CH₂)_n-X2-, preferably of the formula X1- (OCH)₂CH₂)_p-O-(CH₂)_n-X2-, wherein n is selected from 2,3 or 4; p is an integer ranging from 5 to 2000, preferably p is an integer ranging from 10 to 1000, and more preferably p is an integer ranging from 100 to 1000; and X2 is a covalently linked POL of formula (V)^xAnd Hyp²A functional group of (a); and X1 is selected from H, CH₃And C₂H₅。

If m in formula (V) is 1, POL of formula (V) is preferred^xComprising the formula X3- (CH)₂)_n1-(OCH₂CH₂)_p-O-(CH₂)_n2-X2-, preferably of the formula X3- (CH)₂)_n1-(OCH₂CH₂)_p-O-(CH₂)_n2-X2-, wherein n1 and n2 are independently selected from 2,3 and 4; p is an integer in the range of 5 to 2000, preferably p is an integer in the range of 10 to 1000, more preferably p is an integer in the range of 100 to 1000; and X2 and X3 are each covalently linked POL of formula (V)^xAnd Hyp²A functional group of (1).

In a preferred embodiment, mx in formula (V) is 0.

In another preferred embodiment, the POL of formula (V)^xIs a polypeptide (or protein), particularly a non-immunogenic polypeptide as described below.

Preferably, the polymeric moiety POL of formula (V)^xIs a polypeptide which comprises at least about 100 amino acid residues, in particular consists of at least about 100 amino acid residues. In a preferred embodimentIn this case, amino acids selected from alanine, serine and/or proline residues are present, in particular predominantly, and the polypeptide part preferably has a random coil conformation under physiological conditions. It will be appreciated that such a polymeric moiety POL of formula (V)^xThe random coil can be formed temporarily or instantaneously, for example when present as a lyophilizate or a dry composition.

The polypeptide moiety POL of formula (V)^xCan have a random coil conformation and the amino acid sequence consists of up to about 1000 amino acid residues, preferably up to about 900 amino acid residues, more preferably up to about 800 amino acid residues, even more preferably up to about 700 amino acid residues, and particularly preferably up to about 600 amino acid residues. Thus, an amino acid sequence forming a random coil conformation may consist of up to about 500 amino acid residues or up to about 450 amino acid residues.

It is also speculated herein that the amino acid sequence forming the random coil conformation may consist of up to about 1200 and up to about 1500 amino acid residues. Thus, an amino acid sequence that forms a random coil conformation can consist of about 100 to about 1500 amino acid residues.

In particular embodiments, the amino acid sequence forming the random coil conformation consists of about 100 to 1000 amino acid residues characterized herein, i.e., described below including alanine, serine, and/or proline as the predominant or only residue.

In a preferred embodiment, the polypeptide moiety POL of formula (V)^xConsisting essentially of one, two or three amino acid residues alanine, serine and proline, whereby the proline residue preferably represents about 4% to about 40% of the polypeptide moiety POL of formula (V)^x. The alanine and serine residues comprise the remaining at least 60% to 96% of the polypeptide moiety POL of formula (V)^x. However, the polypeptide moiety POL of formula (V) is described in detail herein below^xFurther amino acids other than alanine, serine and proline may also be included, i.e. as minor constituents.

The term "minor constituent" as used herein means that up to 10% (i.e. up to 10 of 100 amino acids) may not be alanine, serine and proline, preferably up to 8% (i.e. up to 8 of 100 amino acids) may not be alanine, serine and proline, more preferably up to 6% (i.e. up to 6 of 100 amino acids) may not be alanine, serine and proline, even more preferably up to 5% (i.e. up to 5 of 100 amino acids) may not be alanine, serine and proline, particularly preferably up to 4% (i.e. up to 4 of 100 amino acids) may not be alanine, serine and proline, more particularly preferably up to 3% (i.e. up to 3 of 100 amino acids) may not be alanine, serine and proline, even more particularly preferably up to 2% (i.e. up to 2 of 100 amino acids) may not be alanine, serine and proline, Serine and proline, and most preferably up to 1% (i.e., up to 1 out of 100 amino acids) may be other than alanine, serine and proline. The amino acids other than alanine, serine and proline may be selected from the group other than alanine, serine and proline, from natural or protein derived amino acids including Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, Val, selenocysteine, selenomethionine and hydroxyproline. The minor component may also be selected from non-naturally occurring amino acids.

The term "at least about 100/150/200/250/300/300/350 (etc.) amino acid residues" is not limited to the exact number of amino acid residues, but also includes amino acid extensions that comprise an additional 10% to 20%, or comprise 10% to 20% fewer residues. For example, "at least about 100 amino acid residues" may also include 80 to 100 and about 100 to 120 amino acid residues without departing from the spirit of the present invention.

In one embodiment, the polypeptide moiety POL of formula (V)^xComprising a plurality of polymer cassettes, wherein said polymer cassettes are composed of one, two or three amino acids selected from Ala, Ser and Pro, and wherein no more than 6 consecutive amino acid residues are identical, and wherein said proline residues constitute more than 4% and less than 40% of said polypeptide moiety POL of formula (V)^xThe amino acid of (1).

The polypeptide moiety POL of formula (V)^xIt may comprise a plurality, in particular 2,3, 4, 5 or more identical polymeric cassettes or a plurality of different polymeric cassettes. Non-limiting examples of polymeric cassettes consist of Ala, Ser, and Pro residues provided below; see SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 14 or peptide fragments or multimers of these sequences. The polymer cassettes may be composed of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein each polymer cassette comprises Ala, Ser and Pro residues.

In one embodiment, the polymeric cassettes of the present invention do not comprise more than 100 amino acid residues. Preferably, the polymer cassette as defined herein comprises more than about 4%, preferably more than about 5%, even more preferably more than about 6%, particularly preferably more than about 8%, more particularly preferably more than about 10%, even more particularly preferably more than about 15% and most preferably more than about 20% proline residues. Such polymer cassettes as defined herein preferably comprise less than about 40% or less than about 35% proline residues.

In a preferred embodiment, the polypeptide moiety POL of formula (V)^xComprising, in particular consisting of, formula (a):

Ser_x[Ala_ySer_z]_n (a)，

wherein the formula further comprises a proline residue as defined herein, and wherein

x is independently selected from the integer 0 to 6,

each y is independently selected from an integer ranging from 1 to 6,

each z is independently selected from an integer ranging from 1 to 6.

n is any integer such that the polypeptide moiety POL of formula (V)^xFrom at least about 100 amino acid residues and in particular toFrom about 100 to about 3000 amino acid residues, preferably to about 2000, more preferably to about 1000 amino acid residues.

In another preferred embodiment, the polypeptide moiety POL of formula (V)^xComprises no more than 5 identical consecutive amino acid residues, more preferably no more than 4 identical consecutive amino acid residues, and most preferably no more than 3 identical consecutive amino acid residues.

As indicated above, in one embodiment the polypeptide moiety POL of formula (V)^xComprising proline residues which constitute more than about 4%, preferably more than about 5%, even more preferably more than about 6%, particularly preferably more than about 8%, more particularly preferably more than about 10%, even more particularly preferably more than about 15% and most preferably more than about 20% of the POLs of formula (V)^xThe amino acid of (1).

In another preferred embodiment, the polypeptide moiety POL of formula (V)^xContains more than about 4% but less than about 50%, preferably more than about 10% but less than about 50%, and most preferably more than about 20% but less than about 50% of the POL polypeptide portion constituting formula (V)^xProline residue of the amino acid of (1).

In a further preferred embodiment, the polypeptide moiety POL of formula (V)^xContains more than about 4% and less than about 50%, preferably more than about 10% but less than about 50%, and most preferably more than about 20% but less than about 50% of the POL polypeptide portion constituting formula (V)^xSerine residue of the amino acid of (1).

Preferably, the polypeptide moiety POL of formula (V)^xPOL comprising a polypeptide moiety of formula (V)^xAbout 35% proline residues, about 50% alanine residues and about 15% serine residues of the amino acids of (a). Alternatively, the polypeptide moiety POL of formula (V)^xMay comprise a polypeptide moiety POL of formula (V)^xAbout 35% proline residues, about 15% alanine residues and about 50% serine residues of the amino acids of (a).

Preferably, the polypeptide moiety POL of formula (V)^xComprising one or more of the following alanine-serine polymer cassettes:

SEQ ID NO:1

AAAASSASSASSSSSAAASA

SEQ ID NO：2

AASAAASSAAASAAAASASS

SEQ ID NO:3

ASASASASASASSAASAASA

SEQ ID NO:4

SAASSSASSSSAASSASAAA

SEQ ID NO：5

SSSSAASAASAAAAASSSAS

SEQ ID NO：6

SSASSSAASSSASSSSASAA

SEQ ID NO:7

SASASASASASAASSASSAS

SEQ ID NO：8

ASSAAASAAAASSAASASSS

if the amino acid sequence produced further comprises proline residues as defined above, multimers of these alanine-serine polymer cassettes can form random coil conformations.

In a preferred embodiment, the polypeptide moiety POL of formula (V)^xComprising one or more of the following polymeric cassettes:

SEQ ID NO:9

ASPAAPAPASPAAPAPSAPA

SEQ ID NO:10

AAPASPAPAAPSAPAPAAPS

SEQ ID No:11

APSSPSPSAPSSPSPASPSS

SEQ ID NO:15

SAPSSPSPSAPSSPSPASPS

SEQ ID NO: 15 corresponding to the altered form of the circular sequence of SEQ ID No: 11, wherein the last serine is removed and another serine is added as the starting amino acid. Thus, multimers of such modified sequences have essentially the same internal repeating units as multimers of unmodified sequences, except for the first and last residue. Thus, SEQ ID NO: 15 can be considered as being a polypeptide moiety POL of formula (V)^xExamples of further polymeric cassettes of (a). It will be clear to the skilled person that other polymer cassettes and (shorter) peptide fragments or amino acid polymers provided herein in the form of cyclic sequence alterations may be used as the polypeptide moiety POL of formula (V)^xThe polymer cartridge of (1).

Further and exemplary amino acid polymers that form a random coil conformation can comprise an amino acid sequence selected from the group consisting of:

SEQ ID NO:12

SSPSAPSPSSPASPSPSSPA

SEQ ID NO:13

AASPAAPSAPPAAASPAAPSAPPA

SEQ ID NO:14

ASAAAPAAASAAASAPSAAA

thus, preferred polypeptide moieties POL of formula (V)^xThe polymer cassette of (a) is selected from the following sequences:

ASPAAPAPASPAAPAPSAPA(SEQ ID NO:9)，

AAPASPAPAAPSAPAPAAPS(SEQ ID NO:10),

APSSPSPSAPSSPSPASPSS(SEQ ID NO:11)，

SSPSAPSPSSPASPSPSSPA(SEQ ID NO:12),

AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 13), and

ASAAAPAAASAAASAPSAAA(SEQ ID NO:14);

or a cyclic sequence alteration or multimer of these sequences as a whole or part of these sequences.

Furthermore, peptide fragments or multimers or cyclic sequence variants of these sequences and the sequences provided above may be used as the polypeptide part POL of formula (V) in the context of the present invention^xThe polymer cartridge of (1). One skilled in the art readily generates other amino acid polymer cassettes in one position that form a random coil conformation under physiological conditions and are composed primarily of alanine, serine, and proline as defined herein. The polypeptide moiety POL for formula (V)^xOther and further examples of amino acid polymer cassettes that form random coil conformations may include, inter alia, combinations of the particular polymer cassettes shown above and/or peptide fragments or cyclic sequence alterations.

Thus, exemplary polymeric cassettes may also be provided for individual peptide fragments, which may be recombined to form further polymeric cassettes.

According to the above, the polypeptide moiety POL of formula (V)^xMay comprise a sequence defined by SEQ id no: 9. 10, 11, 12, 13 or 14, or may comprise a multimer of sequences consisting of more than one amino acid sequence of SEQ ID NO: 9. 10, 11, 12, 13 and 14. Furthermore, it is speculated that peptide fragments or cyclic sequence alterations of these exemplary sequences may also be used to construct the polypeptide moiety POL of formula (V)^xFurther polymer cartridges of (1).

In another embodiment, the polypeptide moiety POL of formula (V)^xMay comprise an amino acid sequence selected from SEQ id nos: 9. 10, 11, 12, 13, 14, 15, or multimers of these (cyclic) permuted sequences.

In another embodiment, the polypeptide moiety POL of formula (V)^xMay comprise an amino acid sequence selected from SEQ id nos: 9. 10, 12,13. 14, 15, or a multimer of these exemplary polymeric cassettes.

For producing the polypeptide part POL of formula (V)^xThe peptide fragments of these sequences of (a) may consist of an amino acid sequence of at least 3, preferably at least 4, more preferably at least 5, even more preferably at least 6, more preferably at least 8, especially preferably at least 10, more especially preferably at least 12, even more especially preferably at least 14, even more especially preferably at least 16 and most preferably at least 18 consecutive amino acids selected from the group consisting of the amino acid sequences of SEQ ID NOs: 9. 10, 11, 12, 13 and 14.

For example, individual peptide fragments of a polymeric cassette of the invention may be combined into further individual polymeric cassettes, so long as the above-identified rules regarding the overall distribution and number of alanines, serines and prolines are observed. Moreover, these polymer cassettes may also comprise further amino acid residues, however, only as a minimum or minor constituent, i.e. at most 10%, preferably at most 2%, of the individual polymer cassettes. In one embodiment of the invention, the POL comprising a moiety of formula (V) of the polymer cassette^xConsisting of at least about 100 amino acid residues. The individual polymer cassettes can be combined to form longer polymers of amino acids forming random coils, whereby the polypeptide moiety POL of formula (V)^xHas a maximum length of about 3000 amino acids.

Preferably, POL of formula (V)^xCovalently linked to Hyp of formula (V)¹And Hyp²In particular by permanent attachment, more preferably by permanent amide attachment.

In the carrier-linked treprostinil prodrugs of the invention, Hyp of formula (V)¹And Hyp²The functional groups being respectively bound to moieties X of formula (I)⁰Attached to moiety Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to moiety Q¹(if carrier-linked treprostinilThe prodrug is of formula (IIaa) or (IIab)), or is linked to the moiety X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)).

A highly branched portion of formula (V) Hyp¹And Hyp²Each independently selected from, in particular consisting of: glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran, hyaluronan (hyaluronans), dilysine, trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecalysine, dodecalysine, tridecysine, tetradecysine, pentadecalysine, hexadecysine, heptadecalysine, octadecalysine, nonadecanonalysine, trionithine, tetraornithine, pentaornithine, hexaornithine, heptaornithine, octaornithine, nonaornithine, decaornithine, undecaornithine, dodecaornithine, tridecanine, tetradecanine, pentadecanine, hexadecanine, mannitol, glycerol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran, hyaloronans (hyaloronans), dilysine, pentalysine, heptadecalysine, nonadecaornithine, Heptadecaornithine, octadecaornithine, nonadecaornithine, tris (diaminobutyric acid), tetrakis (diaminobutyric acid), pentakis (diaminobutyric acid), hexakis (diaminobutyric acid), heptakis (diaminobutyric acid), octakis (diaminobutyric acid), nonakis (diaminobutyric acid), decakis (diaminobutyric acid), undeca (diaminobutyric acid), dodecakis (diaminobutyric acid), tridecafluorobutyric acid, tetradecahydrobutyric acid, pentadecakis (diaminobutyric acid), hexadecahydrobutyric acid, heptakis (diaminobutyric acid), octadeca (diaminobutyric acid), nonakis (diaminobutyric acid), di (glutamic acid), tri (glutamic acid), tetra (glutamic acid), pentakis (glutamic acid), hexakis (glutamic acid), heptakis (glutamic acid), octakis (glutamic acid), nonakis (glutamic acid), decakis (glutamic acid), unde, Thirteen (glutamic acid), fourteen (glutamic acid), fifteen (glutamic acid), sixteen (glutamic acid), seventeen (glutamic acid), eighteen (glutamic acid), nineteen (glutamic acid), two (aspartic acid), three (aspartic acid), four (aspartic acid), five (aspartic acid)Aspartic acid), hexa (aspartic acid), hepta (aspartic acid), octa (aspartic acid), nona (aspartic acid), deca (aspartic acid), undec (aspartic acid), dodeca (aspartic acid), tridecyl (aspartic acid), tetradecyl (aspartic acid), pentadeca (aspartic acid), hexadecane (aspartic acid), heptadeca (aspartic acid), octadecane (aspartic acid), nineteen (aspartic acid), polyethyleneimine and low molecular weight PEI.

In a preferred embodiment, the hyperbranched part of the formula (V) is Hyp¹And Hyp²Each independently selected from, in particular consisting of: in combination, dilysine, trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecalysine, dodecalysine, tridecylline, tetradecylline, pentadecalysine, hexadecylline, heptadecalysine, octadecalysine, nonadecalysine, trionithine, tetraornithine, pentaornithine, hexaornithine, heptaornithine, octaornithine, nonaornithine, decaornithine, undecaornithine, dodecaornithine, tridecylline, tetradecanoline, pentadecaornithine, hexadecanoic ornithine, heptadecaornithine, octadecaornithine, nonadecaornithine, tris (diaminobutyric acid), tetrakis (diaminobutyric acid), pentakis (diaminobutyric acid), hexakis (diaminobutyric acid), heptakis (diaminobutyric acid), octakis (diaminobutyric acid), nonakis (diaminobutyric acid), Deca (diaminobutyric acid), undec (diaminobutyric acid), dodeca (diaminobutyric acid), tridecyl (diaminobutyric acid), tetradecyl (diaminobutyric acid), pentadeca (diaminobutyric acid), hexadecyl (diaminobutyric acid), heptadeca (diaminobutyric acid), octadeca (diaminobutyric acid), nonadeca (diaminobutyric acid), di (glutamic acid), tri (glutamic acid), tetra (glutamic acid), penta (glutamic acid), hexa (glutamic acid), hepta (glutamic acid), octa (glutamic acid), nona (glutamic acid), deca (glutamic acid), undec (glutamic acid), dodeca (glutamic acid), tridecyl (glutamic acid), tetradecyl (glutamic acid), pentadeca (glutamic acid), hexadecadry (glutamic acid), heptadeca (glutamic acid), nona (glutamic acid), di (aspartic acid), tri (aspartic acid), tetra (aspartic acid), Penta (aspartic acid), hexa (aspartic acid), hepta (aspartic acid), octa (aspartic acid), nona (aspartic acid)Acid), deca (aspartic acid), undec (aspartic acid), dodeca (aspartic acid), tridecyl (aspartic acid), tetradecyl (aspartic acid), pentadecyl (aspartic acid), hexadeca (aspartic acid), heptadeca (aspartic acid), octadeca (aspartic acid), nineteen (aspartic acid), polyethyleneimine and low molecular weight PEI.

More preferably, the highly branched portion of formula (V) Hyp¹And Hyp²Independently selected from the following, more preferably consisting of: combined forms of trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecalysine, dodecalysine, tridecysine, tetradecysine, pentadecalysine, hexadecysine and heptadecalysine, even more preferably Hyp¹And Hyp²Independently, the following, more preferably consisting of: a conjugated form of trilysine, heptalysine or pentadecalysine.

More preferably, Hyp of formula (V)¹And Hyp²Independently selected from any one of the following structures:

wherein

The dotted line marked with an asterisk indicates the connection toPOL of formula (V)^x，

The unlabeled dotted lines represent the connections to X, respectively⁰(if the carrier-linked treprostinil prodrug is of formula (I)), attached to Q (if the carrier-linked treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)), and

qx is an integer from 0 to 15, preferably from 3 to 7, and even more preferably 6.

Preferably, Hyp of formula (V)¹And Hyp²Each being a heptalysine group, in particular Hyp of the formula (V)¹And Hyp²Each having a structure of formula (ii-x) above.

Preferably, Hyp of formula (V)¹And Hyp²Have the same structure.

Hyp of formula (V)¹And Hyp²The functional group of (A) is used as Hyp of the formula (V)¹And Hyp²Are each independently of X⁰(if the carrier-linked treprostinil prodrug is of formula (I)), with Q (if the carrier-linked treprostinil prodrug is of formula (II)), with Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or with X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or a direct point of attachment to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)). Are not separately associated with X⁰、Q、Q¹Or X₁The remaining functional groups attached may be blocked independently of each other with suitable blocking agents or may optionally be attached to at least one target moiety, in particular by permanent attachment.

Thus, in the water-soluble carrier-linked prodrug of the invention, the highly linked moiety Hyp of formula (V)¹And Hyp²POL connected to formula (V)^xAnd Hyp of formula (V)¹And Hyp²Are respectively connected to X of the formula (I)⁰Attached to Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)), or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)), a permanent linkage, a targeting moiety and/or a capping group.

In a preferred embodiment, the hyperbranched part of the formula (V) is Hyp¹And Hyp²All functional groups of (A) are independently bound to X of formula (I)⁰Attached to Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or to the rest of the molecule (if the carrier-linked prodrug is of formula (IIba)).

Preferably, the highly branched portion of formula (V) is Hyp¹And Hyp²Independently have a molecular weight in the range of 0.1kDa to 4kDa, more preferably 0.4kDa to 2 kDa. Preferably, the highly branched portion of formula (V) is Hyp¹And Hyp²Each independently has at least 3 branches and each independently is connected to at least 4X's respectively⁰、Q、Q¹、X₁Or the remainder of the molecule, permanently attached and/or end-capped groups, and each independently has up to 63 branches, and each independently is attached to up to 64X's, respectively⁰、Q、Q¹、X₁Or the remainder of the molecule, a permanent attachment and/or a capping group. Preferably a hyperbranched part of the formula (V) Hyp¹And Hyp²Each independently having at least 7 branches and each independently connected to at least 8X's, respectively⁰、Q、Q¹、X₁Or the remainder of the molecule, permanently attached and/or end-capping groups, and each independently have up to 31 branches, and each independently are attached to up to 32X's, respectively⁰、Q、Q¹、X₁Or of moleculesThe remainder, permanently attached and/or end-capping groups.

Preferably, the highly branched portion of formula (V) is Hyp¹And Hyp²Each independently a hyperbranched polypeptide. Preferably, such hyperbranched polypeptides comprise lysine in bound form. Preferably, each highly branched moiety of formula (V) Hyp¹And Hyp²Independently have a molecular weight in the range of 0.1kDa to 4kDa, in particular 0.4kDa to 2 kDa.

Preferably, mx is 0, and POL-Hyp of formula (V)²-is selected from the following structures:

wherein

The dotted lines indicate the connections to X, respectively⁰(if the carrier-linked treprostinil prodrug is of formula (I)), attached to Q (if the carrier-linked treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X¹(if the carrier-linked treprostinil prodrug is of formula (IIac) or (IIad)) provided that one of m1, m2 of formula (I) is 1, and wherein the carrier is covalently linked to the T of formula (I) if m1, m2= 0.

px is an integer from 5 to 2000, preferably from 10 to 1000, in particular from 100 to 1000, and

qx is an integer from 0 to 15, preferably from 3 to 7, more preferably qx is 6.

In another preferred embodiment, Z of formulae (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb), and (IIba)¹A structure having formula (VI):

wherein

B is the core of the branch,

each a is independently a poly (ethylene glycol) based polymeric chain,

each Hyp^yIndependently is a branch portion, and

n is an integer from 3 to 32.

In a preferred embodiment, the branched core B of formula (VI) comprises, preferably consists of, a moiety selected from:

polyols containing at least 2 hydroxyl groups (preferably further comprising functional groups, which are preferably further amino or carboxylic acid groups, more preferably further carboxylic acid groups),

preferably, B is selected from glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran and hyaluronan, erythritol, threitol, arabitol, xylitol, ribitol, dulcitol, iditol; more preferred are glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran, and hyaluronan.

Or a polyamine containing at least 2 amine groups (preferably further comprising functional groups, which are preferably further hydroxyl or carboxylic acid groups, more preferably carboxylic acid groups),

preferably selected from ornithine, dignithine, triglycine, tetraornithine, pentaornithine, hexaornithine, heptaornithine, octaornithine, nonaornithine, decaornithine, undecimine, dodecaornithine, tridecanoic, tetradecanoic, pentadecaornithine, hexadecanoic, heptadecaornithine, octadecaornithine, nonadecaornithine, diaminobutyric acid, bis (diaminobutyric acid), tris (diaminobutyric acid), tetrakis (diaminobutyric acid), pentakis (diaminobutyric acid), hexakis (diaminobutyric acid), heptakis (diaminobutyric acid), octakis (diaminobutyric acid), nonakis (diaminobutyric acid), deca (diaminobutyric acid), undecimetic (diaminobutyric acid), dodeca (diaminobutyric acid), tridecanoic (diaminobutyric acid), tetradecanoic (diaminobutyric acid), pentadeca (diaminobutyric acid), hexadecanoic (diaminobutyric acid), heptakis (diaminobutyric acid), Eighteen (diaminobutyric acid), nineteen (diaminobutyric acid), lysine, dilysine, trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecabysine, dodecalysine, tridecysine, tetradecysine, pentadecalysine, hexadecahysine, heptadecalysine, octadecalysine, nonadecalysine, oligolysine, polyethyleneimine and polyvinylamine;

wherein the polyol or polyamine is in bound form.

In a preferred embodiment, the branched core B of formula (VI) comprises, preferably consists of, pentaerythritol.

Preferably, the poly (ethylene glycol) -based polymeric chain a linked to the branched core B of formula (VI) consists of a linear PEG chain, wherein one end is linked to B of formula (VI) and the other end is linked to Hyp of formula (VI)^y. It is to be understood that the PEG-based chain a of formula (VI) may optionally be terminated in the case of a branched PEG chain, and/or may optionally be interrupted by alkyl or aryl groups in the case of a branched or linear PEG chain, and may optionally be substituted by heteroatoms and/or functional groups.

Each substructure A-Hyp of formula (VI) extending from a branched core B of formula (VI)^yIndependently of one another, are identical or different substructures A-Hyp^y. In a preferred embodiment, all the substructures A-Hyp of the formula (VI)^yAre the same.

Each of the formula (VI)A and each Hyp^yMay be independently selected from other moieties A and Hyp^y. Preferably, all the substructures A-Hyp linked to B of formula (VI)^yHave the same structure.

Preferably, the PEG-based polymeric chain a of formula (VI) is connected to B by a permanent connection.

N in formula (VI) is an integer of 3 to 32. Preferably, n is an integer from 3 to 16, more preferably n is an integer from 4 to 8, and most preferably n is 4.

In a preferred embodiment, n of formula (VI) is 4 and m is 2.

In one embodiment, the PEG-based polymeric chain a of formula (VI) is selected from linear or branched PEG-based polymeric chains. Preferably, a is a linear PEG-based polymeric chain.

Preferably, each A of formula (VI) is independently selected from formula (la)

-X3-(CH₂)_n1-(OCH₂CH₂)_p-O-(CH₂)_n2-X2-,

Wherein

n1 and n2 are independently selected from 1,2, 3 and 4, preferably from 1,2 and 3;

p is an integer in the range of 5 to 2000, preferably p is an integer in the range of 10 to 1000, more preferably p is an integer in the range of 100 to 1000; and is

X3 and X2 are independently covalently linked to B or Hyp, respectively^yA functional group of (1).

Preferably, part A and part Hyp of formula (VI)^yIs a permanent connection, more preferably the permanent connection comprises a linking group comprising, in particular consisting of, a group selected from: amine groups, amide groups, carbamate (carbamate) groups, thioether groups, ether groups, and most preferably part A and part Hyp of formula (VI)^yThe permanent linkage between them is an amide linkage.

In a preferred embodiment, the substructure of formula (VI)Are detailed in the product list of multi-arm PEG derivatives, such as JenKem Technology, USA (available from 2011, 3, 8 th http:// jenkemusa. net/pegproducts2. aspx), such as in particular 4-arm-PEG derivatives comprising a pentaerythritol core, 8-arm-PEG derivatives comprising a hexaglycerol core, and 8-arm-PEG derivatives comprising a tripentaerythritol core. Most preferred are substructures of formula (VI) comprising, in particular consisting of, moieties selected from

4-arm-PEG amine comprising a pentaerythritol core:

n ranges from 400 to 2000;

4-arm-PEG carboxyl group containing pentaerythritol core:

n ranges from 400 to 2000;

8-arm-PEG amine comprising a hexaglycerol core:

n ranges from 400 to 2000; and is

R = hexaglycerol core structure;

8-arm-PEG carboxyl comprising a hexaglycerol core:

n ranges from 400 to 2000; and is

R = hexaglycerol core structure;

8-arm-PEG amine comprising tripentaerythritol:

n ranges from 400 to 2000;

and R = tripentaerythritol core structure;

and 8-arm-PEG carboxyl group comprising tripentaerythritol:

n ranges from 400 to 2000; and is

R = tripentaerythritol core structure;

6-arm-PEG amine containing sorbitol or dipentaerythritol core:

n ranges from 400 to 2000; and is

R = sorbitol or dipentaerythritol;

8-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 400 to 2000; and is

R = sorbitol or dipentaerythritol;

8-arm-PEG amine containing sorbitol or dipentaerythritol core:

n ranges from 400 to 2000;

and R = sorbitol or dipentaerythritol;

and 8-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 400 to 2000; and is

R = sorbitol or dipentaerythritol;

each in a bound form.

Also preferred are the substructures of the following structural formulae

4-arm-PEG amine comprising a pentaerythritol core:

n ranges from 400 to 2000;

4-arm-PEG carboxyl group containing pentaerythritol core:

n ranges from 400 to 2000;

8-arm-PEG amine comprising a hexaglycerol core:

n ranges from 400 to 2000, and

r = hexaglycerol core structure;

8-arm-PEG carboxyl comprising a hexaglycerol core:

n ranges from 400 to 2000, and

r = hexaglycerol core structure;

8-arm-PEG amine comprising a tripentaerythritol core:

n is in the range of 400 to 2000,

and R = tripentaerythritol core structure;

and 8-arm-PEG carboxyl comprising a tripentaerythritol core:

n ranges from 400 to 2000, and

r = tripentaerythritol core structure;

6-arm-PEG amine containing sorbitol or dipentaerythritol core:

n ranges from 400 to 2000, and

r = sorbitol or dipentaerythritol;

6-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 400 to 2000, and

r = sorbitol or dipentaerythritol;

6-arm-PEG amine containing sorbitol or dipentaerythritol core:

n is in the range of 400 to 2000,

and R = sorbitol or dipentaerythritol;

and 6-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 400 to 2000, and

r = sorbitol or dipentaerythritol;

each in a bound form.

Preference is also given to substructures of the formula (VI) comprising, in particular combined from

4-arm-PEG amine comprising a pentaerythritol core:

n ranges from 20 to 500;

4-arm-PEG carboxyl group containing pentaerythritol core:

n ranges from 20 to 500;

8-arm-PEG amine comprising a hexaglycerol core:

n ranges from 20 to 500; and is

R = hexaglycerol core structure;

8-arm-PEG carboxyl comprising a hexaglycerol core:

n ranges from 20 to 500; and is

R = hexaglycerol core structure;

8-arm-PEG amine comprising a tripentaerythritol core:

n ranges from 20 to 500;

and R = tripentaerythritol core structure;

and 8-arm-PEG carboxyl comprising a tripentaerythritol core:

n ranges from 20 to 500; and is

R = tripentaerythritol core structure;

6-arm-PEG amine containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

8-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

8-arm-PEG amine containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500;

and R = sorbitol or dipentaerythritol;

and 8-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

each in a bound form.

Also preferred are substructures of the following formulae

4-arm-PEG amine comprising a pentaerythritol core:

n ranges from 20 to 500;

4-arm-PEG carboxyl group containing pentaerythritol core:

n ranges from 20 to 500;

8-arm-PEG amine comprising a hexaglycerol core:

n ranges from 20 to 500; and is

R = hexaglycerol core structure;

8-arm-PEG carboxyl comprising a hexaglycerol core:

n ranges from 20 to 500; and is

R = hexaglycerol core structure;

8-arm-PEG amine comprising a tripentaerythritol core:

n ranges from 20 to 500;

and R = tripentaerythritol core structure;

and 8-arm-PEG carboxyl comprising a tripentaerythritol core:

n ranges from 20 to 500; and is

R = tripentaerythritol core structure;

6-arm-PEG amine containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

6-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

6-arm-PEG amine containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500;

and R = sorbitol or dipentaerythritol;

and 6-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

each in a bound form.

In a preferred embodiment, the substructure of formula (VI) B- (A)_nHas a molecular weight in the range of 1kDa to 80kDa, more preferably 1kDa to 40kDa, and even more preferably 10kDa to 40 kDa. It will be appreciated that the terminal amine group or carboxyl group, respectively, is used to attach a moiety Hyp of formula (VI)^y。

Partial Hyp of formula (VI)^yAre respectively linked to a moiety X of formula (I)⁰Attached to Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X¹(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)).

In a preferred embodiment, the moiety of formula (VI) Hyp^yAre each linked to a moiety X of formula (I) via a functional group⁰Attached to Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or to the rest of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)), said functional group is selected from the group consisting of an amide group, a carbamate group, an ester group, an ether group, an amine group, a thioether group. Preferably, the moiety of formula (VI) Hyp^yLinked to the moiety X of formula (I) via an amide, sulfide and/or ether group, respectively, even more preferably via an amide group⁰Part Q connected to formula (II), part Q connected to formula (IIaa) or (IIab)¹Or to a moiety X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba).

Optionally, not linked to a moiety X of formula (I)⁰Part of formula (II) Q, formula (IIaa) or (II:)IIab) part Q¹Or a moiety X of formulae (IIac), (IIad), (IIb)₁Or a portion Hyp of formula (VI) of the remainder of the molecule of formula (IIba)^yThe functional group(s) may be capped with a suitable capping reagent and/or may optionally be attached to at least one targeting moiety; in particular by a permanent connection. Thus, a partial Hyp of formula (VI)^yCan be respectively connected to the moiety X of formula (I)⁰Part Q connected to formula (II), part Q connected to formula (IIaa) or (IIab)¹Or to the moiety X of formulae (IIac), (IIad), (IIb)₁Or to the remainder of the molecule of formula (IIba), a capping moiety and/or a targeting moiety. Preferably, the moiety of formula (VI) Hyp^yAre respectively linked to a moiety X of formula (I)⁰Part Q connected to formula (II), part Q connected to formula (IIaa) or (IIab)¹Or to a moiety X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba) and not to the capping moiety and/or targeting moiety. If present, the targeting moiety may be linked directly or indirectly via a spacer moiety to a moiety Hyp of formula (VI)^y。

Examples of suitable end-capping moieties are linear, branched or cyclic C_1-8An alkyl group.

In one embodiment, each branched moiety of formula (VI) Hyp^yDirectly or indirectly connected to at least two moieties X of formula (I), respectively⁰At least two portions Q connected to formula (II), at least two portions Q connected to formula (IIaa) or (IIab)¹Or to at least two moieties X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba). More preferably, each branched moiety of formula (VI) Hyp^yDirectly or indirectly connected to at least three moieties X of formula (I), respectively⁰At least three parts Q connected to formula (II), at least three parts Q of formula (IIaa) or (IIab)¹Or to at least three moieties X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba). Most preferably, each branched moiety of formula (VI) Hyp^yAre directly or indirectly connected to, respectively, a compound of formula (I)Four less parts X⁰At least four moieties Q connected to formula (II), at least four moieties Q connected to formula (IIaa) or (IIab)¹Or to at least four moieties X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba).

A branched moiety Hyp of formula (VI)^yA moiety comprising, preferably consisting of, a binding form moiety selected from the group consisting of:

polyols containing at least 2 hydroxyl groups in bound form (preferably further comprising functional groups, which are preferably further hydroxyl groups or carboxylic acid groups, more preferably further hydroxyl groups),

preferably selected from glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran and hyaluronan, erythritol, threitol, arabitol, xylitol, ribitol, dulcitol, iditol; more preferred are glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran, and hyaluronan.

-or a polyamine containing at least 2 amine groups (preferably further comprising functional groups, which are preferably further amine groups or carboxylic acid groups, more preferably carboxylic acid groups),

preferred are ornithine, dignithine, triglycine, tetraornithine, pentaornithine, hexaornithine, heptaornithine, octaornithine, nonaornithine, decaornithine, undecimine, dodecaornithine, tridecanoic, tetradecanoic, pentadecaornithine, hexadecanoic, heptadecaornithine, octadecaornithine, nonadecaornithine, diaminobutyric acid, bis (diaminobutyric acid), tris (diaminobutyric acid), tetrakis (diaminobutyric acid), pentakis (diaminobutyric acid), hexakis (diaminobutyric acid), heptakis (diaminobutyric acid), octakis (diaminobutyric acid), nonakis (diaminobutyric acid), deca (diaminobutyric acid), undecimetic (diaminobutyric acid), dodeca (diaminobutyric acid), tridecanoic acid, tetradecanoic (diaminobutyric acid), pentadeca (diaminobutyric acid), hexadecanoic (diaminobutyric acid), heptakis (diaminobutyric acid), Octadeca (diaminobutyric acid), nonadeca (diaminobutyric acid), lysine, dilysine, trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecenyl lysine, dodecalysine, tridecysine, tetradecysine, pentadecalysine, hexadecysine, heptadecalysine, octadecalysine, nonadecalysine, trionithine, tetraornithine, pentaornithine, hexaornithine, heptaornithine, octaornithine, nonaornithine, decaornithine, undecenyl ornithine, dodecaornithine, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, heptadecaornithine, octadecaornithine, nonadecaornithine, tris (diaminobutyric acid), tetrakis (diaminobutyric acid), penta (diaminobutyric acid), hexakis (diaminobutyric acid), heptakis (diaminobutyric acid), Octa (diaminobutyric acid), nona (diaminobutyric acid), deca (diaminobutyric acid), undeca (diaminobutyric acid), dodeca (diaminobutyric acid), tridecyl (diaminobutyric acid), tetradecyl (diaminobutyric acid), pentadecyl (diaminobutyric acid), hexadecane (diaminobutyric acid), heptadeca (diaminobutyric acid), octadeca (diaminobutyric acid), nonadeca (diaminobutyric acid),

or a polycarboxylate comprising at least two carboxylate groups in combined form (preferably further comprising functional groups, which are preferably further amino or carboxylic acid groups, more preferably further carboxylic acid groups),

preferably selected from the group consisting of di (glutamic acid), tri (glutamic acid), tetra (glutamic acid), penta (glutamic acid), hexa (glutamic acid), hepta (glutamic acid), octa (glutamic acid), nona (glutamic acid), deca (glutamic acid), undec (glutamic acid), dodeca (glutamic acid), tridecyl (glutamic acid), tetradecyl (glutamic acid), pentadeca (glutamic acid), hexadecadry (glutamic acid), hepta (glutamic acid), octadeca (glutamic acid), nonadeca (glutamic acid), di (aspartic acid), tri (aspartic acid), tetra (aspartic acid), penta (aspartic acid), hexa (aspartic acid), hepta (aspartic acid), octa (aspartic acid), nona (aspartic acid), deca (aspartic acid), undec (aspartic acid), dodeca (aspartic acid), tridecyl (aspartic acid), tetradecyl (aspartic acid), pentadeca (aspartic acid), hexadecadry (aspartic acid, Heptadeca (aspartic acid), octadeca (aspartic acid), nineteen (aspartic acid), polyethyleneimine and polyvinylamine.

In a preferred embodiment, the moiety of formula (VI) Hyp^ySelected from the following, in particular consisting of: in combination, dilysine, trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecalysine, dodecalysine, tridecylline, tetradecylline, pentadecalysine, hexadecylline, heptadecalysine, octadecalysine, nonadecalysine, trionithine, tetraornithine, pentaornithine, hexaornithine, heptaornithine, octaornithine, nonaornithine, decaornithine, undecaornithine, dodecaornithine, tridecylline, tetradecanoline, pentadecaornithine, hexadecanoic ornithine, heptadecaornithine, octadecaornithine, nonadecaornithine, tris (diaminobutyric acid), tetrakis (diaminobutyric acid), pentakis (diaminobutyric acid), hexakis (diaminobutyric acid), heptakis (diaminobutyric acid), octakis (diaminobutyric acid), nonakis (diaminobutyric acid), Deca (diaminobutyric acid), undec (diaminobutyric acid), dodeca (diaminobutyric acid), tridecyl (diaminobutyric acid), tetradecyl (diaminobutyric acid), pentadeca (diaminobutyric acid), hexadecyl (diaminobutyric acid), heptadeca (diaminobutyric acid), octadeca (diaminobutyric acid), nonadeca (diaminobutyric acid), di (glutamic acid), tri (glutamic acid), tetra (glutamic acid), penta (glutamic acid), hexa (glutamic acid), hepta (glutamic acid), octa (glutamic acid), nona (glutamic acid), deca (glutamic acid), undec (glutamic acid), dodeca (glutamic acid), tridecyl (glutamic acid), tetradecyl (glutamic acid), pentadeca (glutamic acid), hexadecadry (glutamic acid), heptadeca (glutamic acid), nona (glutamic acid), di (aspartic acid), tri (aspartic acid), tetra (aspartic acid), Penta (aspartic acid), hexa (aspartic acid), hepta (aspartic acid), octa (aspartic acid), nona (aspartic acid), deca (aspartic acid), undec (aspartic acid), dodeca (aspartic acid), tridecyl (aspartic acid)) Tetradecyl (aspartic acid), pentadecyl (aspartic acid), hexadecyl (aspartic acid), heptadecyl (aspartic acid), octadecanyl (aspartic acid), nineteen (aspartic acid), polyethyleneimine and low molecular weight PEI.

More preferably, the moiety of formula (VI) Hyp^ySelected from the following, more preferably consisting of: combined forms of trilysine, tetralysine, pentalysine, hexalysine, heptalysine, octalysine, nonalysine, decalysine, undecalysine, dodecalysine, tridecysine, tetradecysine, pentadecalysine, hexadecysine and heptadecalysine, even more preferably, the partial Hyp of formula (VI)^ySelected from the following, preferably consisting of: a conjugated form of trilysine, heptalysine or pentadecalysine.

In a preferred embodiment, the moiety of formula (VI) Hyp^yHas a molecular weight in the range of 0.1kDa to 4kDa, more preferably 0.2kDa to 2 kDa.

In a further preferred embodiment, each of the branching moieties Hyp of formula (VI)^yHaving at least 1 branch and respectively connected to at least two moieties X of formula (I)⁰At least two portions Q connected to formula (II), at least two portions Q connected to formula (IIaa) or (IIab)¹Or to at least two moieties X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba); and has a maximum of 63 branches and is connected to a maximum of 64 portions X of formula (I) respectively⁰At least 64 parts Q connected to formula (II), at least 64 parts Q connected to formula (IIaa) or (IIab)¹Or to at least 64 portions X of formulae (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba), more preferably to each branching moiety Hyp of formula (VI)^yHaving at least 1 branch and respectively connected to at least 2 moieties X of formula (I)⁰At least 2 portions Q connected to formula (II), at least 2 portions Q connected to formula (IIaa) or (IIab)¹Or to at least 2 moieties X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba); and has a maximum of 31A plurality of branches and respectively connected to at most 32 portions X of formula (I)⁰At least 32 portions Q connected to formula (II), at least 32 portions Q connected to formula (IIaa) or (IIab)¹Or to at least 32 portions X of formulae (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba).

In a preferred embodiment, Z of formula (VI)¹Comprising a quaternary carbon, in particular a quaternary carbon of a branched core moiety B, wherein B of formula (VI) is pentaerythritol in bound form. Preferably, each A of formula (VI) is independently a PEG-based polymeric chain terminated by a permanently covalently attached branched core moiety-CH of pentaerythritol₂The O-moiety is linked to the quaternary carbon of pentaerythritol and the distal end of the PEG-based polymeric chain is covalently bound to a branching moiety Hyp of formula (VI)^yEach branch part of the formula (VI) Hyp^yAre each linked to a moiety X of formula (I)⁰Part Q connected to formula (II), part Q connected to formula (IIaa) or (IIab)¹Or to a moiety X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba).

In a preferred embodiment, the branching moiety of the formula (VI) Hyp^yComprising, preferably consisting of, branched polyamines containing at least 2 amine groups. Preferably, the branched polyamine containing at least 2 amine groups contains one or more lysine residues in bound form. Preferably, each branch of formula (VI) is a Hyp moiety^yHas a molecular weight in the range of 0.1kDa to 4kDa, in particular 0.2kDa to 2 kDa. In a preferred embodiment, the moiety of formula (VI)(where n =4) are composed of identical or different branching moieties Hyp^yIs composed of, and each part Hyp^yCan be selected independently. In a preferred embodiment, all parts of formula (VI) are Hyp_yAre the same.

In a preferred embodiment, the moiety of formula (VI) Hyp^yComprises, in particular fromConsists of the following components: 1 to 32 bound forms of lysine, preferably 1,3, 7 or 15 bound forms of lysine, more preferably 1,3 or 7 bound forms of lysine. Most preferably, Hyp of formula (VI)^yContaining, in particular consisting of heptalysine groups.

Preferably, moieties of formula (VI)(wherein n is preferably 4) has a molecular weight in the range of 1kDa to 160kDa, more preferably 1kDa to 80kDa, and even more preferably 10kDa to 40 kDa.

Preferred moieties of the formula (VI)Selected from structures (i-y) to (iii-y):

wherein

The dotted lines represent the moieties X respectively attached to formula (I)⁰Part Q connected to formula (II), part Q connected to formula (IIaa) or (IIab)¹Or to a moiety X of formula (IIac), (IIad) or (IIb)₁Or to the remainder of the molecule of formula (IIba), provided that one of m1, m2 of formula (I) is 1, and wherein if m1, m2=0, the carrier is covalently linked to T,

p is an integer from 5 to 2000, preferably from 10 to 1000, more preferably from 10 to 500, most preferably from 100 to 1000,

q is 1 or 2.

In a preferred embodiment, B of formula (VI) is pentaerythritol.

In another preferred embodiment, Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Is a protein carrier comprising, in particular consisting of, an amino acid sequence of at least 100 amino acid residues.

In another preferred embodiment, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Is in a random coil conformation.

In another preferred embodiment, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprising, in particular consisting of alanine, serine and proline residues.

In a preferred embodiment, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹An amino acid sequence comprising at least 100 amino acid residues, in particular consisting of an amino acid sequence of at least 100 amino acid residues, and

wherein the amino acid sequence of at least 100 amino acid residues is in a random coil conformation, and

wherein the amino acid sequence of at least 100 amino acid residues comprises alanine, serine and proline residues.

Preferably, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Consists of the following components: an amino acid sequence comprising at least about 10 amino acid residues, at least 100 amino acid residues consisting of alanine, serine and proline residues, which has a random coil conformation under physiological conditions. It will be appreciated that the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹The random coil can be temporarily or temporarily not formed, for example, when present as a lyophilizate or a dry composition.

In one embodiment, protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb), or (IIba)¹An amino acid sequence having a random coil conformation comprising at most about 3000 amino acid residues, preferably at most about 1500 amino acid residues, more preferably at most about 900 amino acid residues, even more preferably at most about 700 amino acid residues, particularly preferably at most about 600 amino acid residues. Thus, the amino acid sequence forming the random coil conformation is at most about 500 amino acid residues or at most about 450 amino acid residues in length.

Thus, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹In particular the protein carrier Z forming the formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹The random coil conformation of (a) is about 100 to about 3000 amino acid residues in length.

In particular embodiments, the amino acid sequence forming a random coil conformation of about 100 to about 1000 amino acid residues is characterized herein, i.e., comprises alanine, serine and proline as the major or only residues defined below.

A protein carrier moiety Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Consists essentially of the three amino acid residues alanine, serine and proline, and wherein all three amino acids are present in the protein carrier moiety Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Whereby the proline residues preferably represent from about 4% to about 40% of the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹. The alanine and serine residues preferably comprise the remaining at least 60% to 96% of the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac) or (IIad)¹. However, as described in detail below, the protein carrier Z of formula (I), (II), (IIaa), (IIab) or (IIac)¹Amino acids other than alanine, serine and proline may also be further included, i.e., as a minor groupAnd (4) obtaining.

The term "minor constituent" as used herein means that at most 10% (i.e. at most 10 out of 100 amino acids) may be different from alanine, serine and proline, preferably at most 8% (i.e. at most 8 out of 100 amino acids) may be different from alanine, serine and proline, more preferably at most 6% (i.e. at most 6 out of 100 amino acids) may be different from alanine, serine and proline, even more preferably at most 5% (i.e. at most 5 out of 100 amino acids) may be different from alanine, serine and proline, particularly preferably at most 4% (i.e. at most 4 out of 100 amino acids) may be different from alanine, serine and proline, more particularly preferably at most 3% (i.e. at most 3 out of 100 amino acids) may be different from alanine, serine and proline, even more particularly preferably at most 2% (i.e. at most 2 out of 100 amino acids) may be different from alanine, serine and proline, Serine and proline, and most preferably up to 1% (i.e.up to 1 out of 100 amino acids) (encoding the protein vector Z of formula (I), (II), (IIaa), (IIab), (IIac) or (IIad)¹) May be different from alanine, serine and proline. The amino acids other than alanine, serine and proline may be selected from natural or protein-derived amino acids consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr and Val. Minor constituents may also be selected from non-naturally occurring amino acids, such as hydroxyproline or selenomethionine or other modified natural amino acids.

The term "at least about 100/150/200/250/300/300/350 (etc.) amino acid residues" is not limited to the exact number of amino acid residues, but also includes amino acid extensions comprising an additional 10% to 20%, or comprising 10% to 20% fewer residues. For example, "at least about 100 amino acid residues" may also comprise 80 to 100 and about 100 to 120 amino acid residues.

In one embodiment, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac) or (IIad)¹Comprising a plurality of polymer boxes, wherein said polymer boxes consist of Ala, Ser and/or Pro, and wherein formula (I), (II), (IIaa), (IIab), (IIac) or(IIad) protein Carrier Z¹No more than 6 consecutive amino acid residues of the polymeric cassette of (a) are identical, and wherein said proline residue constitutes more than 4% and less than 40% of said protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac) or (IIad)¹The amino acid of (1).

In one embodiment, the protein carrier moiety Z of formula (I), (II), (IIaa), (IIab) or (IIac)¹Comprising, preferably consisting of, a plurality of amino acid repeats,

wherein the repeats consist of Ala, Ser, and Pro residues,

and wherein the carrier moiety Z of formula (I), (II), (IIaa), (IIab), (IIac) or (IIad), (IIb) or (IIba)¹Is identical to no more than 6 consecutive amino acid residues.

In a preferred embodiment, said proline residues constitute more than 4% and less than 40% of the protein carrier moiety Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹The amino acid of (1).

In a further preferred embodiment, the protein carrier moiety Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprising an amino acid sequence of about 100 to 3000 amino acid residues forming a random coil conformation, in particular consisting of an amino acid sequence of about 100 to 3000 amino acid residues forming a random coil conformation.

A protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Multiple identical polymeric cassettes or multiple different polymeric cassettes may be included. Non-limiting examples of polymeric cassettes consisting of Ala, Ser and/or Pro residues are provided below; see SEQ id no: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 14, or peptide fragments or multimers of these sequences. The polymeric cartridges may be made of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein each polymer cassette comprises Ala, Ser and/or Pro residues, preferably Ala, Ser and Pro residues.

In one embodiment, the polymeric cassette does not comprise more than 100 amino acid residues. Preferably, the polymer cassette as defined herein comprises more than about 4%, preferably more than about 5%, even more preferably more than about 6%, particularly preferably more than about 8%, more particularly preferably more than about 10%, even more particularly preferably more than about 15% and most preferably more than about 20% proline residues. Such polymer cassettes as defined herein preferably comprise less than about 40% or less than about 35% proline residues.

In one embodiment, protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb), or (IIba)¹Is of formula (b):

Ser_x[Ala_ySer_z]_v (b)，

the structural formula further comprises a proline residue as defined herein and wherein

x is independently selected from the integer 0 to 6,

each y is independently selected from an integer ranging from 1 to 6,

each z is independently selected from an integer ranging from 1 to 6.

v is any integer, such that the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Consists of at least about 100 amino acid residues and in particular at least about 100 to about 3000 amino acid residues, preferably about 2000 and more preferably about 1000 amino acid residues.

In one embodiment, v Ala of formula (b)_y Ser_zY of formula (b) and z of formula (b) are the same for all monomeric moieties. In another embodiment, v Ala of formula (b)_y Ser_zThe monomeric moiety y of formula (b) and z of formula (b) are not the same.

In a preferred embodiment, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprises no more than 5 identical consecutive amino acid residues, more preferably no more than 4 identical consecutive amino acid residues, and most preferably no more than 3 identical consecutive amino acid residues.

As indicated above, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprising proline residues which constitute more than about 4%, preferably more than about 5%, even more preferably more than about 6%, particularly preferably more than about 8%, more particularly preferably more than about 10%, even more particularly preferably more than about 15% and most preferably more than about 20% of the protein carrier Z constituting formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹The amino acid of (1). These proline residues may be introduced at any position of formula (b). Preferably, the proline residue may be at one or more of the v Ala residues of formula (b)_y Ser_zAre present in the monomers and they may be present in the same or different positions.

In another preferred embodiment, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprising a protein carrier Z comprising the constituent formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹More than about 4% but less than about 50%, preferably more than about 10% but less than about 50% and most preferably more than about 20% but less than about 50% of the amino acids of (a) alanine residues.

In a further preferred embodiment, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprising a protein carrier Z comprising the constituent formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹More than about 4% and less than about 50%, preferably more than about 10% but less than about 50% and most preferably more than about 20% but less than about 50% of the amino acids of (a).

Thus, formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) protein Carrier Z¹Comprising a protein carrier Z comprising the constituent formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹About 35% proline residues, about 50% alanine residues and about 15% serine residues of the amino acids. Alternatively, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Protein carrier Z which may comprise the constituent formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹About 35% proline residues, about 15% alanine residues and about 50% serine residues of the amino acids of (a).

Preferably, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprising one or more of the following alanine-serine polymer cassettes:

SEQ ID NO:1

AAAASSASSASSSSSAAASA

SEQ ID NO:2

AASAAASSAAASAAAASASS

SEQ ID NO:3

ASASASASASASSAASAASA

SEQ ID NO：4

SAASSSASSSSAASSASAAA

SEQ ID NO:5

SSSSAASAASAAAAASSSAS

SEQ ID NO：6

SSASSSAASSSASSSSASAA

SEQ ID NO:7

SASASASASASAASSASSAS

SEQ ID NO：8

ASSAAASAAAASSAASASSS

conditionIs a protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Further comprising proline residues as described herein.

In a preferred embodiment, the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprising, preferably consisting of, one or more of the following polymeric cassettes:

SEQ ID NO:9

ASPAAPAPASPAAPAPSAPA

SEQ ID NO：10

AAPASPAPAAPSAPAPAAPS

SEQ ID No:11

APSSPSPSAPSSPSPASPSS

SEQ ID NO:15

SAPSSPSPSAPSSPSPASPS

SEQ ID NO: 15 corresponding to the circular sequence variants provided herein SEQ ID No: 11, wherein the last serine is removed and another serine is added as the starting amino acid. Thus, multimers of the modified sequences have essentially the same internal repeating units as multimers of the unmodified sequences, except for the first and last residue. Thus, SEQ ID NO: 15 can be considered as protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Examples of further polymeric cassettes of (a). It will be clear to the skilled person that other polymeric cassettes and (shorter) peptide fragments or amino acid polymers provided herein in the form of altered cyclic sequences are also useful as protein carriers Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Of (2) a polymerAnd (5) a box.

Further and exemplary amino acid polymers that form a random coil conformation can comprise an amino acid sequence, which can be selected from the group consisting of:

SEQ ID NO:12

SSPSAPSPSSPASPSPSSPA,

SEQ ID NO:13

AASPAAPSAPPAAASPAAPSAPPA, and

SEQ ID NO:14

ASAAAPAAASAAASAPSAAA.

thus, Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Preferred polymer cassettes of (a) are selected from the following sequences:

ASPAAPAPASPAAPAPSAPA(SEQ ID NO:9),

AAPASPAPAAPSAPAPAAPS(SEQ ID NO:10),

APSSPSPSAPSSPSPASPSS(SEQ ID NO:11)，

SSPSAPSPSSPASPSPSSPA(SEQ ID NO:12),

AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 13), and

ASAAAPAAASAAASAPSAAA(SEQ ID NO:14);

or a circular sequence variant or multimer of such sequences as a whole or part of such sequences.

In one embodiment, the protein carrier moiety Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Comprising at least one amino acid sequence selected from the group consisting of:

ASPAAPAPASPAAPAPSAPA(SEQ ID NO:9),

AAPASPAPAAPSAPAPAAPS(SEQ ID NO:10),

APSSPSPSAPSSPSPASPSS(SEQ ID NO:11),

SSPSAPSPSSPASPSPSSPA(SEQ ID NO:12),

AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 13), and

ASAAAPAAASAAASAPSAAA(SEQ ID NO:14);

and cyclic sequence variants or multimers of these sequences as a whole or part of these sequences.

Furthermore, these sequences in the form of peptide fragments or multimers or cyclic sequence alterations and the sequences provided above can be used as protein carriers Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹The polymer cartridge of (1).

Thus, exemplary polymeric cassettes may also be provided for individual peptide fragments that may be recombined to form further polymeric cassettes.

A protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) according to above¹May comprise a polypeptide consisting of SEQ ID NO: 9. 10, 11, 12, 13 or 14, or may comprise a multimer consisting of more than one of the amino acid sequences of SEQ ID NOs: 9. 10, 11, 12, 13 and 14. Furthermore, it is speculated that these exemplary sequences in the form of peptide fragments or altered circular sequences may also be used to construct a further protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹The polymer cartridge of (1).

In another embodiment, protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb), or (IIba)¹May comprise a polymer comprising, preferably consisting of: selected from the group consisting of SEQ ID NO: 9. 10, 11, 12, 13, 14, 15 and multimers of these (cyclic) permuted sequences.

In another embodiment, formulas (I), (II), (IIaa), (IIab), (IIac), (IIad)Protein Carrier Z of (IIb), (IIb) or (IIba)¹The following may be, preferably consists of: selected from the group consisting of SEQ ID NO: 9. 10, 12, 13, 14, 15 and multimers of these exemplary polymeric cassettes.

A protein carrier Z for the production of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹The peptide fragments of these sequences of (a) may consist of at least 3, preferably at least 4, more preferably at least 5, even more preferably at least 6, more preferably at least 8, particularly preferably at least 10, more particularly preferably at least 12, even more particularly preferably at least 14, even more particularly preferably at least 16 and most preferably at least 18 consecutive amino acids of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 9. 10, 11, 12, 13 and 14.

For example, individual peptide fragments of a polymeric cassette may be combined into further individual polymeric cassettes, so long as the above-identified rules for overall distribution and number of alanines, serines and prolines are observed. Furthermore, these polymer cassettes may also comprise further amino acid residues, however only as a minimum or minor constituent, i.e. at most 10%, preferably at most 2% of the individual polymer cassettes. The single polymeric cassette is composed of at least about 100 amino acid residues. The individual polymer cassettes can be combined to form longer polymers of random coil-forming amino acids, thereby forming the protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹Has a maximum length of about 3000 amino acids. A protein carrier Z of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba)¹The preferred minor constituent of (a) is lysine.

In another embodiment, the vector Z¹Is C_10-24Fatty acids, i.e. carboxylic acids having a linear carbon chain of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms, which are saturatedAnd either partially or fully unsaturated.

In another embodiment, the vector Z¹Having the structure of formula (VII):

wherein

B is the core of the branch,

a is a poly (ethylene glycol) -based polymeric chain, and

n is an integer from 3 to 32.

In a preferred embodiment, the branched core B of formula (VII) comprises, preferably consists of, a moiety selected from:

preferably B is selected from glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran and hyaluronan, erythritol, threitol, arabitol, xylitol, ribitol, dulcitol, iditol; more preferred are glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, hydroxyalkyl starch, polyvinyl alcohol, dextran, and hyaluronan.

wherein the polyol or polyamine is in bound form.

In a preferred embodiment, the branched core B of formula (VII) comprises, preferably consists of, pentaerythritol.

Preferably, the poly (ethylene glycol) -based polymeric chain a linked to the branched core B of formula (VII) consists of a linear PEG chain, one of its ends being linked to B of formula (VII) and the other ends being linked respectively to X of formula (I)⁰Attached to Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)).

It is understood that the PEG-based chain a of formula (VII) may optionally be terminated in the case of a branched PEG chain, and/or may optionally be interrupted by alkyl or aryl groups and may optionally be substituted by heteroatoms and/or functional groups in the case of a branched or linear PEG chain.

Preferably, the vector Z of formula (VII)¹Are multi-arm PEG derivatives, such as those detailed in the product list of JenKemTtechnology, USA (available from 2011, 3, 8 th http:// jenkemusa. net/pegproducts2. aspx), such as in particular 4-arm-PEG derivatives comprising a pentaerythritol core, 8-arm-PEG derivatives comprising a hexaglycerol core, and 8-arm-PEG derivatives comprising a tripentaerythritol core. Most preferred are substructures B (A) of formula (VI) comprising, in particular consisting of_n：

4-arm-PEG amine containing pentaerythritol core:

n ranges from 20 to 500;

4-arm-PEG carboxyl with pentaerythritol core:

n ranges from 20 to 500;

8-arm-PEG amine containing hexaglycerol core:

n ranges from 20 to 500; and is

R = hexaglycerol core structure;

8-arm-PEG carboxyl containing hexaglycerol core:

n ranges from 20 to 500; and is

R = hexaglycerol core structure;

8-arm-PEG amine containing a tripentaerythritol core:

n ranges from 20 to 500;

and R = tripentaerythritol core structure;

8-arm-PEG carboxylic acid containing a tripentaerythritol core:

n ranges from 20 to 500; and is

R = tripentaerythritol core structure;

6-arm-PEG amine with sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

6-arm-PEG carboxyl with sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

6-arm-PEG amine with sorbitol or dipentaerythritol core:

n ranges from 20 to 500;

and R = sorbitol or dipentaerythritol;

6-arm-PEG carboxyl with sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = sorbitol or dipentaerythritol;

4-arm-PEG amine containing pentaerythritol core:

n ranges from 20 to 500;

4-arm-PEG carboxyl with pentaerythritol core:

n ranges from 20 to 500;

8-arm-PEG amine containing hexaglycerol core:

n ranges from 20 to 500; and is

R = hexaglycerol core structure;

8-arm-PEG carboxyl containing hexaglycerol core:

n ranges from 20 to 500; and is

R = hexaglycerol core structure;

8-arm-PEG amine containing a tripentaerythritol core:

n ranges from 20 to 500;

and R = tripentaerythritol core structure;

and 8-arm-PEG carboxyl containing a tripentaerythritol core:

n ranges from 20 to 500; and is

R = tripentaerythritol core structure;

6-arm-PEG amine with sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = comprises a sorbitol or dipentaerythritol core;

6-arm-PEG carboxyl with sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = comprises a sorbitol or dipentaerythritol core;

6-arm-PEG amine with sorbitol or dipentaerythritol core:

n ranges from 20 to 500;

and R = comprises a sorbitol or dipentaerythritol core;

and 6-arm-PEG carboxyl containing sorbitol or dipentaerythritol core:

n ranges from 20 to 500; and is

R = comprises a sorbitol or dipentaerythritol core;

wherein the dotted lines represent X's respectively attached to formula (I)⁰Attached to Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to Q¹(if the carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)), or is linked to X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)).

In a preferred embodiment, the carrier B- (A) of the formula (VII)_nHas a molecular weight in the range of 1kDa to 80kDa, more preferably 1kDa to 40kDa, and even more preferably 10kDa to 40 kDa.

More preferably, the support of formula (VII) has the structure of formula (VIIa):

wherein

t ranges from 80 to 160;

w ranges from 2 to 6;

and the dotted lines represent the remainder of the treprostinil prodrug attached to the carrier linkage, i.e., to X of formula (I), respectively⁰Attached to moiety Q (if the carrier-attached treprostinil prodrug is of formula (II)), attached to moiety Q¹(if the carrier-attached treprostinil prodrug is of formula (IIaa) or (IIab)), or attached to moiety X₁(if the carrier-linked treprostinil prodrug is of formula (IIac), (IIad) or (IIb)) or to the remainder of the molecule (if the carrier-linked treprostinil prodrug is of formula (IIba)).

Preferably, w is 2 or 3.

Most preferably, the carrier-linked treprostinil prodrug has the structure of formula (IIc):

wherein y is 4, and Z¹Representing part (IIca):

wherein the dashed line represents the remainder of the structure connected to formula (IIc), t ranges from 80 to 160, and w is 2 or 3.

In another preferred embodiment, the present invention relates to a pharmaceutical composition comprising a carrier-linked treprostinil prodrug, wherein the pharmaceutical composition is characterized in that the prostacyclin compound releases prostacyclin in a plasma-independent manner. Preferably, the prostacyclin compound releases prostacyclin in an enzyme-independent manner.

The term "plasma independent" means that the prostacyclin release kinetics of the prostacyclin compound measured independently in a buffer at ph7.4 and in 80% buffered plasma at ph7.4 at 37 ℃ does not vary by more than 50%, preferably by more than 40%, more preferably by more than 30%, even more preferably by more than 20% and most preferably by more than 10%.

The term "enzyme independent" means that the prostacyclin release of the prostacyclin compound does not require the presence of an enzyme.

Another aspect of the present invention is a pharmaceutical composition comprising a carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt of the present invention, optionally one or more pharmaceutically acceptable excipients.

The following paragraphs further describe pharmaceutical compositions.

Pharmaceutical compositions comprising the carrier-linked treprostinil prodrugs of the invention may be provided as liquid compositions or as dry compositions. Suitable drying methods are, for example, spray drying and freeze drying (freeze drying). The preferred method of drying is lyophilization.

Preferably, the carrier-linked treprostinil prodrug in the composition is dosed sufficient to provide a therapeutically effective amount of treprostinil for at least 12 hours in a single application. More preferably, one application of a pharmaceutical composition comprising a carrier-linked treprostinil prodrug is sufficient for at least 1 day, e.g. 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, e.g. 2 weeks, 3 weeks or 4 weeks.

In one embodiment, the present invention relates to pharmaceutical compositions

(i) Wherein the carrier-linked treprostinil prodrug of the invention in the pharmaceutical composition is in a dosage sufficient to provide a therapeutically effective amount of treprostinil for at least 12 hours on a single application, and/or

(ii) Wherein a single dose of the pharmaceutical composition comprises about 2 to about 6, preferably about 4mg of treprostinil.

In a preferred embodiment, a single dose of a liquid pharmaceutical composition of the invention has a volume of from about 0.1 to about 10mL, preferably from about 0.5 to about 5mL, even more preferably from about 0.5 to about 2mL, especially about 1 mL.

"about" according to the invention is to be understood as meaning the range of experimental errors, in particular. + -. 5% or. + -. 10%.

The pharmaceutical compositions of the carrier-linked treprostinil prodrugs of the invention optionally comprise one or more excipients.

Excipients may be classified as buffers, isotonic modifiers, preservatives, stabilizers, anti-adsorbents, oxidation protectants, viscosity enhancers/viscosity enhancers, or other adjuvants. In some cases, these components may have dual or triple functions. The pharmaceutical compositions of carrier-linked treprostinil prodrugs of the present invention comprise one or more excipients selected from the group consisting of:

(i) buffering agent: physiologically tolerable buffers that maintain the pH in the desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyruvate. Antacids such as Mg (OH) may also be used₂Or ZnCO₃. Can adjust the buffering energyForce to satisfy the most sensitive condition to pH stability

(ii) Isotonic modifier: pain due to cell damage caused by differences in osmotic pressure at the injection depot is minimized. Glycerol and sodium chloride are examples. The effective concentration can be determined by osmometry using a serum with an osmolality assumed to be 285-315 mOsmol/kg.

(iii) Preservatives and/or antimicrobial agents: multiple dose parenteral formulations require the addition of preservatives in sufficient concentrations to minimize the risk of infection of the patient by injection, and corresponding regulatory requirements have been established. Representative preservatives include m-cresol, phenol, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimersol, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride.

(iv) A stabilizer: stabilization is achieved by strengthening the protein stabilizing power, by destabilization in the denatured state, or by direct binding of the protein by excipients. The stabilizer may be an amino acid such as alanine, arginine, aspartic acid, glycine, histidine, lysine, proline; sugars such as glucose, sucrose, trehalose; polyols such as glycerol, mannitol, sorbitol; salts such as potassium phosphate, sodium sulfate; chelating agents such as EDTA, hexaphosphate; ligands such as divalent metal ions (zinc, calcium, etc.); other salts or organic molecules such as phenol derivatives. Furthermore, oligomers or polymers such as cyclodextrins, dextrans, dendrimers, PEG or PVP or protamine or HSA may be used.

(v) Anti-adsorption agent: primarily ionic or non-ionic surfactants or other proteins or soluble polymers are used to coat or compete for adsorption onto the interior surface of the composition or composition container. Suitable surfactants are, for example, alkyl sulfates, such as ammonium and sodium lauryl sulfates; alkyl ether sulfates such as sodium laureth sulfate and sodium myristyl polyoxyethylene ether sulfate; sulfonates such as dioctyl sodium sulfosuccinate, perfluorooctane sulfonate, perfluorobutane sulfonate, alkylbenzene sulfonate; phosphates such as alkyl aryl ether phosphates and alkyl ether phosphates; carboxylates, such as fatty acid salts (soaps) or sodium stearate, sodium lauroyl sarcosinate, perfluorononanoate, perfluorooctanoate, octenidine dihydrochloride; quaternary ammonium cations such as cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, cetylpyridinium chloride, polyoxyethylene tallow amine, benzalkonium chloride, benzethonium chloride, 5-bromo-5-nitro-1, 3-dioxane, dimethyldioctadecyl ammonium chloride, dioctadecyl dimethyl ammonium bromide; zwitterions, such as 3- [ (3-cholamidopropyl) dimethylammonio ] -1-propanesulfonate, cocamidopropyl hydroxysultaine, amino acids, imide, cocamidopropyl betaine, lecithin; fatty alcohols, such as cetyl alcohol, stearyl alcohol, cetearyl alcohol, oleyl alcohol, polyethylene glycol alkyl ethers, such as octanediol monododecyl ether, pentanediol monododecyl ether; polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers such as decyl glucoside, lauryl glucoside, octyl glucoside; polyoxyethylene glycol octylphenol ethers such as Triton X-100; polyoxyethylene glycol alkylphenol ethers such as nonoxynol-9; glycerol alkyl esters such as glycerol laurate; polyoxyethylene glycol sorbitan alkyl esters such as polysorbates; sorbitan alkyl esters; cocamide MEA and cocamide DEA; dodecyl dimethyl amine oxide; block copolymers of polyethylene glycol and polypropylene glycol, such as poloxamer (Pluronic F-68), PEG lauryl ether (Brij35), polysorbate 20 and 80; other anti-adsorbents are dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA and gelatin. The concentration and type of excipient chosen depends on the effect of the elimination, but usually a monolayer of surfactant is formed at the interface above the CMC value.

(vi) Lyoprotectants and/or cryoprotectants: during freezing or spray drying, excipients may counteract the destabilizing effects of hydrogen bond cleavage and dehydration. Sugars and polyols can be used for this purpose, but corresponding positive effects are also observed for surfactants, amino acids, non-aqueous solvents and other peptides. Trehalose is particularly effective in reducing moisture-induced agglomeration and improves the thermal stability that can result from exposure of hydrophobic groups of proteins to water. Mannitol and sucrose, alone as lyoprotectants or in combination with each other, may also be used, with a higher proportion of mannitol to sucrose being known to enhance the physical stability of the lyophilized cake. Mannitol may also be combined with trehalose. Trehalose may also be combined with sorbitol, or sorbitol is used as a separate protectant. Starch or starch derivatives may also be used.

(vii) Oxidation protective agent: antioxidants such as ascorbic acid, ectoine carboxylic acid (ectoine), methionine, glutathione, monothioglycerol, morin, Polyethyleneimine (PEI), propyl gallate, vitamin E, chelating agents such as citric acid, EDTA, hexaphosphate, thioglycolic acid.

(viii) Spreading or dispersing agent: permeability of the connective tissue is modified by hydrolysis of extracellular matrix components in the intracellular space, such as but not limited to hyaluronic acid, polysaccharides found in the intercellular space of connective tissue. Spreading agents such as, but not limited to, hyaluronidase transiently reduce the viscosity of the extracellular matrix and facilitate dispersion of the injected drug.

(ix) Other adjuvants: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase. Acids and bases such as hydrochloric acid and sodium hydroxide are the adjuvants required for pH adjustment during the preparation.

In a general embodiment, pharmaceutical compositions comprising a carrier-linked treprostinil prodrug of the invention in dry or liquid form are provided as single or multi-dose compositions.

In one embodiment of the present invention, a liquid or dry pharmaceutical composition comprising a carrier-linked treprostinil prodrug is provided as a single dose, meaning that a container is provided containing one dose of the drug.

Alternatively, the liquid or dry pharmaceutical composition comprising the carrier-linked treprostinil prodrug is a multi-dose composition, meaning that a container is provided comprising more than one therapeutic dose, i.e. a multi-dose composition comprising at least 2 doses. Such multi-dose compositions of carrier-linked treprostinil prodrug may be used in different patients in need thereof, or may be used in one patient, where the remaining dose is stored until needed after the first dose is administered.

In another aspect of the invention, the pharmaceutical composition is in a container. Suitable containers for liquid or dry compositions are, for example, syringes, vials with stoppers and seals, ampoules and cartridges. In particular, liquid or dry compositions comprising a carrier-linked treprostinil prodrug of the invention are provided in a syringe. If the pharmaceutical composition comprising the carrier-linked treprostinil prodrug is a dry pharmaceutical composition, the container is preferably a dual chamber syringe. In this embodiment, the dry pharmaceutical composition is provided in a first chamber of a dual chamber syringe and the reconstitution solution is provided in a second chamber of the dual chamber syringe.

The dry composition of the carrier-linked treprostinil prodrug is reconstituted prior to its application to a patient in need thereof. Reconstitution can be carried out in containers, wherein a dry composition of the carrier-linked treprostinil prodrug is provided, for example, in vials, syringes, dual chamber syringes, ampoules, and cartridges. Reconstitution is accomplished by adding a predetermined amount of the reconstitution solution to the dry composition. The reconstitution solution is a sterile liquid, such as water or a buffer, which may contain further additives, such as preservatives and/or antimicrobial agents, such as benzyl alcohol and cresol. Preferably, the reconstitution solution is sterile water. When a dry composition is reconstituted, it is referred to as a "reconstituted pharmaceutical composition" or "reconstituted composition".

Another aspect of the invention relates to methods of administering reconstituted or liquid pharmaceutical compositions comprising a carrier-linked treprostinil prodrug of the invention. Pharmaceutical compositions comprising a carrier-linked treprostinil prodrug may be administered by inhalation, injection or infusion methods, including intradermal, subcutaneous, intramuscular, intravenous, intraosseous and intraperitoneal. Preferably, the pharmaceutical composition comprising the carrier-linked treprostinil prodrug is administered subcutaneously.

For dry pharmaceutical compositions comprising a carrier-linked treprostinil prodrug of the invention, the preferred method of administration is by inhalation.

Thus, in a preferred embodiment, the present invention relates to a carrier-linked treprostinil prodrug of the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the invention for use as a medicament for topical, enteral, parenteral, inhalation, injection or infusion, intra-articular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous and intraperitoneal, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intraventricular or intrasternal administration, preferably subcutaneous administration.

In another embodiment, a first carrier-linked treprostinil prodrug of the invention is administered by a first method of administration and a second carrier-linked treprostinil prodrug of the invention is administered by a second method of administration, either concurrently or sequentially. The first and second methods of administration may be any combination of topical, enteral, parenteral, inhalation, injection or infusion, intra-articular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous and intraperitoneal, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intraventricular or intrasternal administration.

Thus, in another preferred embodiment, the present invention relates to a carrier-linked treprostinil prodrug of the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the invention, wherein such water soluble carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof or pharmaceutical composition is suitable for administration by topical, enteral or parenteral administration as well as by means of external use, inhalation, injection or infusion, including intra-articular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous and intraperitoneal, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intraventricular and intrasternal applications, preferably for administration to a patient by subcutaneous administration.

A further aspect is a method of preparing a reconstituted composition comprising a therapeutically effective amount of a carrier-linked treprostinil prodrug of the invention and optionally one or more pharmaceutically acceptable excipients, the method comprising the steps of:

a pharmaceutical composition comprising a carrier-linked treprostinil prodrug of the invention is contacted with a reconstitution solution.

Another aspect is a reconstituted pharmaceutical composition comprising a therapeutically effective amount of a carrier-linked treprostinil prodrug of the invention and optionally one or more pharmaceutically acceptable excipients.

Another aspect of the invention is a method of making a dry composition of a carrier-linked treprostinil prodrug. In one embodiment, such dry compositions are prepared by the steps of:

(i) the carrier-linked treprostinil prodrug is mixed with one or more excipients,

(ii) the amount corresponding to a single dose or multiple doses is transferred into suitable containers,

(iii) drying the composition in said container, and

(iv) and (4) sealing the container.

Suitable containers are vials, syringes, dual chamber syringes, ampoules and cartridges.

Another aspect of the invention is a kit of parts.

If the administration device is simply a hypodermic syringe, the kit can comprise a syringe, a needle, and a container for use with the syringe of a dry pharmaceutical composition comprising a carrier-linked treprostinil prodrug, and a second container comprising a reconstitution solution.

If the pharmaceutical composition is a liquid composition, the kit can comprise a syringe, a needle, and a container for use with the syringe that contains a carrier-linked liquid composition of the treprostinil prodrug.

In a more preferred embodiment, the injection device is not a simple hypodermic syringe, and thus a separate container of the reconstituted or liquid carrier-linked treprostinil prodrug is suitable for use with an injection device such that, in use, the liquid composition in the container is in fluid connection with the outlet of the injection device. Examples of applicators include, but are not limited to, hypodermic syringes and pen injector devices. A particularly preferred injection device is a pen injector, wherein the container is a cartridge, preferably a disposable cartridge. Optionally, part of the cartridge includes a needle safety device which may be used to cover or cap the used needle to prevent injury.

A preferred kit of parts comprises a needle and a container comprising the composition of the invention and optionally further comprising a reconstitution solution, the container being suitable for use with a needle. Preferably, the container is a dual chamber syringe.

In another aspect, the present invention provides a cartridge of a pharmaceutical composition comprising the carrier-linked treprostinil prodrug described above for use with a pen injector device. The cartridge may contain a single dose or multiple doses of the carrier-linked treprostinil prodrug.

Another aspect of the invention is a carrier-linked treprostinil prodrug of the invention or a pharmaceutical composition of the invention for use as a medicament.

Where the carrier-linked treprostinil prodrugs of the invention contain one or more acidic or basic groups, the invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, particularly their pharmaceutically acceptable salts. Thus, carrier-linked treprostinil prodrugs of the invention comprising acidic groups may be used according to the invention, for example as alkali metal, alkaline earth metal or ammonium salts. More precise examples of such salts include sodium, potassium, calcium, magnesium or salts with ammonia or organic amines such as ethylamine, ethanolamine, triethanolamine or amino acids. Carrier-linked treprostinil prodrugs of the invention comprising one or more basic groups (i.e. groups which may be protonated) may be present and may be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. If the carrier-linked treprostinil prodrug of the invention contains acidic and basic groups in the molecule, the invention also includes inner or betaine (zwitterions) in addition to the mentioned salt forms. The different salts can be obtained by conventional methods known to the person skilled in the art, for example by contacting these with organic or inorganic acids or bases in solvents or dispersants, or by anion exchange or cation exchange with other salts. The invention also includes all salts of the prodrugs which are not directly suitable for use in medicine due to low physiological compatibility, but which may be used, for example, as intermediates in chemical reactions or for the preparation of pharmaceutically acceptable salts.

Another aspect of the present invention is a method of treatment, control, delay of progression or prevention in a mammalian patient, preferably a human, in need of treatment for one or more conditions, diseases or disorders, comprising administering to said patient a therapeutically effective amount of a carrier-linked treprostinil prodrug of the present invention or a pharmaceutical composition comprising a carrier-linked treprostinil prodrug of the present invention or a pharmaceutically acceptable salt thereof.

Such conditions, diseases or disorders that may be prevented and/or treated with a vector-linked treprostinil prodrug of the invention are, for example, pulmonary hypertension, ischemic diseases (e.g., peripheral vascular disease including peripheral arterial disease, raynaud's phenomenon including raynaud's disease and raynaud's syndrome, scleroderma including systemic sclerosis, myocardial ischemia, ischemic shock, renal insufficiency), ischemic ulcers including digital ulcers, heart failure (including congestive heart failure), pulmonary hypertension, interstitial lung disease, idiopathic pulmonary fibrosis, conditions requiring anticoagulation (e.g., post-MI, post-cardiac surgery), thrombotic microangiopathy, extracorporeal circulation, central retinal vein occlusion, atherosclerosis, inflammatory diseases (e.g., COPD, psoriasis), hypertension (e.g., preeclampsia), reproduction and parturition, cancer or other unregulated cell growth conditions, Cell/tissue protection and other emerging therapeutic areas where prostacyclin treatment appears to have a beneficial effect, preferably pulmonary arterial hypertension.

In one embodiment, the present invention relates to a carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the invention for use in a method of treating or preventing a disease or disorder which can be treated and/or prevented by treprostinil.

In a preferred embodiment, the disease or disorder is pulmonary hypertension.

In a further embodiment, the present invention relates to the use of a carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment and/or prevention of a disease or disorder which can be treated and/or prevented by treprostinil.

In a preferred embodiment, the disease or disorder is pulmonary hypertension.

In one embodiment, the carrier-linked treprostinil prodrug of the invention may be administered to the same patient by different routes of administration, such as, but not limited to, subcutaneous injection and inhalation.

In another embodiment, a carrier-linked treprostinil prodrug of the invention may be administered in combination with one or more additional drugs or biologically active moieties of the free form or prodrug. In another embodiment, such one or more additional drugs are administered together with a carrier-linked treprostinil prodrug of the present invention using the same or different routes of administration. It is preferred that one or more additional drugs and the carrier-linked treprostinil prodrug of the invention are administered in a fixed dose combination.

Thus, such one or more additional active agents may be administered separately from the carrier-linked treprostinil prodrug. The particular additional drug or biologically active moiety that may be administered in combination with the carrier-linked treprostinil prodrug may depend on the particular disease or condition that may be treated or prevented by administration of the carrier-linked treprostinil prodrug. In certain instances, the additional drug/bioactive moiety can be a cardiovascular agent, such as a prostacyclin compound, a NO activity modulator, a calcium channel blocker, a phosphodiesterase inhibitor, a diuretic, an endothelial antagonist, or an antiplatelet agent.

In another object of the invention, carrier-linked treprostinil prodrugs can be administered in combination with inhaled prostacyclin.

Another subject of the invention is a method for synthesizing the carrier-linked treprostinil prodrug of the invention or a pharmaceutically acceptable salt thereof. The carrier-linked treprostinil prodrugs of the invention or precursors of these drugs may be prepared by known methods or according to the reaction sequence described below. The starting materials for preparing (synthesizing) the carrier-linked treprostinil prodrug of the invention or a precursor thereof are known or commercially available, or can be prepared by known methods or as described below.

All reactions for the synthesis of carrier-linked treprostinil prodrugs (including precursors) of the invention are well known per se to the skilled person and may be carried out under standard conditions according to or similar to the procedures described in the standard literature of organic chemistry. Depending on the individual case, it may be necessary or advantageous to convert the carrier-linked treprostinil prodrug or its precursor into the desired functional group in a later reaction step, by introducing protecting groups to temporarily block the functional groups and either remove them at a later stage of the synthesis or introduce functional groups in the formation of the precursor group, in order to avoid side reactions during the synthesis of the carrier-linked treprostinil prodrug or its precursor. Suitable such synthetic strategies and protecting and precursor groups individually are known to the skilled worker. If desired, the carrier-linked treprostinil prodrug or precursor thereof may be purified by conventional purification methods, for example by recrystallization or chromatography.

In one embodiment, the carrier-linked treprostinil prodrug (or a pharmaceutically acceptable salt thereof) of the present invention may be prepared by a process comprising the steps of: converting carboxylic acid of treprostinil to a biologically active moiety reagent D-Y, wherein Y is a leaving group, followed by reacting reagent D-Y with a reversible prodrug linker reagent X containing a hydroxyl group⁰-OH reaction to produce a biologically active moiety-reversible prodrug linker conjugate T-X through formal carboxylate linkage⁰. Then, T-X may be added⁰Bound to carrier moiety Z¹Carrier-linked treprostinil prodrugs of the biologically active moiety containing a carboxylic acid group of the present invention are obtained. Alternatively, the carrier moiety Z¹Can already be bound to X⁰-OH。

It is to be understood that functional groups of treprostinil not involved in the synthesis of the carrier-linked treprostinil prodrugs of the invention may be protected with suitable protecting groups known to those skilled in the art.

Y is a leaving group. Suitable leaving groups are known to those skilled in the art. Preferably, if attached to D, Y is chloride, bromide, fluoride, nitrophenoxy, imidazolyl, N-hydroxysuccinimide, N-hydroxybenzotriazole, N-hydroxyazobenzotriazolyl, pentafluorophenoxy, 2-thioxo-thiazolinyl, or N-hydroxysulfosuccinimide.

The carrier-linked treprostinil prodrugs of the invention may be prepared by conventional methods known in the art starting from polymers. Several routes are clear to practitioners in the art. For example, the moiety T-X⁰Can react with the reactive functional groups of the polymer of the support moiety POL. Alternatively, a compound comprising moiety Z may be prepared¹-X⁰For subsequent reaction with the preferably activated bioactive acid D-Y. It should be understood that D is treprostinil.

Figure 1 shows treprostinil release at different time points in buffer and buffered rat plasma expressed as% treprostinil release compared to total treprostinil content (see example 29).

Figure 2 shows the prolonged circulation duration of treprostinil conjugate 25 for more than 2 weeks in monkeys after subcutaneous injection (see example 30).

Figure 3 shows a single dose intravenous injection of compound 25 and subsequent plasma analysis of total treprostinil and carrier content (see example 30).

Figure 4 shows a plasma analysis of a single dose of intravenously injected compound 25 and subsequent free treprostinil (see example 32).

Figure 5 shows a single dose subcutaneous injection of compound 25 and subsequent plasma analysis of free treprostinil (see example 32).

Materials, methods and analysis:

purification of the product

The normal phase purification was carried out on Biotage "Isolera one" purification system Biotage AB in sweden. Biotage KP-Sil silica column. A heptane/ethyl acetate or dichloromethane/methanol gradient was used. The product was detected and collected at 254 and 280 nm.

For preparative RP-HPLC, a Waters600 controller and 2487 Dual-Path absorption Detector were used, equipped with a Waters Xbridge^TMBEH300Prep C185 μm, 150X 10mm, flow rate 6 mL/min, or Waters Xbridge^TMBEH300Prep C1810 μm, 150X 30mm, flow rate 40 mL/min. A gradient 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) was used.

The HPLC fractions containing the product were combined and, if not specifically stated, lyophilized.

Automatic fast chromatography

Automatic flash chromatography was performed on Biotage "Isolera one" purification system Biotage AB in sweden using a Biotage KP-Sil silica column. The product was detected and collected at 254 and 280 nm.

LC/MS analysis

Analytical RP-HPLC/ESI-MS was performed on a Waters instrument consisting of a 2695 sample manager, a 2487 two-way absorption detector and a ZQ4000ESI instrument, equipped with a 5 μm Reprodosil PurODS-3 column (75 × 1.5mm) (dr. maisch, amerbush, germany; flow rate: 350 μ L/min, typical gradient: MeCN in water 10-90% over 5 min, 0.05% TFA) or on a Waters acquisition UPLC tandem Thermo LTQ Orbitrap Discovery high resolution/high accuracy mass spectrometer equipped with acquisition PDA detector, equipped with a Waters acquisition UPLC BEH300C18RP column (2.1 × 50mm,1.7 μm, flow rate: 0.25 mL/min; solvent A: UP-H₂0+0.04% TFA, solvent B: UP-acetonitrile +0.05% TFA).

RP-UPLC/ESI-MS was performed on a Waters/Thermo instrument consisting of a Waters acquisition UPLC tandem Thermo LTQ orbita spectrometer high resolution/high accuracy mass spectrometer equipped with an acquisition PDA detector, equipped with an acquisitionBEH300C18RP column (Waters Corporation,2.1 x 50mm,1.7 μm, flow rate: 0.25 mL/min; solvent A: UP-H₂0+0.04% TFA, solvent B: UP-MeCN +0.05% TFA).

A typical gradient of treprostinil release from the TransCon5kDa PEG linker treprostinil was determined as: flow rate of 0.25mL, gradient: 30-50% B over 10 minutes.

RP-HPLC purification:

for preparative RP-HPLC, a Waters600 controller and 2487 dual-path absorption detector were used, equipped with the following columns: waters Xbridge^TMBEH300Prep C185 μm, 150X 10mm, flow rate 6 mL/min, or Waters Xbridge^TMBEH300Prep C1810 μm, 150X 30mm, flow rate 40 mL/min. A linear gradient 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) was used.

Typical gradients for the purification process are:

flow rate 6 mL/min, solvent a: h₂0+0.05% TFA, solvent B: MeCN +0.05% TFA, typical gradient: 1-95% B over 14 min

Flow rate 6 mL/min, solvent a: h₂0+0.05% TFA, solvent B: MeCN +0.05% TFA, typical gradient: 10-80% B over 14 minutes

Flow rate 40 mL/min, solvent a: h₂0+0.05% TFA, solvent B: MeCN +0.05% TFA, typical gradient: 40-95% B over 14 min

HPLC fractions containing product were combined and lyophilized if not otherwise indicated.

Chemicals and drugs:

trioniac is purchased from Shanghai Techwell Biopharmaceutical Co., Ltd., or Myrica rubra Chirogate International Inc., Shanghai, China. 6- (S-tritylmercapto) hexanoic acid was purchased from Polypeptide, Stosselberg, France. cis-Cyclohexanedicarboxylic anhydride from Alfa Aesar GmbH, Carlsuhe, Germany&Co KG. 2-chlorotrityl chloride resin (1%,DVB) was obtained from Merck Biosciences GmbH, germany. 6- (S-tritylsulfanyl) -hexylamine was synthesized according to WO-A2009/133137. The PEG used in this work was obtained from NOF Europe n.v. belgium herbare. All other chemicals were purchased from dovugxing sigma aldrich GmbH, germany. Analytical RP-HPLC of water and acetonitrilePurchased from Biosolve b.v., and TFA from Thermo scientific.

Example 1:

benzyl protection of 3-hydroxybutyrate 1:

3-Hydroxybutanoic acid 1(434mg, 4.17mmol) was dissolved in THF (10mL) and BnBr (700. mu.L, 5.89mmol) and Cs were added₂CO₃(2.5g, 7.67 mmol). The reaction mixture was refluxed in a sealed tube for 4-6 hours. After cooling to room temperature, the reaction mixture was filtered and the residue was washed several times with EtOAc. The organic solvent was removed and the product was purified by automated flash chromatography on silica (SNAP25g column, flow 30 mL/min, solvent a: DCM, solvent B: MeOH; gradient: 0-5% B over 19CV) in one portion, removing the starting material and affording the desired benzyl-protected 3-hydroxybutyrate 2 as a yellow oil.

Yield: 361mg (45%)

MS：m/z217.1=[M+Na]⁺(MW + Na, calculated = 217.2).

Example 2:

coupling reaction of benzylated 3-hydroxybutyrate 2 with treprostinil:

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

Yield: 8mg (50%)

MS：m/z589.3=[M+Na]⁺(MW + Na, calculated = 589.7).

Example 3:

hydrogenation of benzyl ester 3:

benzyl ester 3(13mg, 0.0229mmol) was dissolved in EtOAc (EtOAc)2mL), and 5% palladium on charcoal (5% Pd, 15mg) was added. Hydrogen was passed through the solution for 30 minutes. The reaction mixture was stirred under hydrogen atmosphere for another 12.5 hours until consumption was complete (analytical RP-HPLC). The mixture was filtered through celite and washed several times with EtOAc. The organic solvent was removed in vacuo and the residue was purified by RP-HPLC (solvent A: H containing 0.05% TFA)₂O, solvent B: MeCN with 0.05% TFA, gradient: 1-95% B, over 20 minutes, flow rate: 6 mL/min). The product containing fractions were combined and lyophilized to give 4 as a white solid.

Yield: 1.9mg (29%).

MS：m/z499.3=[M+Na]⁺(MW + Na, calculated = 499.6).

Example 4:

coupling reaction of linear PEG5kDa amine with linker treprostinil 4:

linker Trionil 4(1.9mg, 3.98. mu. mol) and linear PEG5kDa amine (86mg, 17.2. mu. mol) were dissolved in THF/MeCN (R) ((R))1.5mL:0.5mL) and Et₃To N (40. mu.L), catalytic amounts of DMAP and T3P (50% in EtOAc, 50. mu.L, 73.2. mu. mol) were added sequentially. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was washed with 20. mu. L H₂O dilution and removal of volatile solvent in vacuo. The residue was subjected to RP-HPLC (solvent A: H containing 0.05% TFA)₂O, solvent B: MeCN with 0.05% TFA, gradient: 10-80% B, over 20 minutes, flow rate: 6 mL/min). The product containing fractions were combined and lyophilized to give the TransCon PEG linker treprostinil 5 as a white solid.

Yield: 12.5mg (58%).

MS：m/z1378.6=[M+4H]⁴⁺(calculated =1378.9), there is a representative peak in the polymer distribution.

Example 5:

treprostinil release kinetics of tracon PEG linker treprostinil 5:

TransCon PEG linker treprostinil 5(0.5-1.5mg) was incubated in pH7.4 hydrolysis buffer (60mM sodium phosphate, 3mM EDTA, 0.05% Tween-20, 1mL) at 37 ℃ and aliquots were analyzed for released treprostinil by UPLC at various time points.

Half-life determination of the hydrolysis kinetics of the TransCon PEG linker treprostinil 5:

the percentage of treprostinil released after incubation at ph7.4 and 37 ℃ for a given time was determined by integrating the corresponding peaks (released material versus bound) in the RP-UPLC chromatogram. The data shown in table 1 were then plotted against time. Using a first order kinetic fit, a half-life of 4.20 days for treprostinil release was obtained.

Table 1:

item(s)	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:

synthesis of intermediate 6a/6 b:

6- (S-tritylsulfanyl) -hexylamine (synthesis see WO-A2009/133137) (507mg, 1.35mmol) was dissolved in DCM (4mL) and cis-1, 2-cyclohexanedicarboxylic anhydride (251mg, 1.63mmol) was added to the reaction mixture at RT. DIPEA (0.70mL, 4.06mmol) was added and the mixture was stirred at RT until 6- (S-tritylsulfanyl) -hexylamine (LC/MS) was completely consumed. Removing the volatile solvent in vacuo and dissolving the residue inH₂O/MeCN (6:1, 18mL) and the product was purified by RP-HPLC (solvent A: H with 0.05% TFA)₂O, solvent B: MeCN with 0.05% TFA, gradient: 40-95% B, over 16 minutes, flow rate: 40 mL/min). The combined fractions were treated with saturated NaHCO₃The solution (pH about 6) was neutralized and the organic solvent was removed in vacuo. The remaining aqueous phase was extracted twice with DCM. The combined organic layers were washed with MgSO₄Drying and removal of the solvent in vacuo gave 6a/6b as a racemic mixture.

Yield: 580mg (81%).

MS：m/z552.23=[M+Na]⁺(MW + Na, calculated =552.62 g/mol).

Example 7:

synthesis of intermediate 7a/7 b:

N-Boc-1, 6-hexanediamine (270mg, 1.25mmol) was dissolved in DMF (2ml) and cis-1, 2-cyclohexanedicarboxylic anhydride (231mg, 1.50mmol) was added to the reaction mixture at RT. DIPEA (0.65mL, 3.76mmol) was added and the mixture was stirred at RT until N-Boc-1, 6-hexanediamine (LC/MS) was consumed. Subjecting the reaction mixture to hydrogenation with H₂O/MeCN (9:1) dilution and passage of the product through RP-HPLC (solvent A: H with 0.05% TFA)₂O, solvent B: MeCN with 0.05% TFA, gradient: 10-80% B, over 16 minutes, flow rate: 40 mL/min). The combined fractions were treated with saturated NaHCO₃The solution (pH about 6) was neutralized and the organic solvent was removed in vacuo. The remaining aqueous phase was extracted several times with DCM. The organic layer was washed with MgSO₄Drying and removal of the solvent in vacuo gave 7a/7b as a racemic mixture.

Yield: 410mg (88%).

MS：m/z371.39=[M+H]⁺(MW + H, calculated =371.27 g/mol).

Example 8:

synthesis of Dmob protected treprostinil 8:

treprostinil (61mg, 0.156mmol) was dissolved in toluene (dry, molecular sieves, 2.5mL) and the silylating agent BSA (0.6mL, 0.245mmol) was added. The reaction mixture was stirred at RT for 12 h. The volatile solvent was removed in vacuo and the TMS-protected treprostinil was used without further purification.

TMS protected Trionil was dissolved in DCM (2.5mL) and H₂O (60. mu.L). DMAP (76mg, 0.624mmol), EDC & HCl (119mg, 0.624mmol) and Dmob-ol (105mg, 0.624mmol) in DCM (1mL) were added. The reaction mixture was stirred at RT until the reaction was complete (LC/MS). The solution was diluted with DCM and quenched by addition of 0.1n hcl solution saturated with NaCl. The aqueous phase was extracted several times with DCM. The combined organic layers were washed with MgSO₄Drying and removal of the solvent in vacuo gave crude product 8. The crude product was RP-HPLC (solvent A: H with 0.05% TFA)₂O, solvent B: MeCN with 0.05% TFA, gradient: 35-85% B, over 16 minutes, flow rate: 40 mL/min). By adding saturated NaHCO₃The solution adjusted the combined HPLC fractions to pH about 7. MeCN was removed in vacuo. H is remained₂The O layer was extracted several times with DCM and the combined organic phases were MgSO₄Drying, filtration, and removal of the solvent in vacuo afforded product 8 as a colorless solid.

Yield: 69mg (82%).

MS：m/z563.20g/mol=[M+Na]⁺(MW + Na, calculated =563.67 g/mol).

Example 9:

synthesis of Trinidel linker thiols

Formic acid 6a/6b (147mg, 0.277mmol), EDC. HCl (53mg, 0.277mmol), and DMAP (34mg, 0.277mmol) were dissolved in 0.5mL DCM. Dmob-protected treprostinil 8(43mg, 0.08mmol) was dissolved in 0.5mL DCM and added to the reaction mixture. The mixture was stirred at RT until 8 was completely consumed (overnight. LC/MS). The volatile solvent was removed in vacuo. The residue was dissolved in HFIP (2mL), TFA (100. mu.L) and TES (50. mu.L) and stirred at RT for 30 min (LC/MS). Volatiles were removed in vacuo. The residue is dissolved in H₂O/MeCN (9/1, 0.05% TFA, 2mL), and the mixture of the four possible isomers was subjected to RP-HPLC (solvent A: H with 0.05% TFA)₂O, solvent B: MeCN with 0.05% TFA, gradient: 60-85% B, over 16 minutes, flow rate: 6 mL/min). The product isomer eluted was three separate peaks. Fractions of the peak with the shortest elution time (compound "9 x") were pooled and used in the pegylation step without further treatment. The structural assignment of 9x to the possible isomers 9a, 9b, 9c or 9d was not performed in this experiment. The yield of 9x was determined using the Ellman test.

Yield: 8.1mg (26%)

MS：m/z682.21g/mol=[M+Na]⁺(MW + Na, calculated =682.40 g/mol).

Example 10:

of treprostinil linker aminesSynthesis of

Carboxylic acid 7a/7b (50mg, 0.134mmol), EDC HCl (26mg, 0.134mmol) and DMAP (16mg, 0.134mmol) were dissolved in DCM (0.3 mL). Dmob-protected treprostinil 8(36mg, 0.066mmol) was dissolved in DCM (0.5mL) and added to the reaction mixture. The mixture was stirred at RT until complete consumption (LC/MS). The volatile solvent was removed in vacuo. The residue is dissolved in H₂O/MeCN (9/1, 0.05% TFA, 2mL), and separation of the mono-coupled product (treprostinil coupled one 7a/7b molecule) from the di-coupled product (treprostinil coupled two 7a/7b molecules) by RP-HPLC: thermo Fisher Hypersil Gold PFP column. 150X 10mm, solvent A: h with 0.05% TFA₂O, solvent B: MeCN with 0.05% TFA, gradient: 35-55% B, over 16 minutes, flow rate: 6 mL/min. HPLC fractions containing the mono-coupled product were combined and lyophilized. The lyophilizate was dissolved in HFIP (0.9mL), DCM (0.1mL), TFA (100. mu.L) and TES (20. mu.L) and stirred for 10 min at RT. Volatiles were removed in vacuo and the residue was dissolved in H₂O/MeCN (9/1, 0.05% TFA, 2mL), and the mixture of the four possible isomers was subjected to RP-HPLC (solvent A: H with 0.05% TFA)₂O, solvent B: MeCN with 0.05% TFA, gradient: 35-55% B, over 16 minutes, flow rate: 6 mL/min). The product isomer eluted was three separate peaks. Fractions of the peak of the shortest elution time (compound "10 ×") were pooled and used for the pegylation step without further treatment. The structure assignment of 10x corresponding to the possible isomers 10a, 10b, 10c or 10d was not performed in this experiment. The 10x yield was estimated by HPLC using a treprostinil standard curve (280 nm).

Yield: 3.0 mg.

MS：m/z643.28g/mol=[M+Na]⁺(MW + Na, calculated =643.45 g/mol).

Example 11:

PEGylation reaction of Trionil linker amine with Linear PEG5kDa NHS

To Trinidene linker amine 10X (0.6mg, 1. mu. mol solution, MeCN/H)₂O, 0.05% TFA, 5mL) was added linear PEG5kDa NHS (23mg, 4.6. mu. mol). The solution was neutralized by adding 0.5M pH7.4 buffer (0.5M phosphate, 0.6 mL). Addition of H₂O (1mL), resulting in a clear solution, and the reaction mixture was incubated at RT for 1 hour. The reaction mixture was then passed through RP-HPLC (solvent A: H containing 0.01% HCl)₂O, solvent B: MeCN with 0.01% HCl, gradient: 10-70% B, over 16 minutes, flow rate: 6 mL/min) and lyophilized to give TransCon linear 5kDa PEG treprostinil 11.

Yield: 3 mg.

Example 12:

PEGylation of Trionil linker thiol with Linear PEG40kDa Maleimide

MeCN/H at Trinidene linker thiol 9x (6.2mg, 9.42. mu. mol)₂To a solution of O (0.05% TFA, 87mL) was added linear PEG40kDa maleimide (463mg, 11.3. mu. mol). The solution was neutralized by adding pH7.4 buffer (0.5M phosphate, 4.4 mL). After 1 hour incubation time, another aliquot of linear 40kDa Mal-PEG (73mg, 178. mu. mol) and H was added₂O (5mL), and the reaction solution was incubated for another 1.5 hours. The reaction mixture was passed through RP-HPLC (solvent A: H containing 0.01% HCl)₂O, solvent B: MeCN with 0.01% HCl, gradient: 30-50% B, over 16 minutes, flow rate: 40 mL/min) and lyophilized to give TransCon linear 40kDa PEG treprostinil 12.

Yield: 321mg (82%).

Example 13:

PEGylation reaction of Trionil linker thiol with 4-arm PEG20kDa maleimide

MeCN/H at Trinidene linker thiol 9x (2.54mg, 3.84. mu. mol)₂To a solution of O (0.05% TFA, 5.7mL) was added 4-arm PEG20kDa maleimide (21mg, 0.98. mu. mol). The solution was neutralized by adding pH7.4 buffer (0.5M phosphate, 3.0 mL). Addition of H₂O (3mL) until the reaction mixture again became a clear solution. The reaction mixture was incubated at RT for 2 hours and then by RP-HPLC (solvent A: H containing 0.01% HCl₂O, solvent B: MeCN with 0.01% HCl, gradient: 45-85% B, over 16 minutes, flow rate: 40 mL/min) and lyophilized to give TransCon 4-arm PEG20kDa treprostinil 13.

Yield: 14mg (66%).

Example 14:

TranCon PEG linker treprostinil Release kinetics of treprostinil Compounds 11 and 12 Learning:

the release kinetics were determined according to example 5. Compound 11 and 12 were obtained with a half-life of 4.3 days (± 0.7 days) for release of treprostinil.

Example 15:

treprostinil release kinetics of TransCon PEG linker treprostinil Compound 13

TransCon PEG linker treprostinil 13(2.5mg) was incubated in pH7.4 hydrolysis buffer (60mM sodium phosphate, 3mM EDTA, 0.05% Tween-20, 1mL) at 37 ℃ and aliquots were analyzed for released treprostinil by UPLC at various time points. The percentage of treprostinil released was determined compared to the area of treprostinil after equal portions of complete hydrolysis (50 μ L of hydrolysis solution mixed with 25 μ L of 5N NaOH for 20 minutes 25 μ L of AcOH was added and the resulting solution analyzed by LCMS).

By applying a first order kinetic fit, a 5 day half-life release of treprostinil from 13 was obtained.

Example 16:

synthesis of building Block 14

The building block 14 was synthesized according to the following scheme:

mmt-chloride (3g, 9.71mmol) was dissolved in DCM (20mL) and added dropwise to a solution of ethylenediamine (6.5mL, 97.1mmol) in DCM (20 mL). After 2 hours, the solution was poured into ether (300mL) and washed three times with 30/1(v/v) brine/0.1M NaOH solution (50mL each) and once with brine (50 mL). Passing the organic phase over Na₂SO₄Dried and volatiles were removed under reduced pressure. The Mmt-protected amine (3.18g, 9.56mmol) was used in the next step without further purification.

Mmt-protected amine (3.18g, 9.56mmol) was dissolved in anhydrous DCM (30 mL). 6- (S-tritylmercapto) hexanoic acid (4.48g, 11.47mmol), PyBOP (5.96g, 11.47mmol) and DIPEA (5.0mL, 28.68mmol) were added and the mixture was stirred at RT for 30 min. The solution was diluted with ether (250mL) and washed three times with 30/1(v/v) brine/0.1M NaOH solution (50mL each) and once with brine (50 mL). Passing the organic phase over Na₂SO₄Dried and volatiles were removed under reduced pressure. The amide was purified by flash chromatography eluting with heptane/ethyl acetate containing 0.02% (v/v) diethylmethylamine.

Yield: 5.69g (8.07 mmol).

MS：m/z705.4=[M+H]⁺(MW=705.0)。

The amide (3.19g, 4.53mmol) was dissolved in anhydrous THF (50mL) and BH was added₃THF (1M solution, 8.5mL, 8.5 mmol). The solution was stirred at RT for 16 h. Further adding BH₃THF (1M solution, 14mL, 14mmol) and stirring at RT for a further 16 h. The reaction was quenched by the addition of methanol (8.5 mL). N, N-dimethyl-ethylenediamine (3mL, 27.2mmol) was added, the solution was heated to reflux and stirred for 3 hours. The reaction mixture was cooled to RT, then diluted with ethyl acetate (300mL) and saturated Na₂CO₃Aqueous solution (2X 100mL) and saturated NaHCO₃Aqueous (2X 100mL) wash. Passing the organic phase over Na₂SO₄Drying and volatiles were removed under reduced pressure to give the crude amine intermediate (3.22 g).

The amine intermediate (3.22g) was dissolved in DCM (5 mL). Reacting Boc₂O (2.97g, 13.69mmol) was dissolved in DCM (5mL) and DIPEA (3.95mL, 22.65mmol) was added and the mixture was stirred at RT for 30 min. The Boc-and Mmt-protected intermediates were purified by flash chromatography.

Yield: 3.00g (3.79 mmol).

MS：m/z791.4=[M+H]⁺，519.3=[M-Mmt+H]⁺(MW, calculated = 791.1).

0.4M aqueous HCl (48mL) was added to a solution of Boc-and Mmt-protected intermediates in acetonitrile (45 mL). The mixture was diluted with acetonitrile (10mL) and stirred at RT for 1 hour. Subsequently, the pH of the reaction mixture was adjusted to 5.5 by addition of 5M aqueous NaOH solution. Acetonitrile was removed under reduced pressure and the aqueous solution was extracted with DCM (4 × 100 mL). The combined organic phases are passed over Na₂SO₄Dried and volatiles were removed under reduced pressure. The crude amine 14 was used without further purification.

Yield: 2.52g (3.19 mmol). The MW of the crude amine 14 is assumed to be 791.1 g/mol.

MS：m/z519.3=[M+H]⁺(MW, calculated =519.8 g/mol).

Example 17:

synthesis of linker building blocks 15a, 15b and 15c

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

amine 14(503mg, 0.635mmol, assuming crude 1 MW 791.1g/mol) was dissolved in 4mL DMF (anhydrous, mol. molecular sieves). Fmoc-N-Me-Ala-OH (310mg, 0.95) was added3mmol), COMU (408mg, 0.953mmol) and DIPEA (332. mu.L, 1.906mmol) and the reaction was stirred at RT for 3 h. To the mixture was added 150 μ L piperidine and 150 μ L DBU, and stirring was continued for another 60 minutes. Acetic acid, 400 μ L, was added and the product was purified by HPLC. The HPLC fraction containing the product 15a was diluted with saturated NaHCO₃The solution was neutralized and extracted twice with DCM. The combined organic phases are passed over Na₂SO₄Dried and volatiles were removed under reduced pressure.

Yield: 203mg (0.336 mmol).

MS：m/z604.1=[M+H]⁺(calculated MW =603.9 g/mol).

Connector structural unit 15b

Linker building block 15b was synthesized as described for 15a, except that Fmoc-Aib-OH was used in place of Fmoc-N-Me-Ala-OH.

Yield: 95mg (0.161 mmol).

MS：m/z604.2=[M+H]⁺(calculated MW =603.9 g/mol).

Connector structural unit 15c

Linker building block 15c was synthesized as described for 15a, except that Fmoc-N-Me-Ala-OH was replaced with Fmoc-N-Me-Aib-OH.

Yield: 149mg (0.241 mmol).

MS：m/z619.0=[M+H]⁺(calculated MW =617.9 g/mol).

Example 18:

synthesis of Trionile-linker thiols 16a, 16b, 16c, 16d, 16e and 16f

The treprostinil linker thiol 16a/16b was synthesized according to the following scheme:

a10 mL disposable syringe reactor equipped with PE frits was charged with 2-chlorotrityl chloride (TCP) resin (153mg, charge 1.22mmol/g, 0.186 mmol). A solution of treprostinil (54mg, 0.138mmol) and DIPEA (60 μ L, 0.346mmol) in DCM (anhydrous, mol. molecular sieve) was charged to the reactor. The reactor was stirred at RT for 2 hours. 200 μ L of methanol was added and the reactor was stirred for another 10 minutes. The solution was discarded, and the resin was washed with DCM (5 ×), DMF (5 ×), and DCM (10 ×). The resin was dried under vacuum (1 mbar). On a weight basis, a treprostinil loading of 0.72mmol/g TCP resin was obtained.

mu.L of THF (anhydrous, mol. molecular sieves) and 300. mu.L of a 1M LiOEt solution in THF (300. mu. mol) were charged to 30mg of Triille-loaded TCP resin (21, 6. mu. mol) in a disposable 2mL syringe reactor equipped with PE frits. The reactor was stirred at RT for 40 min. The solution was discarded, and the resin was washed with THF (2 ×). A syringe was charged with bis (pentafluorophenyl) carbonate (100mg, 254. mu. mol) in 1mL THF, which was stirred at RT for 90 min. The solution was discarded, and the resin was washed with THF (5 ×) and DMF (5 ×). A syringe was charged with 300. mu.L of DMF (anhydrous, mol. molecular sieves) solution of linker building block 15a (50mg, 83. mu. mol), DIPEA (50. mu.L, 287. mu. mol) and DMAP (1mg, 8. mu. mol). The syringe was stirred at RT for 3 hours. The solution was discarded, and the resin was washed with DMF (10 ×) and DCM (10 ×). The product was cleaved from the resin by incubation with 500. mu.L of cleavage cocktail HFIP/DCM/TES90/10/2v/v/v for 10 minutes (3X). The resin was washed with 500 μ L DCM (2X). TFA (250. mu.L) was added to the combined cleavage and wash solutions and the mixture was incubated for 10 min at RT. Volatiles were removed under reduced pressure. The residue was subjected to HPLC purification to give the thiol 16a/16b as a mixture of two regioisomers. The HPLC eluate was used in the next step without further treatment.

MS：m/z678.1=[M+H]⁺(calculated MW =678.0 g/mol).

Trionil linker thiol 16c/16d

Trionil linker thiols 16c/16d were synthesized as described for 16a/16b except that linker building blocks 15b were used instead of 15 a. The mercaptan 16c/16d was obtained as a mixture of isomers. The HPLC eluate was used in the next step without further treatment.

MS：m/z678.1=[M+H]⁺(calculated MW =678.0 g/mol).

Trionil linker thiols 16e and 16f

Triille linker thiols 16e and 16f were synthesized as described for 16a/16b, except that linker building block 15c was used instead of 15 a. The two isomers designated as structures 16e and 16f were separated by HPLC. The HPLC eluate was used in the next step without further treatment.

15e MS：m/z693.0=[M+H]⁺(calculated MW =692.0 g/mol).

15f MS：m/z693.0=[M+H]⁺(calculated MW =692.0 g/mol).

Example 19:

synthesis of linker building blocks 17a and 17b

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

L-Fmoc-Dpr (Boc) -OH (100mg, 0.234mmol) was dissolved in 0.5mL DMF (anhydrous, mol. molecular sieves). 6- (S-tritylsulfanyl) -hexylamine (71mg, 0.189mmol), COMU (97mg, 0.227mmol) and DIPEA (66 μ L, 0.378mmol) were added and the mixture was stirred at RT for 1 h. Piperidine (50. mu.L, 0.505mmol) and DBU (40. mu.L, 0.336mmol) were added and stirring was continued for 10 h. Cis-cyclohexane dicarboxylic anhydride (600mg, 3.89mmol) was added and stirring was continued for 1 hour. The solution was quenched with water/acetonitrile and acidified with acetic acid. The building blocks were purified by RP-HPLC. The structural designations of the first eluting diastereomer 17a and the second eluting diastereomer 17b are arbitrary and can be reversed.

Yield: 17a30mg (0.042mmol), 17b42mg (0.059mmol)

MS：m/z716.2=[M+H]⁺(calculated MW =716.0 g/mol).

Example 20:

synthesis of Trinidene linker thiol 18a/18b

Linker building block 17a (11mg, 12. mu. mol), EDC HCl (7.4mg, 38.5. mu. mol) and DMAP (4.7mg, 38.5. mu. mol) were dissolved in 300. mu.L DCM (anhydrous, mol. molecular sieves). The solution was charged to 15mg of treprostinil-loaded TCP resin (10.8 μmol, 0.72mmol/g, see example 3) in a single-use 2mL syringe reactor equipped with frit. The reactor was stirred at RT for 15 hours. The solution was discarded, and the resin was washed with DCM (10 ×). The product was cleaved by incubating the resin with 500. mu.L of HFIP/DCM30/70v/v for 10 minutes (3X). The resin was washed with 500 μ LDCM (2X). To the combined cleavage and wash solution 250 μ L TFA was added and the mixture was incubated at RT for 10 min. Volatiles were removed under reduced pressure. The residue was purified by RP-HPLC to give the thiol 18a/18b as a mixture of two regioisomers. The HPLC eluate was used in the next step without further treatment.

Yield: 18a/18b1.5mg (2. mu. mol), determined quantitatively by mercaptan assay by Ellman.

MS：m/z746.2=[M+H]⁺(calculated MW =746.0 g/mol).

Example 21:

synthesis of PEG-linker-drug conjugates 19a/b, 19c/6d, 19e, 19f and 19g/19h

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

excess linear PEG5kDa maleimides were added to HPLC eluates of the treprostinil linker thiols 16a/16b, 16c/16d, 16e, 16f and 18a/18 b. The mixture was neutralized by adding ph7.4 buffer (0.5M phosphate) and incubated at RT. After complete consumption of the thiol (about 1 hour), the mixture was acidified with acetic acid and separated from excess PEG-maleimide by RP-HPLC. The HPLC eluate was lyophilized to give PEG-linker-drug conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h, respectively.

Example 22:

determination of drug Release half of PEG conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h The stage of aging:

PEG-linker-drug conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h were dissolved in pH7.4 buffer (60mM sodium phosphate, 3mM EDTA, 0.05% Tween-20, 1mL) and incubated at 37 ℃. The amount of released treprostinil was determined by UPLC analysis of aliquots at various time period points, which were plotted against time. Drug release was found to follow first order kinetics. Curve fitting software was used to determine the drug release half-lives of the different conjugates (table 1).

Table 2:

item(s)	PEG-linker-drug conjugates	Half life of drug release
			1	19a/b	31 days
2	19c/19d	17 days
			3	19e	24 days
4	19f	37 days
			5	19g/19h	35 minutes

Example 23:

synthesis of intermediate 20:

the amino group of 6- (S-tritylsulfanyl) -hexylamine was Tmob (2,4, 6-trimethoxybenzyl) protected by adding 2,4, 6-trimethoxybenzaldehyde (4.22g, 21.51mmol) dropwise to a solution of 6- (S-tritylsulfanyl) -hexylamine (6.74g, 17.95mmol) and sodium cyanoborohydride (1.58g, 25.14mmol) in 88mL of methanol/DCM 1/1(v/v) in 44mL of methanol. The mixture was stirred at RT for 1.5 h and quenched with 95ml0.4n aqueous HCl. After stirring for further 30 min at RT, the mixture was extracted with ethyl acetate (4 ×). The combined organic layers were washed with saturated NaHCO₃Aqueous (2 x) and brine wash. Subjecting the organic layer to Na₂SO₄Dried and the solvent removed under reduced pressure.

The Tmob protected amine 20 was purified by flash chromatography eluting with DCM/MeOH containing 0.1% (v/v) triethylamine.

Yield: 5.88g (55%).

MS：m/z556.3=[M+H]⁺(calculated MW =555.79 g/mol).

Example 24:

synthesis of intermediate 21:

(1R,2S) -Cyclohexanedicarboxylic acid 1-methyl ester, CAS No. 88335-92-6 (see R. Manzano et al J. org. chem.2010,75(15),5417-5420) (506mg, 2.72mmol) was dissolved in toluene (11mL, anhydrous). Thionyl chloride (1.09mL, 15.0mmol) was added and the mixture was heated in a pressure tube at 60 ℃ for 1 hour. Volatiles were removed in vacuo. A Tmob protected amine 20(1.66g, 2.99mmol) and DIPEA (1.12mL, 6.43mmol) in DCM (30mL, anhydrous) was added and the mixture was stirred at RT for 2 h. Ethyl acetate was added and the organic layer was washed with 0.1N aqueous HCl (2 ×). Subjecting the organic layer to Na₂SO₄Dried and the solvent removed under reduced pressure. The methyl ester 21 was purified by flash chromatography eluting with ethyl acetate/heptane.

Yield: 1.55g (79%).

MS：m/z746.1=[M+Na]⁺(calculated MW =723.98 g/mol).

Example 25:

synthesis of intermediate 22:

methyl ester 21(3.12g, 4.31mmol) was dissolved in isopropanol (10 mL). 35mL of 1MLiOH aqueous solution was added and the mixture was stirred at RT for 5 days. Ethyl acetate was added and the organic layer was washed with 0.05N aqueous HCl (2 ×) and brine. Subjecting the organic layer to Na₂SO₄Dried and the solvent removed under reduced pressure. 225 was purified by flash chromatography eluting with ethyl acetate/heptane containing 0.1% formic acid (v/v).

Yield: 2.41g (79%).

MS：m/z710.1=[M+H]⁺(calculated MW =709.95 g/mol).

Example 26:

synthesis of intermediate 6 a:

compound 22(1.23g, 1.74mmol) was dissolved in DCM (18 mL). TFA (2mL) and TES (600. mu.L) were added and the mixture was stirred at RT for 40 min. Volatiles were removed in vacuo. The residue was dissolved in DCM (20mL) and trityl chloride (728mg, 2.61mmol) was added. The mixture was stirred at RT for 2 hours. DCM was removed under reduced pressure. Carboxylic acid 6a was purified by flash chromatography using ethyl acetate/heptane with 0.1% formic acid (v/v) as eluent followed by RP-HPLC.

Yield: 615mg (67%).

MS：m/z552.2=[M+Na]⁺(calculated MW =529.75 g/mol).

Example 27:

synthesis of Trinidene linker thiol 24a

Dmob-protected treprostinil 8(100mg, 0.185mmol), carboxylic acid 6a (195mg, 0.368mmol), EDC & HCl (72mg, 0.376mmol) and DMAP (43mg, 0.352mmol) were dissolved in DCM (1.8mL, anhydrous, mol. molecular sieves). The mixture was stirred at RT for 1 day. Ethyl acetate was added and the organic layer was washed with 0.1N aqueous HCl (3 ×) and brine. Subjecting the organic layer to Na₂SO₄Dried and the solvent removed under reduced pressure.

The residue was dissolved in HFIP (5mL), TFA (250. mu.L) and TES (250. mu.L) and stirred at RT for 30 min. The precipitate was filtered off and the filtrate was evaporated in vacuo.

UPLC analysis showed that the ratio of regioisomers 24a and 24b was 4/1 (column: Kinetex 100X 2.1mm, 1.7 μm XB-C18 silica, pore sizePhenomonex Ltd, Ashofburg, 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. The 24a inversion was the same as for compound 9 x.

The residue was dissolved in acetonitrile/water and 24a was purified by RP-HPLC (solvent A: H)₂O +0.01% HCl, solvent B: MeCN +0.01% HCl, gradient: 60-85% B over 16 minutes). Isomer 24a is eluted first followed by isomer 24 b. Fractions containing pure 24a were combined and lyophilized. The mixed fractions containing 24a and 24b were repurified.

Yield 24 a: 29.5mg (24%)

MS：m/z660.3=[M+H]⁺(calculated MW =659.9 g/mol).

¹H-NMR(CDCl₃,δ[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)。

¹³C-NMR(126MHz,CDCl₃,δ[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:

PEGylation of Trionil linker thiol 24a with 4-arm PEG20kDa maleimide Should be taken

A solution of the treprostinil linker thiol 24a (7.5mg, 11.3. mu. mol) in 2mL acetonitrile/water 9/1(v/v) was mixed with a solution of 4-arm PEG20kDa maleimide (53.5mg, 2.54. mu. mol) in 2mL acetonitrile/water 1/1 (v/v). The pH was adjusted to 7.0 by adding pH7.4 buffer (50mM phosphate, 0.8 mL). The reaction mixture was stirred at RT for 1.5H and then purified by RP-HPLC (solvent A: H containing 0.01% HCl₂O, solvent B: MeCN with 0.01% HCl, gradient: 45-85% B over 16 minutes). The product containing fractions were combined and acetonitrile was removed under reduced pressure. The solution was neutralized by adding pH7.4 buffer (phosphate, 0.5M). ConcentratingThe solution, and the buffer was phosphate exchanged by ultrafiltration (Vivaspin centrifugal concentrator, 10kDa cut-off PES membrane) with 10mM pH7.0 containing 46g/L mannitol to give 8.5mL25 of the final solution. UPLC and SEC analysis indicated a homogeneous material. The concentration was determined by quantifying treprostinil content after alkaline hydrolysis: a30 μ L aliquot was treated with 35 μ L0.5M NaOH. After incubation at RT for 30 min, 35 μ L of acetic acid was added. The treprostinil content was determined by UPLC using a treprostinil calibration curve. The total treprostinil content was found to be 2.0mg, corresponding to 30mg 25. Yield: 50% based on the PEG starting material.

Example 29:

treprostinil release kinetics of TransCon PEG linker treprostinil Compound 25

The release kinetics of treprostinil of 25 was determined as described in example 15 and compared to the results obtained for compound 13. No difference in half-life (5 days) was found.

Example 30:

trancon PEG linker treprostinil Compound 13 treprostinil Release in rat plasma Discharge kinetics

mu.L of pH7.5HEPES buffer (1M HEPES, 3mM EDTA) was mixed with 1.2mL rat plasma (WISTAR rat lithium heparin plasma, Innovative Research, Novi, MI, USA). mu.L of TransCon PEG linker Triille 13 solution (0.15mg13 in 1.5mL10mM phosphate 46g/L mannitol buffer pH 7.0) was added. The pH of the mixture was confirmed by pH electrode 7.4. The mixture was incubated at 37 ℃. Aliquots of 100 μ L were taken at the given time points. The released and total treprostinil content in 100 μ L aliquots were analyzed.

To analyze released treprostinil, 20 μ L of internal standard (2.8 μ g/mL tolbutamide in methanol/water 1/1 (v/v)) was added to 100 μ L aliquots and transferred to an Ostro96 well plate (waters gmbh, essboyne, germany). Plasma proteins were precipitated by adding three volumes of pre-cooled (0-5 ℃) acetonitrile containing 1% formic acid. Positive Pressure (4bar, Waters Positive Pressure-96Processor) was applied and the eluate was lyophilized. The lyophilisate was dissolved in 40. mu.L of 10mM ammonium formate pH 4.0/acetonitrile 7/3 (v/v). The solution was centrifuged and the supernatant analyzed for released treprostinil by UPLC-MS/MS.

To analyze the total treprostinil content (sum of released and carrier bound treprostinil), 20 μ L of internal standard (2.8 μ g/mL tolbutamide in methanol/water 1/1 (v/v)) was added in 100 μ L aliquots and 50 μ L0.5M LiOH was added. The mixture was incubated at room temperature for 2 hours in a shaker. After addition of 25. mu.L of 1M HCl, the mixture was transferred to an Ostro96 well plate (Waters GmbH, Eschboyn, Germany). Plasma proteins were precipitated by adding three volumes of pre-cooled (0-5 ℃) acetonitrile containing 1% formic acid. Positive Pressure (4bar, Waters Positive Pressure-96Processor) was applied and the eluate was lyophilized. The lyophilisate was dissolved in 100. mu.L of 10mM ammonium formate pH 4.0/acetonitrile 7/3 (v/v). The solution was centrifuged and the supernatant analyzed for total treprostinil content by UPLC-MS/MS.

Measuring the content of treprostinil by a UPLC-MS/MS method:

quantification of plasma treprostinil concentration was performed with a ThermoLTQ Orbitrap Discovery mass spectrometer connected in series with a Waters Acquity UPLC using an ESI probe and Waters BEH C18(50 × 2.1mM i.d.,1.7 μm particle size) as analytical column (mobile phase a: 10mM ammonium formate ph4.6, mobile phase: methanol, T =22 ℃). The gradient system included a linear gradient from 0.1% B to 95% B over 4 minutes, isocratically washing the phase with 95% B (0.5 min), and recovery phase (2.4 min), at a flow rate of 0.25 mL/min. Ion detection was performed in selective reaction monitoring (SRM, negative ionization) mode, with monitoring switching pairs of: daughter ion m/z331.2 for treprostinil parent ion m/z389.2, and daughter ion m/z170.0 for Internal Standard (IS) tolbutamide parent ion m/z 269.1.

The standard curve isBy combining the areas_TreprostinilArea/area_TolbutamideIs obtained by plotting the extracted peak area ratio of (a) to the calibrated treprostinil concentration of the calibration standard. The results were linearly regressed using standard software.

Area of quantitative test at different time points according to calibration curve_TreprostinilArea/area_TolbutamideThe extraction peak area ratio of (a) is used to calculate the treprostinil content.

The treprostinil release at different time points is expressed as% treprostinil release compared to the total treprostinil content (see figure 1). By using a first order kinetic fit, a half-life of 25 treprostinil release kinetics in 37 ℃ buffered rat plasma was obtained of 4.5 days, which is very consistent with the release kinetics in 37 ℃ ph7.4 buffer (example 15).

Example 31

PK of PEG Trinidene conjugate 25 in monkeys

A given dose level of 0.5mg/kg of 25(3mg/mL in 10mM phosphate pH7.0, 46g/L mannitol) was injected as a single dose to three male cynomolgus monkeys sc and iv. Blood samples were collected at given time points over two weeks. Plasma was analyzed for PEG content and total treprostinil content (total of released and carrier bound treprostinil). As compared to carrier-bound treprostinil, plasma treprostinil levels reflect the presence of treprostinil conjugates, rather than free treprostinil levels, due to the rapid elimination of free treprostinil.

To analyze total treprostinil content, 20 μ L of internal standard (2.8 μ g/mL tolbutamide in methanol/water 1/1 (v/v)) was added to 100 μ L of plasma samples and treprostinil standards in cynomolgus monkey plasma, and 50 μ L0.5M LiOH was added. The mixture was incubated at RT for 2.5 hours in a shaker. After addition of 25. mu.L of 1M HCl, the mixture was transferred to an Ostro96 well plate (Waters GmbH, Eschboyn, Germany). Plasma proteins were precipitated by adding three volumes of pre-cooled (0-5 ℃) acetonitrile containing 1% formic acid. Positive Pressure (4bar, Waters Positive Pressure-96Processor) was applied and the eluate was lyophilized. The lyophilisate was dissolved in 100. mu.L of 10mM ammonium formate pH 4.0/acetonitrile 7/3 (v/v). The solution was centrifuged and the supernatant analyzed for total treprostinil content by UPLC-MS/MS.

Measuring the content of treprostinil by a UPLC-MS/MS method:

quantification of plasma treprostinil concentration was performed with a Waters Acquity UPLC tandem ThermoLTQ Orbitrap spectrometer using ESI probe and Waters BEH C18(50 × 2.1mM i.d.,1.7 μm particle size) as analytical column (mobile phase a: 10mM ammonium formate ph4.6, mobile phase: methanol, T =22 ℃). The gradient system included a linear gradient from 0.1% B to 95% B over 4 minutes, isocratically washing the phase with 95% B (0.5 min), and recovery phase (2.4 min), at a flow rate of 0.25 mL/min. Ion detection is performed in a selective reaction monitoring (SRM, negative ion) mode, with the monitoring switching pair: daughter ion m/z331.2 for treprostinil parent ion m/z389.2, daughter ion m/z170.0 for Internal Standard (IS) tolbutamide parent ion m/z 269.1.

The calibration curve is obtained by dividing the area_TreprostinilArea/area_TolbutamideIs plotted against the calibrated treprostinil concentration of a calibration standard formulated in cynomolgus monkey plasma. The results were linearly regressed using standard software.

To analyze the total PEG content, plasma samples were subjected to alkaline pre-incubation to generate a homogeneous PEG species from 25. This is based on the fact that: after injection 25, different treprostinil carrier materials are produced due to the sequential release of 4 treprostinil from the carrier molecule over time.

50 μ L of plasma samples and 25PEG treprostinil conjugate standards in cynomolgus monkey plasma were diluted with 50 μ L of 200mM HEPES solution (pH7.5) and 50 μ L0.5MLiOH was added. The mixture was incubated at room temperature for 2 hours in a shaker. After addition of 50. mu.L of 1M HCl, the mixture was analyzed using the high sensitivity PEG ELISA kit P-0003 from Life Diagnostics Inc. West Chester, PA, USA according to the manufacturer's instructions.

The calibration curve was obtained by plotting the absorbance at 450nm against the calibration PEG concentration of the calibration standard. The results were fitted to a sigmoidal curve using standard software.

The absorbance at 450nm of the quantitative test at different time points was used to calculate the PEG content according to the calibration curve.

As a result: analysis of total treprostinil content following a single sc injection of 25 indicated that the treprostinil conjugate had prolonged circulation duration in monkeys over two weeks (figure 2).

Single dose iv injection 25 and plasma analysis of total treprostinil content indicated similar cycle duration (figure 3). Apparent first order total treprostinil elimination half-life of 2.9 days (rate constant k) was obtained by applying standard software_{Apparent appearance}：0.239d^-1)。

In contrast, plasma analysis of PEG carrier content showed slower elimination (fig. 3). By fitting to first order kinetics using standard software, a PEG carrier elimination half-life of 6.6 days (rate constant k) was obtained_{PEG elimination}: 0.105 day^-1)。

The same elimination rate constants are assumed for different treprostinil carrier materials, e.g., produced by the sequential linker hydrolysis/release of one to four treprostinils from a PEG carrier.

The apparent faster elimination half-life of total treprostinil compared to PEG carrier is based on the combination of elimination of PEG carrier and treprostinil release by linker hydrolysis. According to the determined value k of the rate constant_{Apparent appearance}(0.239 days)^-1) And k_{PEG elimination}(0.105 days)^-1) By linker cleavage rate constant k_{Connecting body}One may calculate the first order treprostinil release:

exp(-k_{apparent appearance}t)=exp(-k_{PEG elimination}t)*exp(-k_{Connecting body}t)=exp(-t[k_{PEG elimination}+k_{Connecting body}])

After taking the logarithm and rearranging, k can be calculated according to the following formula_{Connecting body}：

K_{Connecting body}=k_{Apparent appearance}-k_{PEG elimination}；k_{Connecting body}Day =0.239^-1Day-0.105^-1=0.134 day^-1

According to equation t_{Half life}= ln (2)/k, treprostinil release half-life by linker hydrolysis calculated as 5.2 days, which is very consistent with linker treprostinil release half-life measured in vitro for 5 days.

Example 32

PEG Trinidene conjugate 25 and PK of free Trinidene in rats

25(3mg/mL buffer (10mM pH7.0 phosphate, 46g/L mannitol)) was injected at a dose level of 5.5mg/kg as a single dose to each male Wistar rat. Three animals received sc injections and three animals received iv injections. Over the course of two weeks, blood samples were taken at given time points. Blood samples (250. mu.L) were directly loaded into collection tubes containing 50. mu.L of acidic citrate buffer (0.5M sodium citrate, pH 4.0). Plasma was analyzed for free treprostinil content and total treprostinil content (total of free and carrier bound treprostinil).

To analyze free treprostinil, 50 μ L of plasma was thawed on ice and combined with 5 μ L of acidic citrate buffer and 10 μ L of internal standard (0.28 μ g/mL tolbutamide in methanol/water 1/1 (v/v)). Samples were transferred to an Ostro96 well plate (Waters GmbH, Eschen, Germany) and plasma proteins were precipitated by rapid addition of 400. mu.L of pre-chilled (0-5 ℃) acetonitrile containing 1% formic acid. A Positive Pressure (4bar, Waters Positive Pressure-96Processor) was applied and the eluate was collected. The plate was then rinsed with 100 μ L of ice cold acetonitrile containing 1vol.% formic acid. The eluate was transferred to a 2mL vial, placed in an Eppendorf Thermomixer (10 ℃) and the eluate was concentrated over 45 minutes under a gentle stream of nitrogen to a final volume of 60-80 μ Ι _. To each vial was added 30. mu.L of a solvent mixture (10mM aqueous ammonium formate adjusted to pH4.0 with formic acid/acetonitrile 7/3(v/v)), and the solution was analyzed by UHPLC-MS/MS.

To prepare calibration standards, treprostinil was added to the blank plasma sample and treated as such.

The content of free treprostinil is determined by a UHPLC-MS/MS method:

quantification of plasma treprostinil concentration was performed in ES-mode using Agilent1290UHPLC tandem Agilent triplequad6460 system (MassHunter Xcalibur software). As analytical column, Waters BEH C18 (50X 2.1mm I.D.,1.7 μm particle size) was used. Mobile phase A: 10mM ammonium formate pH5.7, mobile phase B: methanol. The gradient system included an isocratic wash phase with a linear gradient from 35% B to 99% B, 99% B (0.9 min) over 8 min, and a recovery phase (3 min) at a flow rate of 0.200 mL/min (T =40 ℃).

Ion detection is performed in SRM mode, monitoring conversion pairs, for Trionil parent ion m/z389.1, daughter ion m/z 331.1; and the daughter ion m/z169.9 for Internal Standard (IS) tolbutamide parent ion m/z 269.0.

The calibration curve is obtained by dividing the area_TreprostinilArea/area_TolbutamideIs obtained by plotting the extracted peak area ratio of (a) to the calibrated treprostinil concentration of the calibration standard. The results were linearly regressed using standard software.

Total treprostinil plasma levels were determined as given in example 31.

As a result: analysis of free and total treprostinil content following a single sc injection of 25 showed prolonged circulation of the treprostinil conjugate and non-explosive release of free treprostinil, with more than 4 days in rats following iv (figure 4) or sc (figure 5) injections.

Example 33

Isolation of intermediate 6a by enantiomeric separation of racemic mixture 6a/6b

Racemic mixture 6a/6b (107g) was separated on a Chiralpak IA column (250X 76mm, 20 μm, flow rate 270 mL/min) using acetonitrile/acetic acid 1000/1(v/v) as eluent. The combined eluent of the second eluting enantiomer (6a) was mixed with 5vol% water and evaporated under reduced pressure. The residue was dissolved in DCM (500mL) and extracted with 0.1M HCl (500mL, 2 ×) and brine (500 mL). Passing the organic phase over Na₂SO₄Dried and the solvent removed under reduced pressure.

Yield: 28.9g (27%).

MS：m/z552.2=[M+Na]⁺(calculated MW =529.75 g/mol).

6a/6b enantiomer ratio determined on a Chiralpak IC column (4.5X 250mm, 5 μm, 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

Dmob-protected treprostinil 8(200mg, 0.370mmol), carboxylic acid 23(294mg, 0.555mmol), EDC & HCl (248mg, 1.295mmol), and DMAP (158mg, 1.295mmol) were dissolved in DCM (2.9mL, anhydrous, mol. molecular sieves). The mixture was stirred at RT for 1 day. Ethyl acetate was added and the organic layer was washed with 0.1N aqueous HCl (3 ×) and brine. Subjecting the organic layer to Na₂SO₄Dried and the solvent removed under reduced pressure.

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

UPLC analysis showed that the ratio of regioisomers 24a and 24b was 9/1 (column: Kinetex 100X 2.1mm, 1.7 μm XB-C18 silica, pore sizePhenomonex Ltd, Ashofburg, 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.

The residue was dissolved in acetonitrile/water and separated by RP-HPLC (solvent A: H)₂O +0.01% HCl, solvent B: MeCN +0.01% HCl, gradient: 57-62% B over 15 minutes) to isolate 24 a. The mixed fractions were repurified. Fractions containing pure 24a were combined and lyophilized.

Yield 24 a: 98mg (39%)

MS：m/z660.3=[M+H]⁺(calculated MW =659.9 g/mol).

Abbreviations

AcOH acetic acid

AIB 2-Aminoisobutyric acid

BnBr benzyl bromide

Boc tert-butoxycarbonyl-

BSA N, O-bis- (trimethylsilyl) -acetamide

COMU (1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholino-carbonHexafluorophosphates

d days

DIPEA diisopropylethylamine

DCM dichloromethane

DMAP 4- (dimethylamino) pyridine

DMF N, N-dimethylformamide

Dmob 2, 4-dimethoxybenzyl

DMSO dimethyl sulfoxide

Dpr 2, 3-diaminopropionic acid

EDC N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide

EDTA disodium EDTA dihydrate

EtOAc ethyl acetate

eq equivalent weight

h hours

HFIP 1,1,1,3,3, 3-hexafluoroisopropanol

HPLC high performance liquid chromatography

LC/MS mass spectrum tandem liquid chromatography

Mal Maleimidoamino group

MeOH methanol

MeCN acetonitrile

min for

Mmt 4-Methyltriphenylmethyl

mol. mol

m/z mass/charge

NaOH sodium hydroxide

NHS N-hydroxysuccinimide

PEG polyethylene glycol

Pfp pentafluorophenyl ester

PyBOB benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

PP polypropylene

RT Room temperature

RP inverse

sat, saturated

soln. solution

T temperature

T3P propylphosphonic anhydride

TCP 2-chlorotrityl chloride resin

TES Triethylsilane

Trt trityl radical

Tmob 2,4, 6-trimethoxybenzyl

TMS trimethylsilyl group

TransCon transient binding

THF tetrahydrofuran

TFA trifluoroacetic acid

UPLC ultra-high performance liquid chromatography

UV ultraviolet

Claims

1. A carrier-linked treprostinil prodrug of formula (II):

wherein each T is independently selected from structures (i) to (v):

wherein the dotted line represents the attachment to the rest of the molecule;

y is an integer in the range of 1 to 64, preferably in the range of 1 to 16, more preferably y is selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, even more preferably y is 8;

n is 0 or 1;

q is a spacer moiety;

or a pharmaceutically acceptable salt thereof.

2. The carrier-linked treprostinil prodrug of claim 1, wherein R^a2Is H.

3. The carrier-linked treprostinil prodrug of claim 1 or 2, wherein R^a4Selected from H, C_1-6Alkyl or substituted C_1-6Alkyl, preferably R^a4Is H.

4. The carrier-linked treprostinil prodrug of any one of claims 1 to 3, wherein R^a1And R^a3Taken together with the atoms to which they are attached to form ring a; wherein A is selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c_3-10A cyclic hydrocarbon group; a 4-to 7-membered aliphatic heterocyclic group; or a 9-to 11-membered aliphatic heterobicyclic group, wherein A is unsubstituted or substituted; more preferably ring a is selected from the group consisting of cyclopropane, cyclobutane, cyclopentane, cyclohexane and cycloheptane, even more preferably ring a is cyclohexane.

5. The carrier-linked treprostinil prodrug of any one of claims 1 to 4, wherein T is selected from the group consisting of structures (iii).

6. The carrier-linked treprostinil prodrug of any one of claims 1 to 5, wherein y is 4,6, 8, 10 or 12; preferably 4,6 or 8, more preferably y is 4.

7. The carrier-linked treprostinil prodrug of any of claims 1 to 6, wherein the carrier-linked treprostinil prodrug has the structure of formula (II-a):

wherein

Each T is independently selected from structures (i) to (v):

wherein the dotted line represents the attachment to the rest of the molecule;

q is a spacer moiety;

8. The carrier-linked treprostinil prodrug of any one of claims 1 to 7, wherein Q in formula (II) is selected from COOR^a9；OR^a9；C(O)R^a9；C(O)N(R^a9R^a9a)；S(O)₂N(R^a9R^a9a)；S(O)N(R^a9R^a9a)；S(O)₂R^a9；S(O)R^a9；N(R^a9)S(O)₂N(R^a9aR^a9b)；SR^a9；N(R^a9R^a9a)；OC(O)R^a9；N(R^a9)C(O)R^a9a；N(R^a9)S(O)₂R^a9a；N(R^a9)S(O)R^a9a；N(R^a9)C(O)OR^a9a；N(R^a9)C(O)N(R^a9aR^a9b)；OC(O)N(R^a9R^a9a)；W；C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50Alkynyl radical, wherein W, C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl is optionally substituted by one or more R^a10Substituted, said R^a10Are the same or different, and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl groups are optionally interrupted by one or more groups selected from: -W-; -C (O) O-; -O-; -c (o) -; -C (O) N (R)^a11)-；-S(O)₂N(R^a11)-；-S(O)N(R^a11)-；-S(O)₂-；-S(O)-；-N(R^a11)S(O)₂N(R^a11a)-；-S-；-N(R^a11)-；-OC(O)R^a11；-N(R^a11)C(O)-；-N(R^a11)S(O)₂-；-N(R^a11)S(O)-；-N(R^a11)C(O)O-；-N(R^a11)C(O)N(R^a11a) -; and-OC (O) N (R)^a11R^a11a)；

R^a9、R^a9a、R^a9bIndependently selected from H; w; and C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50Alkynyl radical, wherein W, C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl is optionally substituted by one or more R^a10Substituted, said R^a10Are the same or different, and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl group is optionally interrupted by one or more groups selected from: w; -C (O) O-; -O-; -c (o) -; -C (O) N (R)^a11)-；-S(O)₂N(R^a11)-；-S(O)N(R^a11)-；-S(O)₂-；-S(O)-；-N(R^a11)S(O)₂N(R^a11a)-；-S-；-N(R^a11)-；-OC(O)R^a11；-N(R^a11)C(O)-；-N(R^a11)S(O)₂-；-N(R^a11)S(O)-；-N(R^a11)C(O)O-；-N(R^a11)C(O)N(R^a11a) -; and-OC (O) N (R)^a11R^a11a)；

W is selected from phenyl; a naphthyl group; an indenyl group;indanyl; tetrahydronaphthyl; c_3-10A cyclic hydrocarbon group; 4-to 7-membered heterocyclyl; or 9-to 11-membered heterobicyclic group wherein W is optionally substituted with one or more R^a10Substituted, said R^a10Are the same or different;

9. The carrier-linked treprostinil prodrug of claim 8, wherein each-Q-is independently-Q^1a-Q¹-, wherein the asterisk indicates the link to Z¹And wherein

Q¹Is selected from C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl optionally substituted by one or more R^a10Substituted, optionally interrupted by one or more groups selected from the group consisting of, with the proviso that Q¹Is at least C₂：C_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

10. The carrier-linked treprostinil prodrug of any of claims 1 to 9, wherein the carrier-linked treprostinil prodrug is of formula (IIaa):

wherein

Each T is independently selected from structure (i) or (iii):

wherein the dotted line represents the attachment to the rest of the molecule;

y is an integer ranging from 1 to 64;

11. The carrier-linked treprostinil prodrug of any one of claims 1 to 10, wherein the carrier-linked treprostinil prodrug is of formula (IIab):

wherein

T、R^a2、R^a4、A¹、Q¹、Z¹And y is as defined in claim 10.

12. The carrier-linked treprostinil prodrug of any one of claims 1 to 11, wherein the carrier-linked treprostinil prodrug is of formula (IIac) or (IIad):

wherein

Each T is independently selected from structure (i) or (iii):

y is an integer ranging from 1 to 64,

x is selected from 2,3, 4, 5, 6, 7 or 8,

X₁is selected from C_1-15Alkyl radical, C_2-15Alkenyl and C_2-15Alkynyl optionally substituted or interrupted by one or more groups selected from: c_3-7Cycloalkyl, 4-to 7-membered heterocyclyl,

Wherein

R¹³And R^13aIndependently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6Alkynyl.

13. The carrier-linked treprostinil prodrug of any one of claims 1 to 12, wherein the carrier-linked treprostinil prodrug is of formula (IIb):

wherein

Wherein

R¹³And R^13aIndependently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl or C_2-6An alkynyl group;

y is an integer ranging from 1 to 64;

x is selected from 2,3, 4, 5, 6, 7 or 8;

14. The carrier-linked treprostinil prodrug of claim 12 or 13, wherein x is 6.

15. The carrier-linked treprostinil prodrug of any of claims 1 to 14, wherein the carrier-linked treprostinil prodrug has the structure of formula (IIba):

wherein

y is an integer ranging from 1 to 64; and is

16. The carrier-linked treprostinil prodrug of any one of claims 1 to 15, wherein carrier Z is¹Having the structure of formula (VII):

wherein

B is the core of the branch,

a is a poly (ethylene glycol) -based polymeric chain, and

n is an integer from 3 to 32.

17. The carrier-linked treprostinil prodrug of any one of claims 1 to 16, wherein Z is¹Representing part (IIca):

wherein

t ranges from 80 to 160;

w ranges from 2 to 6; and is

The dashed line represents the remainder of the treprostinil prodrug attached to the carrier.

18. The carrier-linked treprostinil prodrug of claim 17, wherein w is 2 or 3.

19. The carrier-linked treprostinil prodrug of any one of claims 1 to 18, which is of formula (IIc) or a pharmaceutically acceptable salt thereof:

wherein y is 4, and Z¹Representing part (IIca):

20. The carrier-linked treprostinil prodrug of claim 1, wherein R^a1Is C_1-6Alkyl or substituted C_1-6An alkyl group.

21. The carrier-linked treprostinil prodrug of any one of claims 1 to 3, wherein R^a3Is H, C_1-6Alkyl or substituted C_1-6An alkyl group.

22. A carrier-linked treprostinil prodrug of formula (I):

wherein each T is independently selected from structures (i) to (v):

wherein the dotted line indicates a connection to X⁰；

y is an integer in the range of 1 to 64, preferably in the range of 1 to 16, more preferably y is selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, even more preferably y is 8,

each X⁰Independently is (X)^0A)_m1-(X^0B)_m2；

m 1; m2 is independently 0; or 1;

X^0Ais T⁰；

X^0BIs branched or unbranchedC of (A)_1-15Alkylene which is unsubstituted or substituted by one or more R³Substituted, said R³Are the same or different;

T⁰is phenyl; a naphthyl group; a group; an indenyl group; indanyl; c_3-7A cyclic hydrocarbon group; a 3-to 7-membered heterocyclic group; or 8 to 11 membered heterobicyclic group, wherein T⁰Optionally substituted by one or more R⁷Substituted, said R⁷Are the same or different;

R¹¹、R^11a、R^11bIndependently selected from H; c_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl, wherein C_1-6An alkyl group; c_2-6An alkenyl group; and C_2-6Alkynyl is optionally substituted with one or more halo, the halo being the same or different;

or a pharmaceutically acceptable salt thereof.

23. The carrier-linked treprostinil prodrug of claim 22, or a pharmaceutically acceptable salt thereof, wherein Z is¹Comprising a PEG-based polymer.

24. The carrier-linked treprostinil prodrug of claim 22 or 23, or a pharmaceutically acceptable salt thereof, wherein Z¹Comprising a water-soluble polymer.

25. The carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 22 to 24, wherein y is an integer from 4 to 16, preferably y is 8.

26. The carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof of any one of claims 1 to 15 and 20 to 25, wherein Z is¹Having the structure of formula (III):

wherein

The dotted line indicates a connection to X⁰Provided that one of m1, m2 is 1, and wherein if m1, m2=0, the carrier is covalently linked to T,

each m, n and p is independently an integer in the range of 5 to 500,

and q ranges from 2 to 32.

27. The carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof of any one of claims 1 to 15 and 20 to 25, wherein Z is¹Is of formula (VI):

wherein

B is the core of the branch,

each a is independently a poly (ethylene glycol) based polymeric chain,

each Hyp^yIndependently is a branch portion, and

n is an integer from 3 to 32.

28. The carrier-linked treprostinil prodrug of claim 27, or a pharmaceutically acceptable salt thereof, wherein Z is¹Selected from the structures (A), (B), (C), (i-y) to (iii-y):

wherein

The dotted line represents X attached to formula (I)⁰Provided that one of m1, m2 is 1, and wherein if m1, m2=0, the carrier is covalently linked to T;

p is an integer from 5 to 2000, preferably from 10 to 1000, more preferably from 10 to 500, most preferably from 100 to 1000;

q is 1 or 2.

29. The carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof of any one of claims 1 to 15 and 20 to 25, wherein Z is¹Having the structure of formula (V):

Hyp¹ _mx-POL^x-Hyp² (V)，

wherein

Hyp¹and Hyp²Independently a highly branched portion, and

mx is 0 or 1.

30. The carrier-linked treprostinil prodrug of claim 29, or a pharmaceutically acceptable salt thereof, wherein mx is 0 and POL^x-Hyp²Selected from the following structures:

wherein

The dotted line indicates a connection to the part X⁰Provided that one of m1, m2 is 1, and wherein if m1, m2=0, the carrier is covalently linked to T;

qx is an integer from 0 to 15, preferably from 3 to 7, more preferably qx is 6.

31. The carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof of any one of claims 1 to 15 and 20 to 25, wherein Z is¹Comprises at least 100 amino acid residues, and

32. The carrier-linked treprostinil prodrug of any one of claims 22 to 31, or a pharmaceutically acceptable salt thereof, wherein each X⁰Is unsubstituted.

33. A pharmaceutical composition comprising the carrier-linked treprostinil prodrug of any one of claims 1 to 32, or a pharmaceutically acceptable salt thereof, optionally comprising one or more pharmaceutically acceptable excipients.

34. The pharmaceutical composition of claim 33, wherein the first and second active ingredients are selected from the group consisting of,

(i) wherein the carrier-linked treprostinil prodrug in the pharmaceutical composition is in a dosage sufficient to provide a therapeutically effective amount of treprostinil for at least 12 hours for a single application, and/or

35. A carrier-linked treprostinil prodrug of any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 33 or 34 for use as a medicament.

36. A carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 32 or a pharmaceutical composition according to claim 33 or 34 for use in a method of treatment or prevention of a disease or disorder which can be treated and/or prevented by treprostinil.

37. Use of the carrier-linked treprostinil prodrug or a pharmaceutically acceptable salt or pharmaceutical composition thereof according to claim 36, wherein the disease or disorder is pulmonary arterial hypertension.

38. A method of treating, controlling, delaying or preventing in a mammalian patient in need of treatment for one or more conditions, which comprises administering to said patient a diagnostically and/or therapeutically effective amount of a carrier-linked treprostinil prodrug of any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of claim 33 or 34.

39. The water-soluble carrier-linked prodrug of any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 33 or 34 for use as a medicament for topical, enteral, parenteral, inhalation, injection or infusion, intra-articular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous and intraperitoneal, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intraventricular or intrasternal administration.