Treprostinil prodrugs

Abstract

The present invention aims to provide novel prodrugs of treprostinil, and methods of making and using these prodrugs.SOLUTION: Provided is a compound having the formula or a pharmaceutically acceptable salt thereof, wherein X is OR14, R14 is H, one of R2 and R3 is a second drug moiety, and the other of R2 and R3 is H, wherein the second drug moiety is a pain-relieving drug moiety or a non-steroidal anti-inflammatory drug moiety.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority This application claims priority to U.S. Provisional Application No. 62 / 890,839, filed August 23, 2019, and U.S. Provisional Application No. 62 / 976,183, filed February 13, 2020, each of which is incorporated by reference in its entirety.

[0002] Field TECHNICAL FIELD This application relates generally to prodrugs of prostacyclin, and more particularly to prodrugs of treprostinil, and methods of making and using such prodrugs. [Background technology]

[0003] Pulmonary hypertension is a progressive, life-threatening disease characterized by elevated pressure in the pulmonary vasculature that can lead, inter alia, to heart failure.

[0004] Pulmonary hypertension (PH) has previously been classified as primary (idiopathic) or secondary. The World Health Organization (WHO) classifies pulmonary hypertension into five groups: Group 1: pulmonary arterial hypertension (PAH); Group 1': Pulmonary veno-occlusive disease (PVOD) and / or pulmonary capillary hemangiomatosis (PCH) Group 2: PH with left heart disease; Group 3: PH with pulmonary disease and / or hypoxemia; Group 4: PH due to chronic thrombotic and / or embolic disease; and Group 5: Other conditions; unknown multifactorial mechanisms (sarcoidosis, histiocytosis X, lymphangiomatosis, and pulmonary vascular compression, etc.).

[0005] Currently, there are many products approved for specific types of pulmonary hypertension, including group 1 (PAH). These products include products containing treprostinil as an active ingredient, such as Remodulin® (treprostinil) injection. However, subcutaneous administration of treprostinil typically results in site pain. In some cases, the site pain is so severe that patients must discontinue use of subcutaneous treprostinil. Therefore, there is a need for a method of administering treprostinil without causing site pain.

[0006] Once treprostinil is absorbed, regardless of the route of administration, its half-life is short, approximately 1 hour. Therefore, there is a need to extend the half-life of treprostinil.

[0007] Another challenge associated with oral administration of treprostinil is its high first-pass efficacy, measured at approximately 60% in animal studies. Therefore, there is a need to increase the bioavailability of treprostinil, possibly by modifying the first-pass efficacy. Summary of the Invention

[0008] In one embodiment, the compound of the formula:

[0009] [ka]

[0010] or a pharmaceutically acceptable salt thereof, wherein: X is OR 14 , -NR 1 SO2R 1 , -NR 1 CO2H,

[0011] [ka]

[0012] or

[0013] [ka]

[0014] and each R 1 are independently H or C1-C4 alkyl, and R 8 is an optionally substituted C1-C6 alkyl or amino acid side group, or R 1 and R 8 together form a 4- to 7-membered heterocycle, R 14 is H, optionally substituted C1-C6 alkyl, a first drug moiety, or

[0015] [ka]

[0016] and R 11 is absent, optionally substituted C1-C6 alkylene, or -Q 1 -O- and Q 1 is an optionally substituted C1-C6 alkylene, and R 12 and R 13 each is independently H, optionally substituted C1-C6 alkoxy, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, optionally substituted C1-C8 cycloal; R 2 and R 3 each independently represents a second drug moiety, a third drug moiety, H, a phosphorus-containing group, —C(O)R 6 or an -ABC substituent, where A is an optionally substituted C1-C6 alkylene, -NR 6 -, -C(O)-, -C(O)O- or -C(O)NR 6 - and; B is a bond that is optionally substituted C1-C6 alkylene, -C(O)-, -O-, -S-, heterocyclyl; C is optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted cycloalkyl, -(HCH) q -OR 6 , -C(O)N(R 6 )2, -C(O)N(R 18 )2, -C(O)R 6 , -CO2H, -OR 6 , -N(R 18 )2, -N(R 6 )2, or

[0017] [ka]

[0018] and both R 18 forms an optionally substituted 3-8 membered heterocyclyl; Each R 6 are independently H, optionally substituted C-C alkyl, optionally substituted heteroaryl, optionally substituted aryl, or R 6 together form an optionally substituted 4- to 8-membered heterocyclyl or an optionally substituted 5-membered heteroaryl; or R 2 and R 3 are connected together by -C(O)-, -SO2-, in 8-12 membered heterocyclyl,

[0019] [ka]

[0020] Forming Each R 10 is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted aryl; q is 0, 1, 2, 3, 4, 5 or 6 however, If A is -C(O)-, B is not a bond and C is -N(R 6 )2 is not; If A is -C(O)-, B is not a bond and C is -OR 6 isn't it; R 14 , R 2 , R 3 is not H; If X is OH, then R 2 and R 3 is not H; R 8 If H, then R 2 and R 3 At least one of them is not H.

[0021] In another aspect, methods of treating a disease or condition are provided, the methods comprising administering to a subject a compound disclosed herein. In some embodiments, the disease or condition is one or more selected from the group consisting of pulmonary hypertension, congestive heart failure, peripheral vascular disease, Raynaud's phenomenon, scleroderma, renal failure, peripheral neuropathy, digital ulcers, intermittent claudication, ischemic limb disease, peripheral ischemic lesions, pulmonary fibrosis, and asthma. [Brief explanation of the drawings]

[0022] [Figure 1A] FIG. 1 shows selected prodrugs. [Figure 1B] FIG. 1 shows selected prodrugs. [Figure 1C] FIG. 1 shows selected prodrugs. [Figure 1D] FIG. 1 shows selected prodrugs. [Figure 1E] FIG. 1 shows selected prodrugs. [Figure 1F] FIG. 1 shows selected prodrugs. [Figure 1G] FIG. 1 shows selected prodrugs. [Figure 1H] FIG. 1 shows selected prodrugs. [Figure 1I] FIG. 1 shows selected prodrugs. [Figure 1J] FIG. 1 shows selected prodrugs. [Figure 1K] FIG. 1 shows selected prodrugs. [Figure 1L] FIG. 1 shows selected prodrugs. [Figure 1M] FIG. 1 shows selected prodrugs. [Figure 1N] FIG. 1 shows selected prodrugs. [Figure 1O] FIG. 1 shows selected prodrugs. [Figure 1P] FIG. 1 shows selected prodrugs. [Figure 1Q] FIG. 1 shows selected prodrugs. [Figure 1R] FIG. 1 shows selected prodrugs. [Figure 1S] FIG. 1 shows selected prodrugs. [Figure 1T] FIG. 1 shows selected prodrugs. [Figure 1U] FIG. 1 shows selected prodrugs. [Figure 1V] FIG. 1 shows selected prodrugs. [Figure 1W] FIG. 1 shows selected prodrugs. [Figure 1X] FIG. 1 shows selected prodrugs. [Figure 1Y] FIG. 1 shows selected prodrugs. [Figure 2] Mean (±SD) prodrug i.v. (bottom two graphs) and treprostinil (top two graphs) plasma concentration-time profiles after single oral gavage doses of 1, 10, 30 mg / kg (dogs) / 50 mg / kg (rats), prodrug i.v. (left graph) and rats (right graph). [Figure 3]Mean (±SD) treprostinil plasma concentration-time profiles after single oral gavage doses of 1, 10, and 50 mg / kg of prodrug XVII in rats or single oral doses of 10 or 30 mg / kg of prodrug XVII in dogs. [Figure 4] Mean (±SD) treprostinil plasma concentration-time profiles after single oral gavage doses of 1, 10, and 50 mg / kg prodrug VI to rats or a single oral dose of 10 mg / kg prodrug VI to dogs. [Figure 5A] Mean (±SD) prodrug IV (5A) and treprostinil (5B) plasma concentration-time profiles after a single oral gavage dose of 1, 10, or 50 mg / kg of prodrug or a single bolus intravenous injection of 1 mg / kg of prodrug in male rats. [Figure 5B] Mean (±SD) prodrug IV (5A) and treprostinil (5B) plasma concentration-time profiles after a single oral gavage dose of 1, 10, or 50 mg / kg of prodrug or a single bolus intravenous injection of 1 mg / kg of prodrug in male rats. [Figure 6] Mean (±SD) treprostinil plasma concentration-time profiles following single oral gavage doses of prodrug XVI 1, 10, and 50 mg / kg or a single bolus intravenous injection of prodrug XVI 1 mg / kg in male rats. [Figure 7] Mean (±SD) treprostinil plasma concentration-time profiles following single oral gavage doses of 1, 10, and 50 mg / kg of prodrug XVII or a single intravenous bolus of 1 mg / kg of prodrug XVII in male rats. [Figure 8A] Plasma concentration-time profiles of prodrug VI (8A) and treprostinil (8B) following single oral gavage doses of 1, 10, and 50 mg / kg of prodrug VI or a single intravenous bolus of 1 mg / kg of prodrug VI. [Figure 8B]Plasma concentration-time profiles of prodrug VI (8A) and treprostinil (8B) following single oral gavage doses of 1, 10, and 50 mg / kg of prodrug VI or a single intravenous bolus of 1 mg / kg of prodrug VI. [Figure 9] 1 is a plot showing data regarding the conversion of selected treprostinil prodrugs to treprostinil in hepatocytes. [Figure 10] 1 is a plot showing data regarding the conversion of selected treprostinil prodrugs to treprostinil in liver microsomes. [Figure 11] 1 shows plots comparing the activity of selected treprostinil prodrugs with the activity of treprostinil at the IP1 receptor. [Figure 12] 1 shows plots comparing the activity of selected treprostinil prodrugs with the activity of treprostinil at IP1, EP2, and DP1 receptors. DETAILED DESCRIPTION OF THE INVENTION

[0023] As used in this specification and claims, the singular forms include "a," "an," and "the" unless the context clearly dictates otherwise. As used herein, unless otherwise indicated, the terms "comprise," "comprises," and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other unstated integers or groups of integers. The term "or" is inclusive unless modified, for example, by "either." Thus, unless the context requires otherwise, the term "or" means any one member of a particular list and also includes any combination of members of that list. All numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified by the term "about" in all instances, other than in the operating examples or where otherwise indicated.

[0024] Headings are for convenience only and should not be construed as limiting the invention in any way. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The terms used herein are used for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined solely by the claims. In order to make this disclosure more readily understandable, certain terms are first defined. Further definitions are provided throughout the detailed description.

[0025] All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations (+) or (-) varied by 0.05%, 1%, 2%, 5%, 10%, or 20%. It is also to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about," and it is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and equivalents thereof are known in the art.

[0026] "HPLC" refers to high performance liquid chromatography.

[0027] "NMR" refers to nuclear magnetic resonance.

[0028] As used herein, C m -C n , e.g. C1-C 12 , C1-C8, or C1-C6, when used before a group, refers to a group containing mn carbon atoms.

[0029] "Optionally substituted" refers to a group selected from the group and substituted forms of the group. The substituents may include any of the groups defined below. In one embodiment, the substituents are C-C 10 or C1-C6 alkyl, substituted C1-C 10 or C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C10 Aryl, C3-C8 cycloalkyl, C2-C 10 Heterocyclyl, C1-C 10 Heteroaryl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, substituted C6-C 10 Aryl, substituted C3-C8 cycloalkyl, substituted C2-C 10 Heterocyclyl, substituted C1-C 10 It is selected from heteroaryl, halo, nitro, cyano, —CO 2 H, or C 1 -C 6 alkyl esters thereof.

[0030] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms. This term includes, by way of example, straight-chain and branched-chain hydrocarbyl groups such as methyl (CH-), ethyl (CHCH-), n-propyl (CHCH-), isopropyl ((CH)CH-), n-butyl (CHCHCH-), isobutyl ((CH)CHCH-), sec-butyl ((CH)(CHCH)CH-), t-butyl ((CH)C-), n-pentyl (CHCHCHCHCHCH), and neopentyl ((CH)CCH-).

[0031] "Alkenyl" refers to a monovalent straight or branched chain hydrocarbon group having 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and preferably 2 to 4 carbon atoms, and having at least 1, preferably 1 to 2 sites of vinyl (>C=C<) unsaturation. Such groups are exemplified, for example, by vinyl, allyl, and 3-en-1-yl. The term includes cis and trans isomers or mixtures of these isomers.

[0032] "Alkynyl" refers to a straight- or branched-chain monovalent hydrocarbon radical having 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and preferably 2 to 3 carbon atoms, and having at least 1, preferably 1 to 2 sites of acetylenic (-C≡C-) unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CHC≡CH).

[0033] "Substituted alkyl" includes alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyl, cyclo "Cycloalkenyloxy" refers to an alkyl group having 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclyl, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein.

[0034] "Heteroalkyl" refers to an alkyl group in which one or more carbons are substituted with -O-, -S-, SO2, a P-containing moiety as provided herein, -NR Q -,

[0035] [ka]

[0036] refers to an alkyl group substituted with a moiety, where R Qis H or C1-C6 alkyl. Substituted heteroalkyl includes alkoxy, substituted alkoxy, acylamino, acylamino, aminocarbonyloxy, aminocarbonylamino, aminocarbonyloxy, aminosulfonyloxy, aminosulfonyloxy, aminosulfonylamino, aminosulfonyloxy, aryloxy, substituted arylthio, arylthio, substituted arylthio, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyloxy, cycloalkylamino, ... " refers to a heteroalkyl group having from 1 to 5, preferably from 1 to 3, or more preferably from 1 to 2 substituents selected from the group consisting of chloroalkenyloxy, substituted siakenylthio, substituted cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted cycloalkenylthio, hydroxy, substituted guanidino, hydroxy, substituted heteroaryloxy, substituted heteroarylthio, substituted heteroarylthio, substituted heterocyclyloxy, substituted heterocyclyloxy, nitro, SO3H, substituted sulfonyloxy, thioacyl, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein.

[0037] "Substituted alkenyl" includes alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substituted cyano. " refers to an alkenyl group having from 1 to 3, preferably 1 to 2, substituents selected from the group consisting of cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenyl, guanidino, substituted guanidino, halo, hydroxyl, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein, with the proviso that the hydroxyl or thiol substitutions are not attached to a vinyl (unsaturated) carbon atom.

[0038] "Heteroalkenyl" refers to a moiety in which one or more carbons contain -O-, -S-, SO2, P as provided herein, -NRQ-,

[0039] [ka]

[0040] refers to an alkenyl group substituted with R Qis H or C1-C6 alkyl. Substituted heteroalkenyl includes alkoxy, substituted alkoxy, acylamino, acylamino, acylamino, acyloxy, aminocarbonylamino, aminocarbonyloxy, aminocarbonyloxy, aminosulfonyloxy, aminosulfonyloxy, aminosulfonylamino, aminosulfonyloxy, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, substituted arylthio, (carboxyl)oxy, cycloalkyl, substituted cycloalkyloxy, cycloalkenyl, substituted cyclo " refers to a heteroalkenyl having 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkenyloxy, cycloalkenyloxy, substituted siakenylthio, substituted cycloalkenylthio, guanidino, substituted cycloalkenylthio, hydroxy, substituted guanidino, hydroxy, heteroaryloxy, substituted heteroarylthio, substituted heteroarylthio, substituted heterocyclyloxy, substituted heterocyclylthio, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein.

[0041] "Substituted alkynyl" includes alkoxy, substituted alkoxy, acylamino, aminocarbonyloxy, aminocarbonyloxy, aminosulfonyloxy, aminosulfonyloxy, aminosulfonyloxy, aryloxy, substituted arylthio, arylthio, substituted arylthio, carboxyl ester, (carboxyl ester)oxy, cycloalkyl, substituted cycloalkyloxy, cycloalkenyl, substituted cycloalkenyloxy, cycloalkenyloxy, substituted sialkenyloxy, sialkenylthio, substituted cycloalkenyl "A" refers to an alkynyl group having 1 to 3, preferably 1 to 2, substituents selected from the group consisting of thio, guanidino, substituted cycloalkenylthio, hydroxy, substituted guanidino, hydroxy, substituted guanidino, heteroaryloxy, substituted heteroarylthio, substituted heteroarylthio, substituted heterocyclyloxy, substituted heterocyclyloxy, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein any hydroxyl or thiol substitution is not attached to a vinyl (unsaturated) carbon atom.

[0042] "Heteroalkynyl" refers to an alkyl group in which one or more carbons are substituted with -O-, -S-, SO2, a P-containing moiety, -NRQ-, as provided herein,

[0043] [ka]

[0044] refers to an alkynyl group substituted with a moiety, wherein R Qis H or C1-C6 alkyl. Substituted heteroalkynyl includes alkoxy, substituted alkoxy, acylamino, acylamino, aminocarbonyloxy, aminocarbonylamino, aminocarbonyloxy, aminosulfonyloxy, aminosulfonyloxy, aminosulfonyloxy, aminosulfonylamino, aminosulfonyloxy, aryloxy, substituted arylthio, arylthio, substituted arylthio, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyloxy, cycloalkenyloxy, substituted sialic acid, cycloalkenylthio, substituted sialic acid, cycloalkenylthio, substituted sialic acid, cycloalkenylthio, substituted sialic acid, aryl ... " refers to a heteroalkenyl group having 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein.

[0045] "Alkylene" refers to a divalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 3 carbon atoms, which is either straight-chained or branched. This term is exemplified by groups such as methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), isopropylene (-CHCH(CH)- or -CH(CH)CH-), butylene (-CHCHCHCH-), isobutylene (-CHCH(CH-)CH-), sec-butylene (-CHCH(CH-)CH-), and the like. Similarly, "alkenylene" and "alkynylene" refer to alkylene moieties containing one or two carbon-carbon double or triple bonds, respectively.

[0046] "Substituted alkylene" refers to an alkylene group having 1 to 3 hydrogen atoms substituted with a substituent selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, substituted aryloxy, cyano, halogen, nitro, carboxyl, carboxyl ester, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and oxo, where said substituents are defined herein. In some embodiments, the alkylene has 1 to 2 of the foregoing groups, or R Q is substituted with H or a C1-C6 alkyl -O-, -S-, or -NRQ moiety. Note that when an alkylene is substituted with an oxo group, two hydrogens bonded to the same carbon of the alkylene group are replaced with "=O". "Substituted alkenylene" and "substituted alkynylene" refer to alkenylene and alkynylene moieties substituted with the substituents described for substituted alkylene.

[0047] "Alkynylene" refers to a straight- or branched-chain divalent hydrocarbon radical having 2 to 10 carbon atoms, preferably 2 to 6 carbon atoms, and preferably 2 to 3 carbon atoms, and having at least 1, preferably 1 to 2 sites of acetylenic (-C≡C-) unsaturation. Examples of such alkynylene groups include C≡C- and CHC≡C-.

[0048] "Substituted alkynyl" includes alkoxy, substituted alkoxy, acylamino, aminocarbonyloxy, aminocarbonyloxy, aminosulfonyloxy, aminosulfonyloxy, aminosulfonyloxy, aminosulfonyloxy, aminosulfonylamino, aryloxy, substituted arylthio, arylthio, substituted arylthio, carboxyl ester, (carboxyl ester)oxy, cycloalkyl, substituted cycloalkyloxy, cycloalkenyl, substituted cycloalkenyloxy, cycloalkenyloxy, substituted sialic acid, sialic acid, sialic acid, aryl "Alkynyl" refers to an alkynyl group having one to three substituents, preferably one to two substituents, selected from the group consisting of arylthio, substituted cycloalkenylthio, guanidino, substituted cycloalkenylthio, hydroxy, substituted guanidino, hydroxy, substituted heteroaryloxy, substituted heteroarylthio, substituted heteroarylthio, substituted heterocyclyloxy, substituted heterocyclyloxy, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioalkyl, thiol, alkylthio, and substituted alkylthio, wherein the hydroxyl or thiol substitution is not attached to an acetylenic carbon atom.

[0049] "Heteroalkylene" refers to an alkyl group in which one or more carbons are substituted with an alkyl group selected from the group consisting of -O-, -S-, SO2, a P-containing moiety as provided herein, -NR Q -,

[0050] [ka]

[0051] refers to a substituted alkylene group that is a moiety, where R Qis H or C1-C6 alkyl. "Substituted heteroalkylene" refers to a heteroalkynyl group having 1 to 3 substituents, preferably 1 to 2 substituents, selected from the substituents disclosed for the substituted alkylene.

[0052] "Heteroalkenylene" refers to an alkenylene group in which one or more carbons are bonded to an alkyl group such as -O-, -S-, SO2, a P-containing moiety as provided herein, -NRQ-,

[0053] [ka]

[0054] refers to an alkenylene group substituted with a moiety, where R Q is H or C1-C6 alkyl. "Substituted heteroalkenylene" refers to a heteroalkynylene group having 1 to 3 substituents, preferably 1 to 2 substituents, selected from the substituents disclosed for substituted alkenylene.

[0055] "Heteroalkynylene" refers to an alkynyl group in which one or more carbons are substituted with -O-, -S-, SO2, a P-containing moiety as provided herein, -NRQ-, -;

[0056] [ka]

[0057] refers to an alkynylene substituted with the moiety R Q is H or C1-C6 alkyl. "Substituted heteroalkynylene" refers to a heteroalkynylene group having 1 to 3 substituents, preferably 1 to 2 substituents, selected from the substituents disclosed for the substituted alkynylene.

[0058] "Alkoxy" refers to an O-alkyl group where alkyl is defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy.

[0059] "Substituted alkoxy" refers to the group O(substituted alkyl), where substituted alkyl is defined herein.

[0060] "Acyl" means HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, substituted alkenyl-C(O)-, substituted alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, substituted aryl-C(O)-, heteroaryl- "C(O)-", substituted heteroaryl-C(O)-, heterocycle-C(O)-, substituted heterocycle-C(O)-, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Acyl includes the "acetyl" group CHC(O)-.

[0061] "Acylamino" means -NR 4 7C(O) alkyl, -NR 4 7C(O) substituted alkyl, -NR 4 7C(O)cycloalkenyl, -NR 4 7C(O) substituted alkenyl, -NR 4 7C(O)alkynyl, -NR 4 7C(O) substituted alkynyl, -NR 4 7C(O)aryl, -NR 4 7C(O) substituted aryl, -NR 4 7C(O)heteroaryl, -NR 4 7C(O)-substituted heteroaryl, -NR 4 7C(O)-substituted heteroaryl, -NR 4 7C(O)heterocyclic, and NR 4 7C(O)-substituted heterocyclic groups, where R 47, R47 is hydrogen or alkyl, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0062] "Acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, alkenyl-C(O)O-, substituted alkenyl-C(O)O-, alkynyl-C(O)O-, substituted alkynyl-C(O)O-, aryl-C(O)O-, substituted aryl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, cycloalkenyl-C(O)O-, substituted cycloalkenyl-C(O)O-, heteroaryl-C(O )O—, substituted heteroaryl-C(O)O, heterocyclic-C(O)O—, and substituted heterocyclic-C(O)O—, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0063] "Amino" refers to the group NH2.

[0064] "Substituted amino" means R 48 and R 49 R refers to the group 48 and R 49is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, SO2α-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2-substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cycloalkyl, -SO2-cycloalkenyl, -SO2-substituted cycloalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic; R 48 and R 49 If both are not hydrogen, then R 48 and R 49 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. R 48 is hydrogen and R 49 When R is alkyl, the substituted amino group is sometimes referred to herein as alkylamino. 48 and R 49 When R is alkyl, the substituted amino group is sometimes referred to herein as dialkylamino. 48 or R 49 is hydrogen, but not both. When referring to a disubstituted amino, it means that R 48 MoR 49 This means that it is not hydrogen.

[0065] "Aminocarbonyl" refers to the group -C(O)NR 50 R 51 refers to R 50 and R 51is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0066] "Aminothiocarbonyl" refers to the group -C(S)NR 50 R 51 refers to R 50 and R 51 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, wherein R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0067] "Aminocarbonylamino" refers to the group -NR 4 7C(O)NR 50 R 51 refers to R 4 7 is hydrogen or alkyl, and R 50and R 51 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, wherein R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0068] "Aminothiocarbonylamino" refers to the group -NR 4 7C(S)NR 50 R 51 refers to R 4 7 is hydrogen or alkyl, and R 50 and R 51 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, wherein R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0069] "Aminocarbonyloxy" refers to the group -OC(O)NR 50 R51 refers to R 50 and R 51 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, wherein R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0070] "Aminosulfonyl" refers to the group -SO2NR 50 R 51 refers to R 50 and R 51 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, wherein R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0071] "Aminosulfonyloxy" refers to the group -O-SO2NR 50 R 51 refers to R 50and R 51 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, wherein R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0072] "Aminosulfonylamino" refers to the group -NR 4 7SO2NR 50 R 51 refers to R 4 7 is hydrogen or alkyl, and R 50 and R 51 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, wherein R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0073] "Amidino" is the group -C(=NR 52 )NR 50 R51 refers to R 50 , R 51 , and R 52 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, wherein R 50 and R 51 is optionally combined with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0074] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6-14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-1,4-benzoxazinone, 3(4H)-1,7-yl, etc.), provided that the point of attachment is an aromatic carbon atom. Preferred aryl groups include phenyl and naphthyl.

[0075] The term "substituted aryl group" refers to alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio. " refers to an aryl group substituted by 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein.

[0076] "Arylene" refers to a divalent aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring or multiple condensed rings. "Substituted arylene" refers to an arylene having 1 to 5, preferably 1 to 3, or more preferably 1 to 2, substituents as defined for an aryl group.

[0077] "Heteroarylene" refers to a divalent aromatic group of 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. "Substituted heteroarylene" refers to a heteroarylene group substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to 2, substituents selected from the same group of substituents defined for substituted aryl.

[0078] "Aryloxy" refers to the group --O-aryl, where aryl is as defined herein, that includes, by way of example, phenoxy and naphthoxy.

[0079] "Substituted aryloxy" refers to the group --O-(substituted aryl), where substituted aryl is as defined herein.

[0080] "Arylthio" refers to the group --S-aryl, where aryl is as defined herein.

[0081] "Substituted arylthio" refers to the group S(substituted aryl), where substituted aryl is as defined herein.

[0082] "Carbonyl" refers to the divalent group -C(O)- which is equivalent to -C(=O)-.

[0083] "Carboxyl" or "carboxy" refers to COOH or salts thereof.

[0084] A "carboxy ester" or "carboxy esters" refers to the groups -C(O)(O)-alkyl, -C(O)(O)-substituted alkyl, -C(O)O-alkenyl, -C(O)(O)-substituted alkenyl, -C(O)(O)-alkynyl, -C(O)(O)-substituted alkynyl, -C(O)(O)-aryl, -C(O)(O)-substituted-aryl, -C(O)(O)-cycloalkyl, -C(O)(O)-substituted cycloalkyl, -C(O)(O)-cycloalkenyl, -C(O)(O)-substituted cycloalkenyl "Heteroaryl" refers to alkyl, -C(O)(O)-heteroaryl, -C(O)(O)-substituted heteroaryl, -C(O)(O)-heterocyclic, and -C(O)(O)-substituted heterocyclic alkyl, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0085] "(Carboxyl ester) amino" means -NR 4 7C(O)(O)-Alkyl, -NR 4 7C(O)(O)-substituted alkyl, -NR 4 7C(O)O-alkenyl, -NR 4 7C(O)(O) group, -substituted alkenyl, -NR 4 7C(O)(O)-alkynyl, -NR 4 7C(O)(O)-substituted alkynyl, -NR 4 7C(O)(O)-aryl, -NR 4 7C(O)(O)-substituted-aryl, -NR4 7C(O)(O)-cycloalkyl, -NR 4 7C(O)(O)-substituted cycloalkyl, -NR 4 7C(O)(O)-cycloalkenyl, -NR 4 7C(O)(O)-substituted cycloalkenyl, -NR 4 7C(O)(O)-heteroaryl, -NR 4 7C(O)(O)-substituted heteroaryl, -NR 4 7C(O)(O)-heterocyclic, and -NR 47C(O)(O)-substituted heterocyclic rings, where R 4 7 is alkyl or hydrogen, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0086] "(Carboxyl ester)oxy" refers to the groups -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-alkynyl, -OC(O)(O)-substituted alkynyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl, -OC(O)O-substituted cycloalkenyl, - refers to -OC(O)O-heteroaryl, -OC(O)O-substituted heteroaryl, -OC(O)O-heterocyclic, and -OC(O)O-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0087] "Cyano" refers to the radical CN.

[0088] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 10 carbon atoms having single or multiple cyclic rings, including fused, bridged, and spiro ring systems. A fused ring may also be an aryl ring if the non-aryl moiety is connected to the remainder of the molecule. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.

[0089] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group of 3 to 10 carbon atoms having a single ring or multiple cyclic rings and having at least one >C=C< ring unsaturation, preferably having 1 to 2 sites of >C=C< ring unsaturation.

[0090] "Substituted cycloalkyl" and "substituted cycloalkenyl" include oxo, thioxo, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cyano, cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cyano, cycloalkyl, cycloalkylamino, cycloalkyloxy, substituted cyano, cycloalkyl, cycloalkylamino, cycloalkyloxy, cycloalkylthio ... " refers to a cycloalkyl or cycloalkenyl group having from 1 to 5, or preferably 1 to 3, substituents selected from the group consisting of cycloalkylthio, cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein.

[0091] "Cyclopropano" means:

[0092] [ka]

[0093] "Cyclobutano" means:

[0094] [ka]

[0095] "Cycloalkyloxy" refers to -O-cycloalkyl.

[0096] "Substituted cycloalkyloxy" refers to --O-(substituted cycloalkyl).

[0097] "Cycloalkylthio" refers to -S-cycloalkyl.

[0098] "Substituted cycloalkylthio" refers to --S-(substituted cycloalkyl).

[0099] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0100] "Substituted cycloalkenyloxy" refers to --O-(substituted cycloalkenyl).

[0101] "Cycloalkenylthio" refers to -S-cycloalkenyl.

[0102] "Substituted cycloalkenylthio" refers to --S-(substituted cycloalkenyl).

[0103] "Guanidino" refers to the group -NHC(=NH)NH2.

[0104] "Substituted guanidino" refers to -NR 53 C(=NR 53 )N(R 53 )2, where each R 53are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic, and are connected to a common guanidino nitrogen atom by two R 53 groups are optionally joined together with the nitrogen attached thereto to form a heterocyclic or substituted heterocyclic group, provided that at least one R 53 is not hydrogen, and said substituents are as defined herein.

[0105] "Halo" or "halogen" means fluoro, chloro, bromo, and iodo.

[0106] "Hydroxy" or "hydroxyl" refers to the group --OH.

[0107] "Heteroaryl" refers to an aromatic group of 1-10 carbon atoms and 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl), where the condensed rings may or may not be aromatic and / or may contain heteroatoms if the point of attachment is through an atom in the aromatic heteroaryl group. In one embodiment, the nitrogen and / or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties. Specific non-limiting examples include pyridinyl, pyrrolyl, indolyl, thiophenyl, oxazolyl, thizolyl, and furanyl.

[0108] "Substituted heteroaryl" refers to a heteroaryl group that is substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to 2, substituents selected from the same group of substituents defined for substituted aryl.

[0109] "Heteroaryloxy" refers to -O-heteroaryl.

[0110] "Substituted heteroaryloxy" refers to the group --O-(substituted heteroaryl).

[0111] "Heteroarylthio" refers to the group --S-heteroaryl.

[0112] "Substituted heteroarylthio" refers to the group --S-(substituted heteroaryl).

[0113] "Heterocyclic" or "heterocyclic" or "heterocyclic" or "heterocycloalkyl" or "heterocyclyl" refers to a saturated or partially saturated, but not aromatic, group having 1-10 ring carbon atoms and 1-4 ring heteroatoms selected from the group consisting of nitrogen, sulfur, or oxygen. Heterocycles encompass single rings or multiple fused rings, including fused bridged and spiro ring systems. In fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In one embodiment, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide the N-oxide, sulfinyl, or sulfonyl moieties.

[0114] "Substituted heterocyclic" or "substituted heterocycloalkyl" or "substituted heterocyclyl" refers to a heterocyclyl group substituted with 1 to 5, preferably 1 to 3, of the same substituents as defined for substituted cycloalkyl.

[0115] "Heterocyclyloxy" refers to the group --O-heterosil.

[0116] "Substituted heterocyclyloxy" refers to the group --O-(substituted heterocyclyl).

[0117] "Heterocyclylthio" refers to the group -S-heterosyl.

[0118] "Substituted heterocyclylthio" refers to the group --S-(substituted heterocyclyl).

[0119] Examples of heterocycles and heteroaryls include azetidine, pyrrole, furan, thiophene, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isopropyl alcohol, ... These include, but are not limited to, soxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4 tetrahydroisoquinoline, 4,5,6,7 tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1 dioxothiomorpholinyl, piperidinyl, pyridine, and tetrahydrofuran.

[0120] "Nitro" refers to the -NO2 group.

[0121] "Oxo" refers to the atom (=O).

[0122] "Phenylene" refers to a divalent aryl ring containing 6 carbon atoms.

[0123] Substituted phenylene includes alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cyclo " refers to phenylene substituted with 1 to 4, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkenyl, substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H, substituted sulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio, wherein said substituents are as defined herein.

[0124] "Spirocycloalkyl" and "spiro ring system" refer to a divalent ring radical of 3-10 carbon atoms having a cycloalkyl or heterocycloalkyl ring with a spiro union (a union formed by a single atom that is the only common member of the rings), as exemplified by the following structure:

[0125] [ka]

[0126] "Sulfonyl" refers to the divalent group -S(O)2-.

[0127] "Substituted sulfonyl" refers to the group -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2-substituted alkenyl, SO2-cycloalkyl, -SO2-substituted cycloalkyl, -SO2-cycloalkenyl, -SO2-substituted cycloalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, -SO2-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Substituted sulfonyl includes groups such as methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0128] "Substituted sulfonyloxy" refers to the groups -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2-alkenyl, -OSO2-substituted alkenyl, OSO2-cycloalkyl, -OSO2-substituted cycloalkyl, -OSO2-cycloalkenyl, -OSO2-substituted cycloalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2-heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocyclic, -OSO2-substituted alkyl, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0129] "Thioacyl" refers to the groups HC(S)-, alkyl-C(S)-, substituted alkyl-C(S)-, alkenyl-C(S)-, substituted alkenyl-C(S)-, alkynyl-C(S)-, substituted alkynyl-C(S)-, cycloalkyl-C(S)-, substituted cycloalkyl-C(S)-, cycloalkenyl-C(S)-, substituted cycloalkenyl-C(S)-, aryl-C(S)-, substituted aryl-C(S)-, and heteroaryl-C(S)-. -, substituted heteroaryl-C(S)-, heterocyclic-C(S)-, and substituted heterocyclic-C(S)-, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0130] "Thiol" refers to the SH group.

[0131] "Thiocarbonyl" refers to the divalent group -C(S)- which is equivalent to -C(=S)-.

[0132] "Thioxo" refers to the atom (=S).

[0133] "Alkylthio" refers to an S-alkyl group, where alkyl is as defined herein.

[0134] "Substituted alkylthio" refers to the group --S-(substituted alkyl), where substituted alkyl is as defined herein.

[0135] Substituted rings may be substituted with one or more fused and / or spiro rings. Such fused rings include fused cycloalkyl, fused heterocyclyl, fused aryl, and fused heteroaryl rings, each of which may be unsubstituted or substituted. Such spiro rings include fused cycloalkyl and fused heterocyclyl, each of which may be unsubstituted or substituted.

[0136] It is understood that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with five fluoro groups), such impermissible substitution patterns being well known to those of skill in the art.

[0137] It is understood that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with five fluoro groups), such impermissible substitution patterns being well known to those of skill in the art.

[0138] "Pharmaceutically acceptable salts" refers to salts of compounds that are suitable for pharmaceutical use and are derived from various organic and inorganic counterions well known in the art, including, by way of example only, when the compound contains an acidic functional group, such as sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, tartrate, acetate, maleate, and oxalate (see Stahl and Wermuth, eds., "Handbook of Pharmaceutically Acceptable Salts," (2002), Verlag Helvetica Chimica Acta, Zurich, Switzerland, for a discussion of pharmaceutical salts, their selection, preparation, and use).

[0139] "Pulmonary hypertension" refers to all forms of pulmonary hypertension, WHO Groups 1-5. Pulmonary arterial hypertension, also known as PAH, refers to WHO Group 1 pulmonary hypertension. PAH includes idiopathic, hereditary, drug- or toxin-induced, and persistent pulmonary hypertension of the newborn (PPHN).

[0140] Generally, a pharmaceutically acceptable salt is a salt that retains substantially one or more of the desired pharmacological activities of the parent compound and is suitable for in vivo administration. Pharmaceutically acceptable salts include acid addition salts formed with inorganic or organic acids. Inorganic acids suitable for forming pharmaceutically acceptable acid addition salts include, but are not limited to, hydrohalic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid, phosphoric acid, etc.

[0141] Organic acids suitable for forming pharmaceutically acceptable acid addition salts include, for example, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanedionate, glycolic acid, oxalic acid, pyruvic acid, lactic acid, succinic acid, malic acid, maleic acid, fumaric acid, citric acid, tartaric acid, palmitic acid, benzoic acid, 3-(4-hydroxybenzoic acid)benzoic acid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethane-sulfonic acid, 2-hydroxyethanesulfonic acid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, etc.), glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like.

[0142] Pharmaceutically acceptable salts also include salts formed when an acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion), or an ammonium ion (e.g., ammonium ions derived from organic bases such as ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, dimethylamine, diethylamine, triethylamine, and ammonia).

[0143] Treprostinil, the active ingredient in Remodulin® (treprostinil) injection, Tivas® (treprostinil) inhalation, and Orenitram® (treprostinil) sustained-release tablets, is described in U.S. Patent No. 4,306,075. Methods for producing treprostinil and other prostacyclin derivatives are described in Moriarty, et al., J. Org. Chem. 2004, 69, 1890-1902, Drug of the Future, 2001, 26(4), 364-374, US6,441,245, 6,528,688, 6,700,025, 6,809,223, 6,756,117, 8,461,393, 8,481,782; 8,242,305, 8,497,393, 8,940,930, 9,029,607, 9,156,786, and 9,388,154, 9,346,738; USP 2012-0 197041, 2013-0331593, 2014-0024856, 2015-0299091, 2015-0376106, 2016-0107973, 2015-0315114, 2016-0152548, and 2016-0175319; PCT International Publication Nos. WO2016 / 0055819 and WO2016 / 081658.

[0144] Treprostinil for various uses and / or in various forms is described, for example, in U.S. Patents 5,153,222, 5,234,953, 6,521,212, 6,756,033, 6,803,386, 7,199,157, 6,054,486, 7,417,070, 7,384,978, 7,879,909, 8,563,614, 8,252,839, 8,536,363, 8,410,169, 8 ,232,316, 8,609,728, 8,350,079, 8,349,892, 7,999,007, 8,658,694, 8,653,137, 9,029,607, 8,765,813, 9,050,311, 9,199,908, 9,278,901, 8,747,897, 9,358,240, 9,339,507, 9,255,064, 9,278,902, 9,278,9 03, 9,758,465; 9,422,223; 9,878,972; 9,624,156; U.S. Patent Application Publications 2009-0036465, 2008-0200449, 2008-0280986, 2009-0124697, 2014-0275616, 2014-0275262, 2013-0184295, 2014-0323567, 2016-0030371, 2016-005 1505, 2016-0030355, 2016-0143868, 2015-0328232, 2015-0148414, 2016-0045470, 2016-0129087, 2017-0095432; 2018-0153847, and PCT application WO00 / 57701, WO20160105538, WO2016038532, and WO2018 / 058124.

[0145] The chemical formula of treprostinil is:

[0146] [ka]

[0147] The term "effective amount" may refer to the amount of a treprostinil prodrug that may be required to treat a disease or condition. In some embodiments, an effective amount of a treprostinil prodrug may be the same as or similar to an effective amount of treprostinil for treating the same disease or condition. In some embodiments, an effective amount of a treprostinil prodrug may be different from an effective amount of treprostinil for treating the same disease or condition. One skilled in the art will be able to determine and determine the "effective amount" of a treprostinil prodrug based, for example, on the relevant disease or condition, the amount of treprostinil known to treat, ameliorate, or prevent the disease or condition, and the rate at which the prodrug is converted to treprostinil in vivo.

[0148] In some embodiments, the prodrug may be a prodrug disclosed in U.S. Patent Nos. 7,384,978, 7,417,070, 7,544,713, 8,252,839, 8,410,169, 8,536,363, 9,050,311, 9,199,908, 9,278,901, 9,422,223, and 9,624,156.

[0149] In some embodiments, the prodrug may be a prodrug disclosed in U.S. Patents 9,371,264, 9,394,227, 9,505,737, and 9,643,911, which are incorporated herein by reference in their entireties.

[0150] In some embodiments, the prodrug may be a prodrug disclosed in U.S. Patent Application Publication No. 2018-0153847.

[0151] In some embodiments, the prodrug can be one of the prodrugs described below.

[0152] Prodrug Compounds In one embodiment, the compound of the formula:

[0153] [ka]

[0154] or a pharmaceutically acceptable salt thereof, wherein: X is OR 14 , -NR 1 SO2R 1 , -NR 1 CO2H,

[0155] [ka]

[0156] or [ka]

[0157] and Each R 1 are independently H or C1-C4 alkyl, and R 8 is an optionally substituted C1-C6 alkyl or a side group of an amino acid, or R 1 and R 8 together form a 4- to 7-membered heterocycle, R 14 is H, optionally substituted C1-C6 alkyl, a first drug moiety, or:

[0158] [ka]

[0159] and R 11 is absent, optionally substituted C1-C6 alkylene, or -Q 1 -O- and Q 1 is an optionally substituted C1-C6 alkylene, and R 12 and R 13each independently represents H, optionally substituted C1-C6 alkoxy, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, optionally substituted C1-C8 cycloalkyl, optionally substituted C1-C 10 is alkyl, R 2 and R 3 each independently represents a second drug moiety, a third drug moiety, H, a phosphorus-containing group, —C(O)R 6 or an -ABC substituent, where A is an optionally substituted C1-C6 alkylene, -NR 6 -, -C(O)-, -C(O)O- or -C(O)NR 6 - and B is a bond, optionally substituted C1-C6 alkylene, -C(O)-, -O-, -S-, heterocyclyl; C is optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted cycloalkyl, -(OCH2CH2) q -OR 6 , -C(O)N(R 6 )2, -C(O)N(R 18 )2, -C(O)R 6 , -CO2H, -OR 6 , -N(R 18 )2, -N(R 6 )2, or

[0160] [ka]

[0161] where both R 18 together form an optionally substituted 3-8 membered heterocyclyl; Each R 6 are independently H, optionally substituted C-C alkyl, optionally substituted heteroaryl, optionally substituted aryl, or R 6together form a 4-8 membered optionally substituted heterocyclyl or a 5 membered optionally substituted heteroaryl; or R 2 and R 3 are bonded together to form -C(O)-, -SO2-, and 8-12 membered heterocyclyl.

[0162] [ka]

[0163] Forming Each R 10 is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted aryl; q is 0, 1, 2, 3, 4, 5 or 6; however, When A is -C(O)-, B is not a bond and C is -N(R 6 )2 instead; When A is -C(O)-, B is not a bond and C is -OR 6 Not; R 14 , R 2 , R 3 is not H; When X is OH, R 2 and R 3 is not H; R 8 If is H, then R 2 and R 3 At least one of them is not H.

[0164] In one embodiment, the compound of the formula:

[0165] [ka]

[0166] or a pharmaceutically acceptable salt thereof, X is OR 14 , -NR 1 SO2R 1 , -NR 1 CO2H,

[0167] [ka]

[0168] or [ka]

[0169] and Each R 1 are independently H or C1-C4 alkyl, and R 8 is a C1-C6 alkyl or a side group of an amino acid, or R 1 and R 8 together form a 4- to 7-membered heterocycle, R 14 is H, C1-C6 alkyl, a first drug moiety, or

[0170] [ka]

[0171] where R 11 is absent, C1-C6 alkylene, or -Q 1 -O(in the formula, Q 1 is C1-C6 alkylene; R 12 and R 13 each independently represents H, OH, C1-C6 alkoxy, C1-C6 alkyl, C1-C6 alkenyl, C1-C8 cycloalkyl, C1-C 10 aryl; R 2 and R 3 each independently represents a second drug moiety, a third drug moiety, H, a phosphorus-containing group, —C(O)R 6 or an -ABC substituent; A is C1-C6 alkylene, -NR 6 -, -C(O)-, -C(O)O-, or -C(O)NR 6 - and B is a bond, C1-C6 alkylene, -C(O)-, -O-, -S-, or heterocyclyl; C is heterocyclyl, heteroaryl, aryl, cycloalkyl, -(OCH2CH2) q -OR 6 , -C(O)N(R 6 )2, -C(O)N(R 18 ) 2、 -C(O)R 6 , -CO2H, -OR 6 , -N(R 18 )2, -N(R 6 )2, or

[0172] [ka]

[0173] and both R 18 together form a 3- to 8-membered heterocyclyl, Each R 6 are independently 6 H, C1-C6 alkyl, heteroaryl, aryl, or both form a 4-8 membered heterocyclyl or a 5 membered heteroaryl; or R 2 and R 3 is bonded to -C(O)-, -SO2-, 8-12 membered heterocyclyl,

[0174] [ka]

[0175] Each R 10 is H, C1-C6 alkyl, C1-C6 alkenyl, cycloalkyl, heteroaryl, or aryl; q is 0, 1, 2, 3, 4, 5 or 6. however, When A is -C(O)-, B is not a bond and C is -N(R 6 )2 instead; When A is -C(O)-, B is not a bond and C is -OR 6 Not; R 14 , R 2 , R 3 is not H; If X is OH, then R 2 and R 3 is not H; R 8 If H, then R 2 and R 3 At least one of them is not H.

[0176] In some embodiments, X is OR 14 and R 14 is H or a first drug moiety, and R 2 is H or a second drug moiety, and R 3 is H or a third drug moiety, with the proviso that R 14 , R 2 and R 3 and each of R is not H. In some embodiments, R 14 is H and R 2 and R 3 One of the is H and the other is R 2 and R 3 The other of R is a drug moiety. 2 is H and R 3 is a third drug moiety. In some embodiments, R 2 is a second drug moiety and R 3 is a third drug moiety. In some embodiments, R 12 , R 13 , R 2 and R 3 are each H, and R 11 is C1-C4 alkylene.

[0177] In some embodiments, R 14 R is a C1-C4 alkyl optionally substituted with a terminal hydroxyl group or a carboxy group. 14When C1-C4 alkyl is substituted with a terminal carboxy group, R 14 can be carboxymethyl, carboxyethyl, carboxypropyl, 4-carboxybutyl, 2-methyl-3-carboxypropyl. -

[0178] Each drug moiety (first, second, and third) may be independently selected. In some embodiments, the drug moiety is a pain-relieving drug moiety. In some embodiments, the drug moiety is a nonsteroidal anti-inflammatory drug (NSAID) moiety. The drug moiety may be selected from any pain-relieving or NSAID drug known in the art to be attached to a compound. Conjugation may include direct covalent bonding or attachment through a linker group. The linker may include an optionally substituted alkylene group, an optionally substituted arylene or heteroarylene group, a peptide, or other linker known in the art for drug attachment. Exemplary pain relief medications include opioids (e.g., morphine, hydrocodone, oxycodone, oxymorphone, hydromorphone, fentanyl, thiofentanyl, tapentadol, methadone, or meperidine); local anesthetics (e.g., lidocaine, prilocaine, tetracaine, articaine, benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine, piperocaine, propoxycaine, proparacaine, saxitoxin, neosaxitoxin, non-limiting examples of nonsteroidal anti-inflammatory drugs (NSAIDS) include aspirin, ibuprofen, celecoxib, or any COX1 or COX2 inhibitor, or naproxen.

[0179] The second drug moiety may form an ester bond with the carboxyl group of treprostinil and / or the hydroxyl group (e.g., one or both). 2 or R 3 is H of treprostinil. For example, if the second drug moiety contains a hydroxyl group, it can form an ester bond with a carboxyl group of treprostinil. If the second drug moiety contains a carboxyl group, it can form an ester bond with one of the hydroxyl groups of treprostinil.

[0180] In some embodiments, R 2 and R 3 and R is a phosphorus-containing group. 2 and R 3 are phosphorus-containing groups. In some embodiments, each phosphorus-containing group independently has the formula:

[0181] [ka]

[0182] and R 31 is absent and is optionally substituted C1-C6 alkylene, or -QO-, where Q is optionally substituted C1-C6 alkylene; and R 32 and R 33 is independently selected from H, optionally substituted C-C alkoxy, optionally substituted C-C alkenyloxy, optionally substituted C-C cycloalkoxy, and optionally substituted aryloxy. In some embodiments, the phosphorus-containing group has the formula:

[0183] [ka]

[0184] In some embodiments, R 31 is C1-C6 alkylene, and R 32 and R 33 Each of is H.

[0185] In some embodiments, X is OH, —CH 2 OPO 3 H 2 ,

[0186] [ka]

[0187] or -NHSO2CH3; where R 8 is C1-C2 alkyl optionally substituted with OH or -CO2H. In some embodiments, X is

[0188] [ka]

[0189] where R 8 is methyl. In some embodiments, R 8 is methyl substituted with OH. In some embodiments, R 8 is methyl substituted with -COH. In some embodiments, R 8 is a side group of an amino acid as defined herein. In some embodiments, R 1 and R 8 together form pyrrolidine, piperidine, aziridine, azepane, or azetidine. 1 and R 8 together form pyrrolidine.

[0190] In some embodiments, R 2 is -C(O)R 17 , -OPO3H2 or -ABC, A is -C(O)-, -C(O)O-, CH2, or -C(O)NR 6 - and B is -CHR 16 -or-(CH2) q - and; C is C1-C3 alkoxy, heterocyclyl, OR 6 , OPO3H2, CO2H, OH, NH2, -C(O)R 6 , -C(O)N(R 18 )2, or -C(O)N(R 6 )2, R 16 is H or C1-C3 alkyl, R 17is C1-C3 alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and q is 0, 1, or 2.

[0191] In some embodiments, R 3 is -C(O)R 17 , -OPO3H2 or -ABC, A is -C(O)-, -C(O)O-, CH2, or -C(O)NR 6 - and B is -CHR 16 -or-(CH2) q - and; C is heterocyclyl, OR 6 , OPO3H2, CO2H, OH, NH2, -C(O)R 6 , -C(O)N(R 18 )2, or -C(O)N(R 6 )2, R 16 is H or C1-C3 alkyl, R 17 is C1-C3 alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and q is 0, 1, or 2.

[0192] In some embodiments, R 2 and / or R 3 is -C(O)-CHR 19 -N(R 6 )2, where each R 19 and R 6 are independently selected, and R 19is a side group of an amino acid or its enantiomer, such as methyl (for alanine), isopropyl (for valine), etc. Exemplary amino acids for which the side group can be used include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the amino acid is alanine, valine, or glycine. In some embodiments, R 2 and R 3 Only one of the groups is -C(O)-CHR 19 -N(R 6 )2 and R 2 and R 3 and the other is H. In some embodiments, R 19 is not H.

[0193] In some embodiments, R 8 is a side group of an amino acid or its enantiomer, such as methyl (for alanine), isopropyl (for valine), etc. Exemplary amino acids for which the side group can be used include arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the amino acid is alanine, valine, or glycine. In some embodiments, R 8 is not H.

[0194] "Amino acid" can refer to a D-isomer amino acid or an L-isomer amino acid. In certain aspects, an amino acid can be a naturally occurring amino acid. However, in some embodiments, an amino acid can be an artificial amino acid. Specific side groups of the above amino acids include -CH3 (alanine), -CH2COH2NH (arginine), -CH2COOH (aspartic acid), -CH2COOH (cysteine), -(CH2)2COH2 (glutamic acid), -(CH2)2COOH (glutamic acid), -HCH (glycine), -CHCH3CH2CH3 (isoleucine), -CH2CH2(CH3)2 (leucine), -(CH2)4NH2 (lysine), -(CH2)2SCH3 (methionine), -CH2Ph ​​(phenylalanine), -CH2OH (serine), -CHOHCH3 (threonine), -CH(CH3)2 (valine),

[0195] [ka]

[0196] -(CH2)3NHCONH2- (citrulline) or -(CH2)3NH2 (ornithine), where Ph represents a phenyl group.

[0197] In some embodiments, R 2 is the ABC part of the following: A and B are CH2, C is CO2H, amino, C(O)N(R 18 )2, or -C(O)N(R 6 )2.

[0198] In some embodiments, R 3 is the ABC part of the following: A and B are CH2, C is CO2H, amino, C(O)N(R 18 )2, or -C(O)N(R 6 )2.

[0199] In some embodiments, R 2is an ABC moiety of formula -C(O)-C, where C is an optionally substituted aryl or an optionally substituted heteroaryl. In some embodiments, C is optionally substituted phenyl, optionally substituted piperidinyl, optionally substituted morpholino, optionally substituted azepanyl, optionally substituted aziridinyl, optionally substituted azetidinyl, optionally substituted azetidinyl, optionally substituted pyrrolidinyl, in some embodiments, C is phenyl, piperidinyl, morpholino, azepanyl, aziridinyl, azetidinyl, pyrrolidinyl, or piperazinyl.

[0200] In some embodiments, R 3 is an ABC moiety of formula -C(O)-C, where C is an optionally substituted aryl or an optionally substituted heteroaryl. In some embodiments, C is optionally substituted phenyl, optionally substituted piperidinyl, optionally substituted morpholino, optionally substituted azepanyl, optionally substituted aziridinyl, optionally substituted azetidinyl, optionally substituted azetidinyl, optionally substituted pyrrolidinyl, in some embodiments, C is phenyl, piperidinyl, morpholino, azepanyl, aziridinyl, azetidinyl, pyrrolidinyl, or piperazinyl.

[0201] In some embodiments, R 2 is an ABC moiety of formula -C(O)-CHCH-C, where C is optionally substituted aryl or optionally substituted heteroaryl. In some embodiments, C is optionally substituted phenyl or optionally substituted naphthyl. In some embodiments, C is phenyl optionally substituted with C-C alkyl or naphthyl optionally substituted with methoxy.

[0202] In some embodiments, R 3is an ABC moiety of formula -C(O)-CHCH-C, where C is optionally substituted aryl or optionally substituted heteroaryl. In some embodiments, C is optionally substituted phenyl or optionally substituted naphthyl. In some embodiments, C is phenyl optionally substituted with C-C alkyl or naphthyl optionally substituted with methoxy.

[0203] In some embodiments, R 2 is —C(O)—X—CH2CO2H, where X is O or NR 1 In some embodiments, R 3 is —C(O)—X—CH2CO2H, where X is O or NR 1 In some embodiments, R 2 -C(O)-(℃H2CH2)q-OR 6 where R 6 is C1-C6 alkyl. In some embodiments, R 6 In some embodiments, q is 1.

[0204] In some embodiments, R 3 is —C(O)—X—CH2CO2H, where X is O or NR 1 In some embodiments, R 3 is —C(O)—X—CH2CO2H, where X is O or NR 1 In some embodiments, R 3 -C(O)-(℃H2CH2)q-OR 6 where R 6 is C1-C6 alkyl. In some embodiments, R 6 In some embodiments, q is 1.

[0205] In some embodiments, R 2 is —C(O)—(CH)COH or —C(O)—(CHCH)—C, where C is an optionally substituted aryl or an optionally substituted heteroaryl. 3is -C(O)-(CH2)2CO2H or C(O)-(CHCH3)-C, where C is an optionally substituted aryl or an optionally substituted heteroaryl. In some embodiments, the optionally substituted aryl is phenyl or naphthyl. In some embodiments, the optionally substituted phenyl or naphthyl is substituted with C1-C6 alkyl or C1-C6 alkoxy. In some embodiments, the optionally substituted phenyl or naphthyl is substituted with methoxy.

[0206] In some embodiments, X is OH and R 2 and R 3 together form a carbonyl-containing group or a phosphorus-containing group. In some embodiments, R 2 and R 3 are bonded together to form -C(O)-, -SO2-, and 8-12 membered heterocyclyl,

[0207] [ka]

[0208] In some embodiments, the compound is of formula:

[0209] [ka]

[0210] or a pharmaceutically acceptable salt thereof.

[0211] In another aspect, there is provided a compound of one of the following formulas:

[0212] [ka]

[0213] [ka]

[0214] [ka]

[0215] [ka]

[0216] [ka]

[0217] [ka]

[0218] or a pharmaceutically acceptable salt thereof.

[0219] In addition to, or as an alternative to, reducing site pain compared to administration of treprostinil or a salt thereof, these prodrugs may have one or more advantages over treprostinil. For example, some of these prodrugs may have improved stability or greater tolerability, at least in some patient populations.

[0220] At least some of these prodrugs may have a half-life in human plasma of less than 150 minutes, less than 120 minutes, less than 90 minutes, less than 60 minutes, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 12 minutes, or less than 10 minutes.

[0221] At least some of these prodrugs may have a plasma half-life of at least 3.5 hours, or at least 4.5 hours, or at least 4.5 hours, or at least 5 hours, or at least 6 hours, or at least 7 hours, or at least 8 hours, or at least 9 hours, or at least 11 hours, or at least 12 hours, or at least 14 hours, or at least 15 hours, or at least 16 hours, or at least 18 hours, or at least 19 hours, or at least 21 hours, or at least 23 hours, or at least 24 hours upon oral administration.

[0222] At least some of these prodrugs may have an oral bioavailability of at least 15%, at least 15%, at least 20%, at least 22%, at least 24%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.

[0223] At least some prodrugs may exhibit a dose-proportional increase in Cmax and AUC0-24hr following oral administration.

[0224] In some embodiments, the prodrug may be such that it does not convert to treprostinil before being administered to a subject, such as a human. For example, the prodrug may be such that it does not convert to treprostinil during storage. Furthermore, the prodrug may be such that it does not convert to treprostinil in a pharmaceutical formulation, such as an injectable formulation, e.g., a subcutaneous formulation, before the formulation is administered to a subject. The prodrug may be such that it does not convert to treprostinil when it comes into contact with the subcutaneous tissue of a subject upon injection, such as subcutaneous injection, of a pharmaceutical formulation containing the prodrug into a subject. The prodrug may be such that it converts to treprostinil only upon reaching the blood and / or liver of the subject. For example, a prodrug formulation, such as a subcutaneous prodrug formulation, may essentially not contain treprostinil itself before administration. In other words, prior to administration, the concentration of treprostinil itself in a prodrug formulation, such as a parenteral prodrug formulation, is less than 0.5%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.02%, less than 0.01%, or less than 0.005%, or less than 0.003%, or less than 0.002%, or less than 0.001%. Preferably, prior to administration, the concentration of treprostinil itself in a prodrug formulation, such as a parenteral prodrug formulation, which may be a subcutaneous prodrug formulation, is not detectable by high performance liquid chromatography (HPLC).

[0225] In some embodiments, the prodrug can be made to not convert to treprostinil when stored for at least 1 week, at least 2 weeks, or at least 3 weeks, or at least 3 weeks, or at a temperature of 30° C. to 45° C., or 35° C. to 45° C., or 37° C. to 43° C., or about 40° C., at a pH ranging from 5 to 9, or 5.5 to 8.5, or 6 to 8. For example, the prodrug formulation, such as a subcutaneous prodrug formulation, can be essentially free of treprostinil itself after such storage. In other words, the concentration of treprostinil itself in a prodrug formulation, such as a parenteral prodrug formulation, after storage is less than 0.5%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.02%, less than 0.01%, less than 0.005%, less than 0.003%, or less than 0.002%, or less than 0.001%, and preferably, the concentration of treprostinil itself in a prodrug formulation, such as a parenteral prodrug formulation, after storage, may be a subcutaneous prodrug formulation, is undetectable by high performance liquid chromatography (HPLC).

[0226] In some embodiments, the prodrug may be such that when the prodrug is administered to a subject, the prodrug is not detectable in the subject's blood or plasma. This may be, for example, oral administration or injection, for example, intravenous or subcutaneous injection. For example, the plasma concentration of the prodrug may be less than 2 ng / ml, or less than 1 ng / ml, or less than 0.7 ng / ml, or less than 0.5 ng / ml, or less than 0.3 ng / ml, or less than 0.2 ng / ml, or less than 0.1 ng / ml at any time after administration of the prodrug.

[0227] In certain embodiments, the prodrug may be such that the metabolic products of the in vivo conversion of the prodrug consist essentially of treprostinil. This may mean that treprostinil constitutes at least 90%, or at least 95%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9% of the metabolic products. In certain embodiments, the prodrug may be such that metabolic products of the in vivo conversion of the prodrug, other than treprostinil, cannot be detected in the blood or plasma of a subject. For example, the plasma concentration of non-treprostinil products of in vivo conversion of the prodrug may be less than 2 ng / ml, or less than 1 ng / ml, or less than 0.7 ng / ml, or less than 0.5 ng / ml, or less than 0.3 ng / ml, or less than 0.2 ng / ml, or less than 0.1 ng / ml at any time after administration of the prodrug.

[0228] In some embodiments, the prodrug can be such that it can provide a detectable plasma treprostinil concentration at least 24 hours after oral administration of the prodrug. For example, the plasma treprostinil concentration 24 hours after oral administration of the prodrug can be at least 1 ng / ml, or at least 1.5 ng / ml, or at least 1.5 ng / ml, or at least 2 ng / ml, or at least 3 ng / ml, or at least 4 ng / ml, or at least 5 ng / ml, or at least 6 ng / ml, or at least 7 ng / ml, or at least 8 ng / ml, or at least 9 ng / ml, or at least 10 ng / ml.

[0229] In some embodiments, the treprostinil prodrug may have an equilibrium aqueous solubility of at least 1 mg / ml, or at least 2 mg / ml, or at least 3 mg / ml, or at least 4 mg / ml, or at least 5 mg / ml, or at least 6 mg / ml. In some embodiments, the treprostinil prodrug may have an equilibrium aqueous solubility of 3-40 mg / ml, or 3-35 mg / ml, or 5-15 mg / ml, or any value or subrange within these ranges. The solubility of the prodrug may be greater if the pH is increased in the vehicle used for the solubility measurement and / or if one or more salts are removed from the vehicle.

[0230] In certain embodiments, the prodrug may have an equilibrium aqueous solubility of at least 7 mg / ml, or at least 8 mg / ml, or at least 9 mg / ml, or at least 10 mg / ml, or at least 20 mg / ml, or at least 30 mg / ml, or at least 50 mg / ml, or at least 70 mg / ml, or at least 100 mg / ml, or at least 200 mg / ml, or at least 300 mg / ml. For oral administration, more soluble prodrugs may be preferred.

[0231] In some embodiments, the prodrug has an equilibrium aqueous solubility of 1 mg / ml or less, or 0.5 mg / ml or less, or 0.2 mg / ml or less, or 0.1 mg / ml or less, or 0.05 mg / ml or less, or 0.02 mg / ml or less, or 0.01 mg / ml or less, or 0.005 mg / ml or less, or 0.002 mg / ml or less, or 0.001 mg / ml or less. In some embodiments, the poorly water-soluble prodrug can be formulated by making a solid dispersion, such as an amorphous solid dispersion. For methods of making solid dispersions, such as amorphous solid dispersions, of poorly water-soluble compounds, see, for example, Newman, Developing Solid Oral Dosage Forms (Second Edition), Pharmaceutical Theory and Practice, 2017, Pages 497-518 and Paudel et al., International Journal of Pharmaceutics 453 (2013) 253-284, which are incorporated herein by reference in their entirety. In some embodiments, low water solubility prodrugs can be used in the form of salts that allow for increased water solubility.

[0232] Pharmaceutical Composition The treprostinil prodrug can be provided in the form of a pharmaceutical composition, which may contain pharmaceutically acceptable carriers, excipients, binders, diluents, etc. Such pharmaceutical compositions can be prepared by methods known in the art, such as granulating, mixing, dissolving, encapsulating, lyophilizing, emulsifying, or suspending processes, among others. The composition may be in the form of, for example, granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, and solutions. The composition can be formulated for a number of different administration routes, such as oral, transmucosal, rectal, transdermal, or subcutaneous administration, as well as intrathecal, intravenous, intramuscular, intraperitoneal, intranasal, intraocular, or intracerebroventricular injection. The treprostinil prodrug can be administered by any of the above routes, for example, as an injection or as a sustained-release formulation, for local rather than systemic administration.

[0233] In one embodiment, the pharmaceutical composition comprises a treprostinil prodrug and a carrier such as sterile water. In some embodiments, the treprostinil prodrug is formulated for subcutaneous administration, and such formulations may or may not contain m-cresol or another preservative.

[0234] The treprostinil prodrugs described herein can be used to treat pulmonary hypertension. In certain embodiments, the treprostinil prodrugs can be used to treat PAH. In some embodiments, the treprostinil prodrugs can be used to treat one or more WHO Group 1-5 pulmonary hypertension. Similarly, the treprostinil prodrugs described herein can be used to treat diseases or conditions for which treprostinil is indicated or useful. The treprostinil prodrugs can be administered as the sole therapeutic agent or in addition to other active agents, including treprostinil.

[0235] For oral, buccal, or sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable solid dosage forms, which can be prepared, for example, by mixing one or more treprostinil prodrugs or pharmaceutically acceptable salts thereof with at least one additive or excipient, such as starch or other additive. Suitable additives or excipients include sucrose, lactose, cellulose sugars, mannitol, maltitol, dextran, sorbitol, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides, methylcellulose, hydroxypropylmethylcellulose, and / or polyvinylpyrrolidone. If desired, oral dosage forms may contain other ingredients to aid administration, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens or sorbic acid, antioxidants such as ascorbic acid, tocopherol, or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, or perfumes. Additionally, dyes or pigments may be added for identification. Tablets may be further treated with suitable coating materials known in the art.

[0236] Liquid dosage forms for oral administration can be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, slurries, and solutions, which may contain inert diluents such as water. Pharmaceutical preparations can be prepared as liquid suspensions or solutions using sterile liquids such as, but not limited to, oils, water, alcohols, and combinations thereof. Pharmaceutically suitable surfactants, suspending agents, and emulsifying agents can be added for oral or parenteral administration.

[0237] As mentioned above, the suspension may contain oil. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. The suspension may also contain fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. The suspension may also contain, but is not limited to, alcohols such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol. Ethers such as poly(ethylene glycol), mineral oil, and petroleum hydrocarbons such as Vaseline; and water may also be used in the suspension.

[0238] Injectable dosage forms generally include aqueous or oily suspensions, which can be prepared using suitable dispersants or wetting agents and suspending agents. The injectable forms may be in solution phase or in the form of a suspension prepared with a solvent or diluent. Acceptable solvents or vehicles include sterile water, Ringer's solution, or isotonic saline solution. Alternatively, sterile oils may be used as solvents or suspending agents. Preferably, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono- and diotriglycerides.

[0239] For injection, the pharmaceutical preparation may be a powder suitable for dissolution in an appropriate solution, as described above. Examples of these include, but are not limited to, freeze-dried, rotary-dried, or spray-dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulation may optionally contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof. The compound may be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion. Unit dosage forms for injection may be contained in ampoules or multi-dose containers. In addition to the representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and can be used. Such excipients and carriers are described, for example, in "Remington's Pharmaceutical Sciences," Mack Pub. Co., New Jersey (1991), incorporated herein by reference.

[0240] Treprostinil prodrugs can be prepared in preparations suitable for parenteral administration, which may include a sterile aqueous preparation of the treprostinil prodrug or a pharmaceutically acceptable salt thereof, in which case the preparation may be isotonic with the blood of the intended recipient. These preparations can be administered by subcutaneous injection, although administration can also be by intravenous, intramuscular, or intradermal injection. Such preparations can conveniently be prepared by mixing the compound with water or a glycine or citrate buffer and rendering the resulting solution sterile and isotonic with blood. Injectable formulations can contain 0.1-5% w / v of treprostinil by weight in the prodrug and can be administered at a rate of 0.1 ml / min / kg. Alternatively, the prodrug can be administered at a rate of 0.625-50 ng / kg / min, based on the weight of treprostinil in the prodrug. Alternatively, the prodrug can be administered at a rate of 10-15 ng / kg / min, based on the weight of treprostinil in the prodrug.

[0241] In certain embodiments, the concentration of the treprostinil prodrug in a formulation for parenteral administration, such as intravenous infusion or subcutaneous infusion (including continuous subcutaneous infusion), can be 0.0005-30 mg / mL, or 0.0007-50 mg / mL, or 0.001-15 mg / mL, or any value or subrange within these ranges. Exemplary concentrations can include 0.1 mg / mL, 1 mg / mL, 2.5 mg / mL, 5 mg / mL, or 10 mg / mL.

[0242] In some embodiments, formulations of treprostinil prodrugs for parenteral administration, such as intravenous infusion or subcutaneous infusion (including continuous subcutaneous infusion), can be prepared by mixing the prodrug with a vehicle, such as a buffer solution. In some embodiments, the vehicle can be a phosphate-containing vehicle, i.e., at least one phosphate salt, for example, a dibasic phosphate salt, such as dibasic sodium phosphate or dibasic potassium phosphate, or a tribasic phosphate salt, such as tribasic sodium phosphate or potassium phosphate. In some embodiments, the vehicle can also contain a halogen salt, such as a chloride salt, for example, sodium chloride or potassium chloride. A halogen salt, such as sodium chloride, can be used to adjust the osmolality of the vehicle. In some embodiments, it may be preferable for the phosphate salt and the halogen salt to have the same cation. For example, when the phosphate salt is sodium phosphate, for example, sodium trihydrogen phosphate or sodium trihydrogen phosphate, the halogen salt can be a halogen sodium salt, such as sodium chloride. Similarly, when the phosphate salt is potassium phosphate, for example, tribasic potassium phosphate or tribasic potassium phosphate, the halogen salt can be a potassium halogen salt, such as potassium chloride. The solvent in the vehicle can include water. In some embodiments, water may be the only solvent in the vehicle. Furthermore, in certain embodiments, the vehicle may contain one or more additional solvents in addition to water. In some embodiments, the additional solvent may be a preservative, such as m-cresol.

[0243] Preferably, the vehicle is isotonic with the blood of a patient, such as a human. The term "isotonic" can mean that the osmolality and ionic concentration of the vehicle match those of a patient, such as a human. Non-limiting examples of vehicles include phosphate buffered saline, which is a water-based salt solution containing disodium hydrogen phosphate, sodium chloride, and, in some formulations, potassium chloride and potassium dihydrogen phosphate. Other examples include a vehicle containing 20 mM dibasic sodium phosphate with 125 mM sodium chloride, and a vehicle containing 15 mM tribasic sodium phosphate, 125 mM sodium chloride, and 0.3 w / m-cresol.

[0244] Treatment method In some embodiments, methods of treating a disease or condition are provided, the methods comprising administering to a subject a compound (e.g., a prodrug) or composition disclosed herein. In some embodiments, the disease or condition is one or more selected from the group consisting of pulmonary hypertension, congestive heart failure, peripheral vascular disease, Raynaud's phenomenon, scleroderma, renal failure, peripheral neuropathy, digital ulcers, intermittent claudication, ischemic limb disease, peripheral ischemic lesions, pulmonary fibrosis, and asthma. In some embodiments, the disease is pulmonary hypertension.

[0245] In some embodiments, the subject has a detectable treprostinil plasma level for at least 24 hours after administration. In some embodiments, the subject has a detectable treprostinil plasma level for at least 30 hours after said administration. In some embodiments, the subject has a detectable treprostinil plasma level for at least 36 hours after administration. In some embodiments, the subject has a detectable treprostinil plasma level for at least 42 hours after administration. In some embodiments, the subject has a detectable treprostinil plasma level for at least 48 hours after administration.

[0246] Administration can be via the routes described above, or, for example, orally, intravenously, intraarterially, intramuscularly, intranasally, rectally, intravaginally, or subcutaneously. In some embodiments, the composition is administered by injection. In some embodiments, administration is orally. In some embodiments, administration is subcutaneously.

[0247] In some embodiments, the administration results in no or less pain at the injection site compared to administering treprostinil. Pain or its reduction can be assessed by medically recognized methods known in the art, such as, for example, numerical rating scales, visual analog scales, Wong-Baker Pain Scale, FLACC Scale, CRIES Scale, COMFORT Scale, McGill Pain Scale, Manoski Scale, or other categorical scales. Compared to injection of treprostinil, pain upon injection of the prodrug, as measured by a medically recognized method, can be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% less pain.

[0248] The subject to be treated may be a human, dog, cat, bird, non-human primate, cow, or horse. In some embodiments, the subject is a human. In some embodiments, the subject is an uncooperative or fearful human being, e.g., a child or an elderly subject with dementia. In some embodiments, a method of treating a disease or condition is provided, the method comprising administering a prodrug of treprostinil to a subject, wherein, upon administration of the prodrug, the prodrug converts to a metabolite consisting essentially of treprostinil. The prodrug may be any of the compounds disclosed herein. In some embodiments, the metabolite consists of treprostinil.

[0249] The embodiments described herein are further illustrated by, but not limited to, the following examples. [Example]

[0250] Example 1: Synthesis of prodrug XXI (10) To study the stability and chemical feasibility of this prodrug, we synthesized a treprostinil side-chain phosphonoxyethyl prodrug. This prodrug was synthesized in nine steps from the side-chain THP benzinetriol (1), as shown in Scheme 1.

[0251] Side-chain THP benzinedentriol (1) was silylated with tert-butyldimethylsilyl trifluoromethanesulfonate in the presence of 2,6-lutidine to give di-TBDMS THP benzinedentriol (2) in 97.6% yield. Treatment of the protected triol (2) with magnesium bromide removed the THP group to give di-TBDMS benzinedentriol (3) in 89.6% yield. Di-TBDMS triol (3) was coupled with 2-benzyloxyethyl triflate in the presence of bis(trimethylsilyl)amide to give di-TBDMS benzinedentriol benzyloxyethyl ether (4) in 49.1% yield. Selective deprotection of the phenolic TBDMS moiety in ether (4) with lithium acetate dihydrate at 70 °C gave TBDMS benzinedentriol benzyloxyethyl ether (5) in 74.6% yield. The benzyl ether of TBDMS benzyl tribenzyloxyethyl ether (5) was hydrogenolyzed using palladium on carbon and hydrogen gas to give the TBDMS benzyl triol side-chain glycol ether (6) in 88.4% yield. The phenol group of glycol ether (6) was O-alkylated with benzyl bromoacetate in the presence of potassium carbonate to give the TBDMS side-chain glycol ether treprostinil benzyl ester (7) in 87.7% yield. The primary alcohol group of benzyl ester (7) was phosphitylated with dibenzyl N,N-diisopropylphosphoramidite in the presence of 1H-tetrazole, followed by oxidation with 3-chloroperbenzoic acid in the same pot to give the TBDMS side-chain dibenzylphosphonooxyethyl treprostinil benzyl ester (8) in 92.5% yield. Desilylation of phosphonooxyethyl treprostinil benzyl ester (8) with hydrogen fluoride-pyridine complex gave the side-chain dibenzyl phosphonooxyethyl treprostinil benzyl ester (9) in 88.9% yield. Hydrogenolysis of the side-chain dibenzyl phosphonooxyethyl treprostinil benzyl ester (9) with palladium on carbon in the presence of hydrogen gas gave the side-chain phosphonoxyethyl treprostinil prodrug (10) in 97.8% yield.

[0252] Scheme 1: Synthesis of Treprostinil Side Chain Phosphonooxyethyl Prodrugs

[0253] [ka]

[0254] experiment: Synthesis of di-TBDMS THP benzindentriol (2): Reaction scheme:

[0255] [ka]

[0256] [Table 1]

[0257] Testing Procedure: A solution of side-chain THP benzinetriol (1) (3.0 g, 7.20 mmol) and 2,6-lutidine (3.7 mL, 31.69 mmol) in dichloromethane (30 mL) was cooled to 0 °C in an ice bath under argon. To this mixture, tert-butyldimethylsilyl trifluoromethanesulfonate (3.6 mL, 15.84 mmol) in dichloromethane (10 mL) was added dropwise over 30 min, and the mixture was stirred while the temperature was allowed to rise to ambient temperature. After 3 h, the reaction was found to be complete based on TLC (1:9 EtOAc / hexanes). The reaction was quenched with water (30 mL), and the organic layer was separated, washed with brine, dried over sodium sulfate, and evaporated in vacuo to give crude product (2). This was purified by silica gel column chromatography using ethyl acetate and hexane (0-3%) to give pure di-TBDMS THP benzinetriol (2) (4.53 g) in 97.6% yield. 1 H NMR and 13 It was characterized by C NMR.

[0258] Synthesis of di-TBDMS benzindentriol (3): Reaction scheme:

[0259] [ka]

[0260] [Table 2]

[0261] Testing Procedure: To a solution of di-TBDMS THP benzinetriol (2) (4.43 g, 6.87 mmol), magnesium bromide (7.6 g, 41.20 mmol) was added and stirred at ambient temperature under argon. After 7 h, the reaction was found to be complete based on TLC (1.5:8.5 EtOAc / hexanes). The reaction was carefully quenched with water (exothermic), and the organic layer was separated. The aqueous layer was extracted with tert-butyl methyl ether (100 mL) and separated. The combined organic layers were washed with brine, dried over sodium sulfate, and evaporated under reduced pressure to give crude product (3). This was purified by silica gel column chromatography using ethyl acetate and hexane (0-5%) to give pure di-TBDMS benzinetriol (3) (3.45 g) in 89.6% yield. This product was 1 H NMR and 13 It was characterized by C NMR.

[0262] Synthesis of di-TBDMS benzindentriol benzyloxyethyl ether (4): Reaction scheme:

[0263] [ka]

[0264] [Table 3]

[0265] Testing Procedure: To a solution of di-TBDMS benzinetriol (3) (0.94 g, 1.68 mmol) in anhydrous tetrahydrofuran (15 mL), sodium bis(trimethylsilyl)amide solution (1.0 M in THF) (2.0 mL, 2.01 mmol) was added over 5 min at −30° C. under argon. The solution was stirred at 30° C. for 1 h, and then 2-benzyloxyethyl triflate solution (1.43 g / 1.43 mL) was added dropwise over 10 min. The reaction progress was monitored by TLC (1:9 EtOAc / hexane). After 18 h, the reaction mixture was evaporated in vacuo, and the residue was partitioned between ethyl acetate (30 mL) and water (20 mL). The organic layer was separated, washed with brine, dried over sodium sulfate, and evaporated under reduced pressure to give crude product (3). This was purified by silica gel column chromatography using ethyl acetate and hexane (0-6%) to give pure di-TBDMS benzindentriol tribenzyloxyethyl ether (4) (570 mg) in 49.1% yield. 1 It was characterized by 1 H NMR and LC-MS.

[0266] Synthesis of TBDMS benzindentriol benzyloxyethyl ether (5): Reaction scheme: [ka]

[0267] [Table 4]

[0268] Testing Procedure: To a solution of di-TBDMS benzidene tribenzyloxyethyl ether (4) (0.5 g, 0.719 mmol) in N,N-dimethylformamide (10 mL) and water (0.2 mL) was added lithium acetate dihydrate (22 mg, 0.216 mmol). The reaction mixture was heated to 70° C. and stirred under argon. The progress of the reaction was monitored by TLC (1:9 EtOAc / hexanes), and the reaction was found to be complete after 7 h. The reaction mixture was quenched with saturated ammonium chloride solution (10 mL). It was extracted with ethyl acetate (3×15 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and evaporated under reduced pressure to give crude product (5). This was purified by silica gel column chromatography using ethyl acetate and hexane (0-8%) to give pure TBDMS benzidene triol tribenzyloxyethyl ether (5) (311 mg) in 74.6% yield. This product was 1 It was characterized by H NMR.

[0269] Synthesis of TBDMS benzindentriol side-chain glycol ether (6): Reaction scheme:

[0270] [ka]

[0271] [Table 5]

[0272] Testing Procedure: To a solution of TBDMS benzindenol tribenzyloxyethyl ether (5) (0.25 g, 0.430 mmol) in ethyl acetate (5 mL) was added palladium on carbon (50 mg). The mixture was evacuated and replaced with hydrogen gas (three times). The mixture was stirred at ambient temperature under a hydrogen atmosphere. The progress of the reaction was monitored by TLC (2:8 EtOAc / hexanes), and the reaction was found to be complete after 4 h. The reaction mixture was filtered through Celite to remove the palladium on carbon, and the resulting filtrate was evaporated under reduced pressure to give crude product (6). This was combined with another batch and purified by silica gel column chromatography using ethyl acetate and hexanes (0-18%) to give pure TBDMS benzindenol tribenzyloxyethyl ether (6) (221 mg) in 88.4% yield. This product was: 1 It was characterized by H NMR.

[0273] Synthesis of TBDMS side chain glycol ether treprostinil benzyl ester (7): Reaction scheme:

[0274] [ka]

[0275] [Table 6]

[0276] Testing Procedure: To a solution of TBDMS benzindentriol side-chain glycol ether (6) (206 mg, 0.419 mmol) in acetone (4 mL) was added powdered potassium carbonate (145 mg, 1.049 mmol) and benzyl bromoacetate (86 μL, 0.545 mmol). The reaction mixture was stirred at ambient temperature under argon. Reaction progress was monitored by TLC (2:3 EtOAc / hexane). After 48 h, the reaction was found to be complete based on TLC. The reaction mixture was filtered to remove potassium carbonate, and the filtrate was evaporated under reduced pressure to give crude product (7). This was purified by silica gel column chromatography using ethyl acetate and hexane (0-13%) to give pure TBDMS side-chain glycol ether treprostinil benzyl ester (7) (235 mg) in 87.7% yield. The pure product was characterized by H NMR.

[0277] Synthesis of TBDMS side chain dibenzylphosphonooxyethyl treprostinil benzyl ester (8): Reaction scheme:

[0278] [ka]

[0279] [Table 7]

[0280] Testing Procedure: To a solution of TBDMS side chain glycol ether treprostinil benzyl ester (7) (0.2 g, 0.313 mmol) in dichloromethane (3 mL) was added tetrazole solution (0.45 M in acetonitrile) (2.1 mL, 0.939 mmol) and dibenzyl N,N-diisopropylphosphoramidite (210 μL), and the reaction mixture was stirred at ambient temperature for 2 h. The progress of the reaction was monitored by TLC (EtOAc / hexane 3:7), and the reaction showed some starting material. Additional tetrazole solution (1.0 mL, 0.469 mmol) and dibenzyl N,N-diisopropylphosphoramidite (105 μL, 0.313 mmol) were added, and the reaction was stirred for 1 h. At this stage, the starting material was completely consumed. The reaction mixture was cooled to −78° C. and the 3-chloro-N,N-diisopropylphosphoramidite in dichloromethane (1 mL) was added. A solution of peroxybenzoic acid (217 mg, 0.970 mmol) was added. The mixture was stirred for 1.5 hours while the temperature was allowed to rise. The reaction was found to be complete based on TLC (3:7 EtOAc / hexane). The reaction mixture was quenched with 10% aqueous sodium sulfite solution (6 mL) and stirred for 15 minutes. The organic layer was separated and the aqueous layer was extracted with dichloromethane (2 x 15 mL). The combined organic layers were washed with saturated water. The sodium bicarbonate solution was dried over sodium sulfate and evaporated under reduced pressure to give crude product (8). This was purified by silica gel column chromatography using ethyl acetate and hexane (0-22%) to give pure TBDMS side-chain dibenzylphosphonooxyethyl treprostinil benzyl ester (8) (260 mg) in 92.5% yield. The pure product was 1 H NMR and 31 It was characterized by P NMR.

[0281] Synthesis of side chain dibenzylphosphonooxyethyl treprostinil benzyl ester (9): Reaction scheme:

[0282] [ka]

[0283] [Table 8]

[0284] Testing Procedure: To a solution of TBDMS side-chain dibenzylphosphonooxyethyl prostinyl benzyl ester (8) (120 mg, 0.133 mmol) in anhydrous tetrahydrofuran (6 mL) was added hydrogen fluoride pyridine (0.9 mL). The reaction mixture was stirred at ambient temperature. The reaction progress was monitored by TLC (4:1 EtOAc / hexane). After 3 h, the reaction was found to be complete based on TLC. The reaction mixture was quenched by the dropwise addition of saturated aqueous sodium bicarbonate solution (25 mL) and stirred for 15 min. The mixture was extracted with ethyl acetate (3 × 15 mL), and the organic layer was separated. The combined organic layers were dried over sodium sulfate and evaporated under reduced pressure to give crude product (9). This was combined with another 120 mg batch and purified by silica gel column chromatography using ethyl acetate and hexane (0-52%) to give pure side-chain dibenzylphosphonooxyethyl prostinyl benzyl ester (9) (186 mg) in 88.9% yield. The pure product was 1 H NMR, 13 C NMR and 31 It was characterized by P NMR.

[0285] Synthesis of Treprostinil Side Chain Phosphonoxyethyl Prodrug (10): Reaction scheme:

[0286] [ka]

[0287] [Table 9]

[0288] Testing Procedure: To a solution of the side-chain dibenzylphosphonoxyethyl treprostinil benzyl ester (9) (167 mg, 0.213 mmol) in ethyl acetate (8 mL) was added palladium on carbon (50 mg) and water (2 mL). The mixture was evacuated and replaced with hydrogen gas (3 times). It was stirred at ambient temperature under a hydrogen atmosphere. The progress of the reaction was monitored by TLC (3:7 EtOAc / hexanes), and the reaction was found to be complete after 6 h. The reaction mixture was filtered through Celite (ethyl acetate (10 mL) and water (5 mL), and the resulting filtrate was evaporated under reduced pressure to give the crude product (6). This was dissolved in tetrahydrofuran (5 mL) and filtered through cotton to remove scum to give the pure treprostinil side-chain phosphonooxyethyl prodrug (10) (106 mg) in 97.2% yield. The pure product was 1 H NMR, 13 C NMR, 31 It was characterized by P NMR, LC-MS and IR. The HPLC purity of the compound was found to be 97.48%.

[0289] [Table 10]

[0290] Example 2: Synthesis of Treprostinil Side Chain Ethyl Carbonate (Prodrug XVI) Scheme 2: Synthesis of Treprostinil Side Chain Ethyl Carbonate (Prodrug XVI)

[0291] [ka]

[0292] experiment: Synthesis of TES-Treprostinil Benzyl Ester Ethyl Carbonate (2) Reaction scheme:

[0293] [ka]

[0294] [Table 11]

[0295] Testing Procedure: To a solution of mono-TES-treprostinil benzyl ester (1) (2.4 g, 4.03 mmol) in anhydrous dichloromethane (35 mL), N,N,N',N'-tetramethylethylenediamine (0.73 mL, 4.84 mmol) was added. The clear solution was cooled to -78-70 °C, and then ethyl chloroformate (0.77 mL, 8.07 mmol) was added dropwise over 5 minutes under argon. After complete addition, the reaction mixture was stirred while the temperature was allowed to rise to room temperature. After 4 hours, the reaction was complete based on TLC (ethyl acetate / hexane, 1:4). The reaction mixture was quenched with water (15 mL). The organic layer was separated, washed with brine (10 mL), dried over sodium sulfate, and evaporated under reduced pressure to give the crude product (3.1 g). The crude compound was purified by column chromatography on silica gel using 0-7% EtOAc / hexane as the mobile phase to give pure TES-treprostinil benzyl ester ethyl carbonate (2) (2.79 g, 103% yield, residual solvent). 1 It was characterized by 1 H NMR.

[0296] Synthesis of Treprostinil Benzyl Ester Ethyl Carbonate (3) Reaction scheme:

[0297] [ka]

[0298] [Table 12]

[0299] Testing Procedure: To a solution of TES-treprostinil benzyl ester ethyl carbonate (2) (2.6 g, 3.89 mmol) in tetrahydrofuran (30 mL) was added 2N hydrochloric acid solution (3.9 mL, 3.89 mmol) (water (1.1 mL) used for rinsing) and the reaction mixture was stirred at room temperature for 1 h. The reaction was found to be complete based on TLC (ethyl acetate / hexane, 2:3). The reaction mixture was neutralized with triethylamine (1.1 mL, 7.79 mmol). The organic volatiles were evaporated in vacuo, and the residue was partitioned between MTBE (20 mL) and water (10 mL). The organic layer was separated, washed with brine, dried (NaSO), and concentrated in vacuo to give the crude product (2.4 g). The crude product was purified by silica gel column chromatography using 0-25% EtOAc / hexane as the mobile phase to give pure treprostinil benzyl ester ethyl carbonate (3) (2.24 g, 104% yield, residual solvent). 1 It was characterized by 1 H NMR.

[0300] Synthesis of Treprostinil Side Chain Ethyl Carbonate (4) Reaction scheme:

[0301] [ka]

[0302] [Table 13]

[0303] Testing Procedure: To a solution of treprostinil benzyl ester ethyl carbonate (3) (2.0 g, 3.62 mmol) in ethyl acetate (20 mL) was added palladium on carbon (5 wt %, 50% water) (0.4 g). The mixture was stirred, evacuated under house vacuum, and replaced with hydrogen (packed in a balloon). This process was repeated three times. The mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction was found to be complete based on TLC (ethyl acetate / hexane, 2:3). The reaction mixture was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to give treprostinil side-chain ethyl carbonate (prodrug XVI) (4) (1.58 g, 94.6% yield). This compound was characterized by spectral data (IR, 1 H NMR, 13 It was fully characterized by C NMR and LC-MS) and HPLC with a purity of 99.63%.

[0304] Scheme 3: Synthesis of Treprostinil Side Chain Isopropyl Carbonate (Prodrug XVII)

[0305] [ka]

[0306] experiment: Synthesis of TES-Treprostinil Benzyl Ester Isopropyl Carbonate (2) Reaction scheme:

[0307] [ka]

[0308] [Table 14]

[0309] Testing Procedure: To a solution of mono-TES-treprostinil benzyl ester (1) (3.57 g, 6.00 mmol) in anhydrous dichloromethane (40 mL) was added N,N,N',N'-tetramethylethylenediamine (1.07 mL, 7.20 mmol). The clear solution was cooled to -78-70°C, and then isopropyl chloroformate solution (1.0 M in toluene) (12 mL, 12.00 mmol) was added dropwise over 10 minutes under argon. After complete addition, the reaction mixture was stirred while the temperature was allowed to rise to room temperature. After 4 hours, the reaction was complete based on TLC. The reaction mixture was quenched with water (20 mL). The organic layer was separated, washed with brine (10 mL), dried over sodium sulfate, and evaporated under reduced pressure to give the crude product (4.86 g). The crude compound was purified by column chromatography on silica gel using 0-8% EtOAc / hexane as the mobile phase to give pure TES-treprostinil benzyl ester isopropyl carbonate (2) (3.88 g, 95.1% yield). 1 It was characterized by 1 H NMR.

[0310] Synthesis of Treprostinil Benzyl Ester Isopropyl Carbonate (3) Reaction scheme:

[0311] [ka]

[0312] [Table 15]

[0313] Testing Procedure: To a solution of TES-treprostinil benzyl ester isopropyl carbonate (2) (3.77 g, 5.54 mmol) in tetrahydrofuran (40 mL) was added 2N hydrochloric acid solution (5.6 mL, 5.54 mmol) (used as a rinse) and water (1.5 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction was found to be complete based on TLC (ethyl acetate / hexane 2:3). The reaction mixture was neutralized with triethylamine (1.6 mL, 11.08 mmol). The organic volatiles were evaporated in vacuo, and the residue was partitioned between MTBE (30 mL) and water (15 mL). The organic layer was separated, washed with brine, dried over NaSO, and concentrated in vacuo to give the crude product (3.88 g). The crude product was purified by silica gel column chromatography using 0-26% EtOAc / hexane to give pure treprostinil benzyl ester isopropyl carbonate (3) (3.0 g, 95.5% yield). The pure compound (3) was obtained. 1 It was characterized by 1 H NMR.

[0314] Synthesis of Treprostinil Side Chain Isopropyl Carbonate (4) Reaction scheme:

[0315] [ka]

[0316] [Table 16]

[0317] Testing Procedure: To a solution of treprostinil benzyl ester isopropyl carbonate (3) (2.9 g, 5.12 mmol) in ethyl acetate (30 mL) was added palladium on carbon (5 wt %, 50% water) (0.58 g). The mixture was stirred, evacuated under house vacuum, and replaced with hydrogen (packed in a balloon). This process was repeated three times. The mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction was found to be complete based on TLC (ethyl acetate / hexane, 2:3). The reaction mixture was filtered through a pad of Celite. The filtrate was concentrated in vacuo to give treprostinil side chain isopropyl carbonate (prodrug XVII) (4) (2.38 g, 97.5% yield). This compound was characterized by spectral data (IR, 1 H NMR, 13 It was fully characterized by C NMR and LC-MS) and HPLC with a purity of 96.53%.

[0318] Scheme 4: Synthesis of Treprostinil Side Chain Phosphate (Prodrug VI)

[0319] [ka]

[0320] experiment: Synthesis of Treprostinil Benzyl Ester (3): Reaction scheme:

[0321] [ka]

[0322] [Table 17]

[0323] Testing Procedure: To a 2 L round-bottom flask equipped with an air-driven mechanical stirrer, 150 g of triol (1) in 800 mL of acetone was added, followed by 124 g of benzyl bromoacetate (2) in 200 mL of acetone. To this stirred solution, 137.3 g of powdered potassium carbonate was added, and the mixture was stirred at room temperature. The reaction was monitored by TLC (MeOH / DCM, 1:10). After completion of the reaction, the mixture was filtered and washed with 2 x 100 mL of acetone. The filtrate was concentrated under reduced pressure to give 260.7 g of crude product. The product was dissolved in 35 mL of ethyl acetate and transferred to a 5 L three-neck flask equipped with an air-driven mechanical stirrer. The mixture was stirred in a water bath at 50 °C. To this solution, 1 L of hexane was added with stirring. The mixture was left at room temperature for 30 minutes, and the supernatant was decanted. 25 mL of ethyl acetate was added, stirred in a water bath at 50 °C, and 750 mL of hexane was added slowly. After stirring for 40 minutes, stirring was stopped at room temperature for 30 minutes, and the supernatant was decanted. This process was repeated once more to obtain a white solid. It was filtered and the solid was washed with hexane (2 x 100 ml). The solid was air-dried overnight and weighed 209.7 g (96.7% yield) (HPLC purity 96.70%). This compound was 1 It was characterized by 1 H NMR and MS.

[0324] Synthesis of Treprostinil MonoTES Benzyl Ester (4): Reaction scheme:

[0325] [ka]

[0326] [Table 18]

[0327] Testing Procedure: A round-bottom flask equipped with a magnetic stir bar was charged with treprostinil benzyl ester (3) (10.0 g), imidazole (1.41 g), DMAP (0.25 g), and anhydrous DCM (200 ml). The mixture was stirred at room temperature under argon, and TES-Cl (3.5 ml) was added. After stirring for 1 hour, the reaction was monitored by TLC (1:4 EtOAc / Hex). The reaction was quenched with water (150 ml). The organic layer was washed with brine and dried over sodium sulfate. It was filtered, and the solvent was removed under reduced pressure to give the crude product, which was purified by silica gel column chromatography using 0-11% ethyl acetate in hexane to give the desired pure treprostinil mono-TES benzyl ester (4) (6.68 g, 54% yield). 1 It was characterized by 1 H NMR.

[0328] Synthesis of mono-TES treprostinil benzyl ester dibenzyl phosphate (6): Reaction scheme:

[0329] [ka]

[0330] [Table 19]

[0331] Testing Procedure: A round-bottom flask equipped with a magnetic stir bar was charged with mono-TES treprostinil benzyl ester (4) (1.08 g), dibenzyl diisopropyl phosphoramidite (5) (1.26 g), and 1H-tetrazole (12.1 ml, 0.45 M in acetonitrile) in anhydrous DCM (50 ml). The mixture was stirred at room temperature under argon for 2 hours and checked by TLC (EtOAc / Hex, 1:4). It was cooled to -78 °C, and m-CPBA (0.94 g, <77% purity) was then added in one portion. The resulting suspension was stirred at that temperature for 2 hours and checked by TLC (EtOAc / Hex, 1:4). After the reaction was complete, 10% NaSO solution (20 ml) and DCM (20 ml) were added and stirred for 10 minutes. The DCM layer was tested with peroxide 100 test paper to confirm the absence of peroxide in the solution (if peroxide was present, it was further washed with NaSO solution). The DCM layer was washed with water (20 ml) and saturated. Sodium bicarbonate (20 ml), brine (20 ml), and dried over sodium sulfate. It was filtered, and the solvent was removed under reduced pressure to give crude product 6 (2.32 g). Purification by silica gel column chromatography using 5-45% ethyl acetate / hexane gave pure treprostinil mono-TES benzyl ester dibenzyl phosphate 6 (1.42 g, 92% yield). 1 It was characterized by 1 H NMR and MS.

[0332] Synthesis of Treprostinil Benzyl Ester Dibenzyl Phosphate (7): Reaction scheme:

[0333] [ka]

[0334] [Table 20]

[0335] Testing Procedure: A round-bottom flask equipped with a magnetic stir bar was charged with treprostinil mono-TES benzyl ester dibenzyl phosphate (6) (1.40 g) in THF (20 ml) and water (4 ml). To this stirred solution, hydrochloric acid (2 M) (1.22 ml) was added, and the reaction mixture was stirred at room temperature for 1 hour and checked by TLC (EtOAc / Hex, 1:2). After completion of the reaction, water (20 ml) and ethyl acetate (20 ml) were added, stirred for 10 minutes, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (2 x 20 ml). The combined organic extracts were washed with water (20 ml), sodium bicarbonate (20 ml), brine (20 ml), and dried over sodium sulfate (20 g). It was filtered and the solvent removed in vacuo to give the crude product (1.53 g), which was purified by silica gel column chromatography using 5-70% ethyl acetate in hexane to give pure treprostinil benzyl ester dibenzyl phosphate (7), (1.12 g, 92% yield) (96.16% HPLC purity). 1 It was characterized by 1 H NMR and MS.

[0336] Synthesis of Treprostinil Side Chain Phosphate (Prodrug VI) Reaction scheme:

[0337] [ka]

[0338] [Table 21]

[0339] Testing Procedure: A two-necked round-bottom flask equipped with a magnetic stir bar and a three-way connector to a hydrogen balloon was charged with treprostinil benzyl ester dibenzyl phosphate (7) (1.00 g) in ethyl acetate (50 ml) and water (2.5 ml). To this stirred solution at room temperature, palladium on carbon (5 wt%) (300 mg) was added. The system was evacuated and purged with hydrogen (repeated two more times). The flask was then connected to a hydrogen balloon and stirred at room temperature for 4 hours, followed by TLC (MeOH / DCM, 1:4). After completion of the reaction, the mixture was evacuated and purged with air and then filtered through a pad of Celite (-2 g). The filter was washed with ethyl acetate (3 × 10 ml). The filtrate was concentrated in vacuo to give treprostinil side chain phosphate (prodrug VI) as a white solid (0.57 g, 90% yield) (99.93% HPLC purity). 1 H, 13 C. 31 It was characterized by P NMR, IR, and MS.

[0340] Large-scale synthesis of treprostinil side chain phosphate (prodrug VI) Scheme 4': Large-scale synthesis of treprostinil side chain phosphate (prodrug VI)

[0341] [ka]

[0342] Treprostinil side chain phosphate (prodrug VI) was synthesized from mono-TES-treprostinil benzyl ester (1) in three steps as shown in Scheme 4'. 1 was phosphitylated with dibenzyl N,N-diisopropylphosphoramidite in the presence of 1H-tetrazole, followed by oxidation with 3-chloroperbenzoic acid to give TES-treprostinil benzyl ester dibenzyl phosphate (2). Desilylation of 2 with 2N hydrochloric acid in aqueous tetrahydrofuran gave treprostinil benzyl ester dibenzyl phosphate (3). Pure compound (3) was hydrogenolyzed using 5% palladium on carbon and hydrogen to give treprostinil side chain phosphate (prodrug VI) (4) as a white solid. UT-30 was characterized by spectral data and purity by HPLC.

[0343] experiment: Synthesis of TES-Treprostinil benzyl ester dibenzyl phosphate (2): Testing Procedure: To a solution of mono-TES-treprostinil benzyl ester (1) (46.33 g, 77.88 mmol) in anhydrous dichloromethane (800 mL), a solution of 1H-tetrazole (0.45 M in acetonitrile) (519 mL, 233.69 mmol) was added over 15 minutes at room temperature under argon. The mixture was stirred at room temperature for 1 hour, and then a solution of dibenzyl N,N-diisopropylphosphoramidite (53.80 g, 155.75 mmol) in anhydrous dichloromethane (120 mL) was added. The reaction mixture was stirred at room temperature for 1 hour and then cooled to -60 ± 3 °C. To this cooled mixture, 3-chloroperbenzoic acid (approximately 77%) (54.11 g, 247.2 mmol) was added in portions. The reaction mixture was stirred at this temperature for 1 hour, and the reaction was complete (TLC, EtOAc / hexane, 1:4). The reaction mixture was treated with 10% sodium sulfite in water (1250 mL) and stirred overnight at room temperature. The organic layer was separated from the mixture, and the aqueous layer was extracted with dichloromethane (2 x 200 mL). The combined organic extracts were washed with saturated sodium bicarbonate solution (400 mL), dried (NaSO), filtered, and concentrated in vacuo to give the crude product. Chromatography of the crude product on silica gel using ethyl acetate in hexanes gave TES-treprostinil benzyl ester dibenzyl phosphate (2) as a viscous liquid (54.1 g). The product was 1 It was characterized by 1 H NMR and HPLC with a purity of 97.43%.

[0344] Synthesis of Treprostinil Benzyl Ester Dibenzyl Phosphate (3): Testing Procedure: To a solution of TES-treprostinil benzyl ester dibenzyl phosphate (2) (53.8 g, 62.91 mmol) in a mixture of tetrahydrofuran (540 mL) and water (108 mL) was added a solution of 2N hydrochloric acid (48 mL) at room temperature. The reaction mixture was stirred at room temperature for 30 minutes, and the reaction was complete (TLC, EtOAc / hexane, 1:1). The reaction mixture was treated with ethyl acetate (100 mL), and the aqueous layer was separated. The aqueous layer was extracted with ethyl acetate (2 × 200 mL). The combined organic extracts were washed with water (1 × 350 mL), saturated sodium bicarbonate (1 × 200 mL), brine (1 × 70 mL), dried (NaSO), filtered, and concentrated in vacuo to give the crude product. Chromatography of the crude product on silica gel with ethyl acetate in hexane gave treprostinil benzyl ester dibenzyl phosphate (3) as a viscous liquid (34.1 g) and 99.47% purity (HPLC).

[0345] Synthesis of Treprostinil Side Chain Phosphate (Prodrug VI) (4): Testing Procedure: To a solution of treprostinil benzyl ester dibenzyl phosphate (3) (34.0 g, 45.89 mmol) in a mixture of ethyl acetate (1500 mL) and water (75 mL) was added 5% palladium on carbon (50% water) (8.5 g). The mixture was evacuated under house vacuum at room temperature and replaced with hydrogen (packed in a balloon). This process was repeated two more times. The reaction mixture was then stirred under an atmosphere of hydrogen at room temperature for 4 hours. The reaction was complete (TLC, EtOAC / hexane, 6:4). The reaction mixture was filtered through a Celite pad, and the pad was washed with ethyl acetate and water. The filtrate was evaporated under vacuum to give a white solid. The solid was treated with ethyl acetate (500 mL) and filtered through a Buchner funnel. The solid treprostinil side chain phosphate (prodrug VI) (4) was air-dried overnight. The dried prodrug VI weighed 19.22 g and was 99.93% pure by HPLC.

[0346] Under similar reaction conditions, 40.79 g and 25.15 g of prodrug VI were also synthesized. These three lots were combined to give 85.15 g of prodrug VI. Prodrug VI was characterized by spectroscopic data (IR, 1 H, 13 C and 31 It was fully characterized by 1 P NMR, melting point and purity of 99.95% by HPLC.

[0347] Mono-TES-treprostinil benzyl ester (1) was prepared from benzinetriol by alkylation, followed by silylation, and chromatography as described above.

[0348] The synthetic process for preparing prodrug VI can be used to prepare prodrug VI in large batches while maintaining high purity of prodrug VI in such batches. For example, prodrug VI can be prepared in batch sizes of at least 20 g, at least 30 g, at least 40 g, at least 60 g, at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least 110 g, at least 130 g, at least 140 g, at least 150 g, at least 160 g, at least 170 g, at least 180 g, at least 190 g, at least 200 g, at least 300 g, at least 500 g, at least 1000 g, or at least 2000 g. Such batches may have a purity of at least 98.0%; at least 98.5%; at least 98.8%; at least 99.1%; at least 99.2%; at least 99.3%; at least 99.4%; at least 99.5%; at least 99.6%; at least 99.7%; at least 99.8%; or at least 99.9%.

[0349] In the synthesis process of prodrug VI, as well as in the synthesis process of other prodrugs VI, TES can be replaced with another silyl ester, such as, for example, trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, phenyldimethylsilyl, while benzyl can be substituted with one or more substituents, which can be independently selected from the group consisting of substituted benzyl, i.e., -NO, -CN, -halogen (e.g., -F, -Cl, -Br, or -I), (C-C)alkyl, halo(C-C)alkyl, (C-C)alkoxy, and halo(C-C)alkoxy.

[0350] Scheme 5: Synthesis of treprostinil side chain piperidine carbamate (prodrug XIX)

[0351] [ka]

[0352] experiment: Synthesis of TES-Treprostinil Benzyl Ester Piperidine Carbamate (2) Reaction scheme:

[0353] [ka]

[0354] [Table 22]

[0355] Testing Procedure: To a solution of mono-TES-treprostinil benzyl ester (1) (1.5 g, 2.52 mmol) in anhydrous tetrahydrofuran (15 mL) was added pyridine (0.62 mL, 7.56 mmol) at room temperature under argon. The clear solution was cooled to 0°C (ice / water bath), and then a solution of 4-nitrophenyl chloroformate (0.76 g, 3.78 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise over 15 minutes, maintaining the temperature below 5°C under argon. After the addition was complete, the reaction mixture (cloudy white) was stirred at 0°C-room temperature for 5 hours. The reaction was partially complete based on TLC (1:4 EtOAc / hexane) and was stored at 2-8°C overnight. The next day, a solution of piperidine (0.75 mL, 7.56 mmol) in tetrahydrofuran (5 mL) was added dropwise over 10 minutes at 0°C. After 6 hours, the reaction mixture was checked by TLC (1:4 EtOAc / hexanes) to confirm completion. The reaction mixture was filtered to remove the precipitate, and the resulting filtrate was concentrated under reduced pressure to give the crude product (2.3 g). The crude compound was purified by silica gel column chromatography using 0-9% EtOAc / hexanes to give pure TES-treprostinil benzyl ester piperidine carbamate (2) (1.49 g, 84.6% yield). Pure compound (2) was obtained. 1 It was characterized by 1 H NMR.

[0356] Synthesis of Treprostinil Benzyl Ester Piperidine Carbamate (3) Reaction scheme:

[0357] [ka]

[0358] [Table 23]

[0359] Testing Procedure: To a solution of TES-treprostinil benzyl ester piperidine carbamate (2) (1.45 g, 2.07 mmol) in a mixture of tetrahydrofuran (12 mL) and water (1 mL) was added 2N hydrochloric acid solution (1.0 mL, 2.07 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours and checked by TLC (EtOAc / hexane, 1:1). The reaction was found to be complete. The reaction mixture was neutralized with triethylamine (0.58 mL, 0.58 mmol), and then the organic volatiles were evaporated. The residue was dissolved in EtOAc (30 mL), washed with water (20 mL), brine (20 mL), dried over NaSO, and concentrated in vacuo to give the crude product (1.28 g). The crude product was chromatographed on a silica gel column using 0-20% EtOAc / hexane to give pure treprostinil benzyl ester piperidine carbamate (3) (1.15 g, 94.3% yield). 1 It was characterized by 1 H NMR.

[0360] Synthesis of Treprostinil Side Chain Piperidine Carbamate (4) Reaction scheme:

[0361] [ka]

[0362] [Table 24]

[0363] Testing Procedure: To a solution of treprostinil benzyl ester piperidine carbamate (3) (1.1 g, 1.86 mmol) in ethyl acetate (15 mL) was added palladium on carbon (5 wt %, 50% water) (0.22 g). The mixture was stirred, evacuated under house vacuum, and replaced with hydrogen (filled in a balloon). This process was repeated three times. The mixture was stirred under a hydrogen atmosphere at room temperature for 4 hours and checked by TLC (2:3 EtOAc / hexane). The reaction was found to be complete. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated in vacuo to give treprostinil side-chain piperidine carbamate (4) (0.86 g, 92.3% yield). This compound was characterized by spectral data (IR, 1 H NMR, 13 It was fully characterized by C NMR and MS and HPLC with a purity of 99.06%.

[0364] Scheme 6: Synthesis of Treprostinil Side Chain Succinate (Prodrug XX)

[0365] [ka]

[0366] Synthesis of Treprostinil Mono-TES Benzyl Ester Side Chain Succinic Acid Benzyl Ester (3): Reaction scheme:

[0367] [ka]

[0368] [Table 25]

[0369] Experimental procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with anhydrous DCM (50 ml) and treprostinil mono-TES benzyl ester (1) (2.33 g). To this stirred solution, under argon at room temperature, was added succinic acid monobenzyl ester (2) (0.90 g), triethylamine (655 μl), and DMAP (48 mg). After stirring for 10 min, EDCI.HCl (0.82 g) was added, and the mixture was stirred overnight under argon at room temperature and checked by TLC (EtOAc / Hex, 1:4). Water (20 ml) was added, and the aqueous layer was extracted with DCM (2 × 20 ml). The combined organic extracts were washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (10 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (3.78 g). Purification by silica gel column chromatography using 1-10% ethyl acetate / hexane gave the desired pure treprostinil mono-TES benzyl ester side chain succinic acid benzyl ester (3) (2.13 g, 69% yield) (96.94% HPLC purity), which was characterized by H NMR.

[0370] Synthesis of Treprostinil Benzyl Ester Side Chain Succinic Acid Benzyl Ester (4) Reaction scheme:

[0371] [ka]

[0372] [Table 26]

[0373] Experimental procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with treprostinil mono-TES benzyl ester side chain succinic acid benzyl ester (3) (1.79 g) in THF (40 ml) and water (8 ml). To this stirred solution, 2 M HCl solution (1.14 ml) was added, and the mixture was stirred at room temperature for 30 minutes and checked by TLC (EtOAc / Hex, 1:4). Triethylamine (0.5 ml) was added, stirred for 10 minutes, and water (20 ml) and EtOAc (20 ml) were added. The aqueous layer was extracted with ethyl acetate (2 x 20 ml). The organic layer was washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (approximately 10 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (2.29 g). Purification by silica gel column chromatography using 1-25% ethyl acetate / hexane gave the desired pure treprostinil benzyl ester side chain succinic acid benzyl ester (4) (1.54 g, 92% yield) (HPLC purity 99.60%). 1 It was characterized by 1 H NMR.

[0374] Synthesis of Treprostinil Side Chain Succinate (Prodrug XX): Reaction scheme:

[0375] [ka]

[0376] [Table 27]

[0377] Experimental Procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with ethyl acetate (50 ml) and treprostinil benzyl ester side chain succinic acid benzyl ester (4) (1.25 g). To this stirred solution at room temperature, palladium on carbon (5 wt %, 50% water, 0.25 g) was added. The system was evacuated and replaced with hydrogen (this process was repeated two more times). The system was connected to a hydrogen balloon and stirred at room temperature for 4 hours, followed by TLC (EtOAc / Hex, 1:2). The system was evacuated and replaced with air. The mixture was filtered through a Celite pad (-5 g) and the filter was washed with ethyl acetate (20 ml). The solvent was removed under vacuum to give treprostinil side chain succinic acid (UT-33) (0.77 g, 84% yield) (96.74% HPLC purity). This compound was purified by HPLC. 1 H, 13 It was characterized by C NMR, IR and LC-MS.

[0378] Scheme 7: Synthesis of treprostinil cyclopentyl succinate (prodrug XXII) [ka]

[0379] Synthesis of Treprostinil Side Chain TBDMS Benzyl Ester Succinic Acid Benzyl Ester (3): Reaction scheme:

[0380] [ka]

[0381] [Table 28]

[0382] Experimental procedure: A 100 ml round-bottom flask equipped with a stir bar was charged with anhydrous DCM (30 ml) and treprostinil side-chain TBDMS benzyl ester (1) (0.82 g). To this stirred solution, under argon at room temperature, was added succinic acid monobenzyl ester (2) (0.32 g), triethylamine (385 μl), and DMAP (34 mg). After stirring for 10 min, EDCI.HCl (0.30 g) was added, and the mixture was stirred overnight under argon at room temperature and checked by TLC (EtOAc / Hex, 1:2). Water (20 ml) was added, and the aqueous layer was extracted with DCM (2 × 10 ml). The combined organic extracts were washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (10 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (1.56 g). Purification by silica gel column chromatography using 1-15% ethyl acetate / hexane gave treprostinil side chain TBDMS benzyl ester succinic acid benzyl ester (3) (0.81 g, 75% yield) (99.01% HPLC purity). 1 It was characterized by 1 H NMR. Reaction scheme:

[0383] Synthesis of Treprostinil Benzyl Ester Cyclopentylsuccinic Acid Benzyl Ester (4)

[0384] [ka]

[0385] [Table 29]

[0386] Experimental procedure: A 100 ml round-bottom flask equipped with a stir bar was charged with IPA (20 ml) and treprostinil benzyl ester side chain TBDMS succinic acid benzyl ester (3) (0.77 g). To this stirred solution, 2 M HCl solution (1.25 ml) was added, and the mixture was stirred at room temperature for 7 hours and checked by TLC (EtOAc / Hex, 1:2). Triethylamine (1 ml) was added, stirred for 10 minutes, and water (10 ml) and EtOAc (20 ml) were added. The aqueous layer was extracted with ethyl acetate (2 × 10 ml). The combined organic layers were washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (approximately 10 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (1.05 g). Purification by silica gel column chromatography using 1-40% ethyl acetate / hexane gave treprostinil benzyl ester cyclopentyl succinic acid benzyl ester (4) (0.52 g, 79% yield) (HPLC purity 99.51%). 1 It was characterized by 1 H NMR.

[0387] Synthesis of Treprostinil Succinate (Prodrug XXII): Reaction scheme:

[0388] [ka]

[0389] [Table 30]

[0390] Experimental Procedure: A 100 ml round-bottom flask equipped with a stir bar was charged with ethyl acetate (20 ml) and treprostinil benzyl ester cyclopentyl succinic acid benzyl ester (4) (0.49 g). To this stirred solution, palladium on carbon (100 mg) was added at room temperature. The system was evacuated and replaced with hydrogen (this process was repeated two more times). The flask was connected to a hydrogen balloon and stirred at room temperature for 4 hours, followed by TLC (EtOAc / Hex, 1:2). The system was evacuated and replaced with air. The mixture was filtered through a Celite pad (approximately 2 g) and the filter was washed with ethyl acetate (10 ml). The solvent was removed under vacuum to give treprostinil cyclopentyl succinate (prodrug XXII) (0.35 g, 97% yield) (98.15% HPLC purity). This compound was 1 H, 13 It was characterized by C NMR, IR and LC-MS.

[0391] Scheme 8: Synthesis of Treprostinil Side Chain Bipiperidine Carbamate (Prodrug XXIII)

[0392] [ka]

[0393] experiment: Synthesis of TES-Treprostinil Benzyl Ester Bipiperidine Carbamate (2) Reaction scheme:

[0394] [ka]

[0395] [Table 31]

[0396] Experimental procedure: To a solution of mono-TES-treprostinil benzyl ester (1) (1.46 g, 2.46 mmol) in anhydrous toluene (20 mL) was added pyridine (0.6 mL, 7.37 mmol) at room temperature under argon. This solution was added dropwise to an ice-cold solution of triphosgene (1.1 g, 3.68 mmol) in toluene (10 mL). After the addition was complete, the reaction mixture (cloudy white) was stirred at room temperature for 1 h. The intermediate formation was complete based on TLC (EtOAc / hexane, 1:4). To this was added a suspension of 4-piperidinopiperidine (0.62 g, 3.68 mmol) in toluene (5 mL), and dichloromethane (18 mL) was used for washing. After 3 h, TLC (MeOH / DCM 1:9) indicated the reaction was complete. The reaction mixture was concentrated in vacuo, and the residue was partitioned between ethyl acetate (30 mL) and water (30 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (15 mL). The combined organic layers were washed with brine, dried over sodium sulfate, and evaporated under reduced pressure to give the crude product (1.8 g). The crude compound was purified by silica gel chromatography using 0-100% EtOAc / hexane and 1-4% MeOH / DCM to give pure TES-treprostinil benzyl ester bipiperidine carbamate (2) (1.32 g, 68.0% yield). Pure compound (2) was obtained by HPLC. 1 It was characterized by 1 H NMR.

[0397] Synthesis of Treprostinil Benzyl Ester Bipiperidine Carbamate (3) Reaction scheme:

[0398] [ka]

[0399] [Table 32]

[0400] Experimental procedure: To a solution of TES-treprostinil benzyl ester bipiperidine carbamate (2) (1.26 g, 1.6 mmol) in a mixture of tetrahydrofuran (15 mL) and water (1 mL) was added 2N hydrochloric acid solution (1.7 mL, 3.4 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 h and checked by TLC (EtOAc / hexane, 1:1). The reaction was found to be complete. The reaction mixture was neutralized with triethylamine (0.94 mL, 6.72 mmol), and then the organic volatiles were evaporated. The residue was dissolved in EtOAc (20 mL), washed with water (10 mL), brine (20 mL), dried over NaSO, and concentrated in vacuo to give the crude product (1.34 g). The crude product was chromatographed on a silica gel column using 0-11% MeOH / DCM to give pure treprostinil benzyl ester bipiperidine carbamate (3) (0.61 g) and some impure compound (3) (0.34 g), for an overall yield of 88.0% pure compound (3). 1 It was characterized by 1 H NMR.

[0401] Synthesis of Treprostinil Side Chain Bipiperidine Carbamate (4) Reaction scheme:

[0402] [ka]

[0403] [Table 33]

[0404] Experimental procedure: To a solution of treprostinil benzyl ester bipiperidine carbamate (3) (0.49 g, 0.73 mmol) in methanol (15 mL) was added palladium on carbon (5 wt %, 50% water) (0.1 g). The mixture was stirred, evacuated under house vacuum, and replaced with hydrogen (filled in a balloon). This process was repeated three times. The mixture was stirred under a hydrogen atmosphere at room temperature for 2 hours and checked by TLC (MeOH / EtOAc, 1:1). The reaction was found to be complete. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated in vacuo to give treprostinil side-chain bipiperidine carbamate (4) (0.43 g, 102% yield with residual solvent). This compound was characterized by spectral data (IR, 1 H NMR, 13 It was fully characterized by C NMR and MS and HPLC with a purity of 98.01%.

[0405] Scheme 9: Synthesis of treprostinil cyclic carbonate (prodrug XXIV)

[0406] [ka]

[0407] experiment: Synthesis of Treprostinil Benzyl Ester Side Chain Chloroformate (3): Reaction scheme:

[0408] [ka]

[0409] [Table 34]

[0410] Experimental procedure: To a solution of mono-TES treprostinil benzyl ester (1.15 g, 1.93 mmol) in anhydrous toluene (15 mL) was added pyridine (0.167 g, 0.17 mL) at room temperature under argon. To this clear solution, a cold solution (pre-cooled at 0 °C before addition) of triphosgene (0.39 g, 1.31 g) in anhydrous toluene (15 mL) was added at room temperature. The reaction mixture became cloudy with a white precipitate and was stirred at room temperature for 6 hours and checked by TLC (EtOAc / hexane, 1:4). Since the reaction was not complete, additional triphosgene (0.21 g, 0.71 mmol) (total amount, 0.60 g) was added to the mixture and stirred at room temperature overnight. After 17 hours, the reaction was complete (TLC, EtOAc, 1:4), forming TES-treprostinil benzyl ester side-chain chloroformate (2) along with some treprostinil benzyl ester side-chain chloroformate (3). The reaction mixture was treated with hexane (60 mL), stirred for 10 minutes, and then passed through a silica gel (30.8 g) column. The compound was eluted with 5-40% ethyl acetate / hexane to give the pure treprostinil benzyl ester side-chain chloroformate (3), as shown by spectral data (IR, 1 H NMR, 13 It was fully characterized by C NMR, DEPT, MS) and HPLC with a purity of 95.28%.

[0411] Synthesis of treprostinil benzyl ester cyclic carbonate (4): Reaction scheme:

[0412] [ka]

[0413] [Table 35]

[0414] Experimental procedure: To a clear solution of treprostinil benzyl ester side-chain chloroformate (3) (0.86 g, 1.58 mmol) in anhydrous dichloromethane (10 mL) was added anhydrous pyridine (10 mL) at room temperature under argon. The clear reaction mixture was stirred at room temperature for 45 minutes and checked by TLC (3:7 EtOAc / hexanes) for completion. The mixture was evaporated in vacuo to remove organic volatiles (DCM and pyridine) to give crude cyclic carbonate (4) as a white solid (0.97 g) along with pyridine hydrochloride. The crude product was chromatographed on silica gel (35.4 g) using 5-10% ethyl acetate / hexanes to give pure treprostinil benzyl ester cyclic carbonate (4) as a white solid (0.59 g, 73.7%). The pure cyclic carbonate (4) was analyzed by spectral data (IR, 1 H NMR, 13 It was fully characterized by C NMR, DEPT, MS) and purity of 99.54% (HPLC).

[0415] Synthesis of Treprostinil Cyclic Carbonate (Prodrug XXIV) (5): Reaction scheme:

[0416] [ka]

[0417] [Table 36]

[0418] Experimental Procedure: To a solution of treprostinil benzyl ester cyclic carbonate (4) (0.51 g, 1.01 mmol) in ethyl acetate (30 mL) was added palladium on carbon, 5% by weight, approximately 50% water (0.21 g). The mixture was evacuated under house vacuum and replaced with hydrogen gas (filled in a balloon). This process was repeated three times. The reaction mixture was then stirred under a hydrogen atmosphere at room temperature for 2 hours and checked by TLC (EtOAc / hexane, 3:7 and 7:3). The reaction was complete. The mixture was passed through a Celite pad (4.25 g) in a disposable filter funnel, and the solid was washed with ethyl acetate (3 × 30 mL). The filtrate was concentrated under reduced pressure to give a jelly product, which was dissolved in tetrahydrofuran (10 mL) and filtered (Note: The product was more soluble in tetrahydrofuran than in acetone, acetonitrile, and ethyl acetate). The filtrate was concentrated under reduced pressure to give treprostinil cyclic carbonate (5) as a white solid (0.43 g, 100%). Treprostinil cyclic carbonate (prodrug XXIV) (5) was analyzed by spectral data (IR, 1 H NMR, 13 Fully characterized by C NMR, DEPT, MS), melting point 184-186°C, and purity 99.05% by HPLC.

[0419] Scheme 10: Synthesis of treprostinil cyclopentyl naproxen ester (prodrug XXV)

[0420] [ka]

[0421] Synthesis of Treprostinil Side Chain CBZ Benzyl Ester Cyclopentyl Naproxen Ester (3): Reaction scheme:

[0422] [ka]

[0423] [Table 37]

[0424] Experimental procedure: A 100 ml round-bottom flask equipped with a stir bar was charged with anhydrous DCM (20 ml) and treprostinil side-chain CBZ benzyl ester (1) (0.53 g). To this stirred solution, under argon at room temperature, was added naproxen (2) (0.24 g), triethylamine (230 μl), and DMAP (17 mg). After stirring for 10 minutes, EDCI.HCl (0.20 g) was added, and the mixture was stirred overnight under argon at room temperature and checked by TLC (EtOAc / Hex, 1:4). Water (10 ml) was added, and the aqueous layer was extracted with DCM (10 ml). The combined organic extracts were washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (10 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (1.18 g). Purification by silica gel column chromatography using 1-30% ethyl acetate / hexane gave prostinil side chain CBZ benzyl ester cyclopentyl naproxen ester (3) (0.33 g, 46% yield) (98.15% HPLC purity), which was characterized by H NMR.

[0425] Synthesis of Treprostinil Cyclopentyl Naproxen Ester (Prodrug XXV): Reaction scheme:

[0426] [ka]

[0427] [Table 38]

[0428] Experimental Procedure: A 100 ml round-bottom flask equipped with a stir bar was charged with ethyl acetate (10 ml) and treprostinil benzyl ester side-chain CBZ cyclopentyl naproxen ester (3) (0.31 g). To this stirred solution, palladium on carbon (50 mg) was added at room temperature. The system was evacuated and replaced with hydrogen (this process was repeated two more times). The flask was then connected to a hydrogen balloon and stirred at room temperature for 1 hour, followed by TLC (EtOAc / Hex, 1:2). The system was evacuated and replaced with air. The mixture was filtered through a pad of Celite (-2 g) and washed with ethyl acetate (2 x 5 ml). The solvent was removed under vacuum to give treprostinil cyclopentyl naproxen ester (prodrug XXV) (0.23 g, 99% yield) (97.08% HPLC purity). This compound was purified by HPLC. 1 H, 13 It was characterized by C NMR, IR and LC-MS.

[0429] Scheme 11: Synthesis of Treprostinil Side Chain Ibuprofen Ester (Prodrug XXVI)

[0430] [ka]

[0431] Synthesis of Treprostinil Mono-TES Benzyl Ester Side Chain Ibuprofen Benzyl Ester (3): Reaction scheme:

[0432] [ka]

[0433] [Table 39]

[0434] Experimental procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with anhydrous DCM (50 ml) and treprostinil mono-TES benzyl ester (1) (3.60 g). To this stirred solution, under argon at room temperature, was added ibuprofen (2) (1.50 g), triethylamine (1.7 ml), and DMAP (150 mg). After stirring for 10 minutes, EDCI.HCl (1.40 g) was added, and the mixture was stirred under argon at room temperature for 6 hours and checked by TLC (EtOAc / Hex, 1:4). Water (50 ml) was added, and the aqueous layer was extracted with DCM (2 × 20 ml). The combined organic extracts were washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (10 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (6.04 g). Purification by silica gel column chromatography using 1-10% ethyl acetate / hexanes gave the desired pure treprostinil mono-TES benzyl ester side chain ibuprofen ester (3) (4.06 g, 85% yield) (97.65% HPLC purity). 1 It was characterized by 1 H NMR.

[0435] Synthesis of Treprostinil Benzyl Ester Side Chain Ibuprofen Ester (4): Reaction scheme:

[0436] [ka]

[0437] [Table 40]

[0438] Experimental procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with THF (80 ml), water (16 ml), and treprostinil mono-TES benzyl ester side chain ibuprofen ester (3) (4.06 g). To this stirred solution, 2.60 ml of HCl solution (2 M) was added, and the mixture was stirred at room temperature for 1 hour and checked by TLC (EtOAc / Hex, 1:4). Triethylamine (2.0 ml) was added, stirred for 10 minutes, and water (20 ml) and EtOAc (20 ml) were added. The aqueous layer was extracted with ethyl acetate (2 x 20 ml). The combined organic layers were washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (approximately 30 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (5.07 g). Purification by silica gel column chromatography using 1-25% ethyl acetate / hexanes gave the desired pure treprostinil benzyl ester side chain ibuprofen ester (4) (3.43 g, 98% yield) (99.76% HPLC purity). 1 It was characterized by 1 H NMR.

[0439] Synthesis of Treprostinil Side Chain Ibuprofen Ester (Prodrug XXVI): Reaction scheme:

[0440] [ka]

[0441] [Table 41]

[0442] Experimental procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with ethyl acetate (100 ml) and treprostinil benzyl ester side-chain ibuprofen ester (4) (2.48 g). To this stirred solution, palladium on carbon (0.50 g) was added at room temperature. The system was evacuated and replaced with hydrogen (this process was repeated two more times). The system was connected to a hydrogen balloon and stirred at room temperature for 4 hours, followed by TLC (EtOAc / Hex, 1:2). The system was evacuated and replaced with air. The mixture was filtered through a pad of Celite (approximately 5 g) and washed with ethyl acetate (2 x 5 ml). The solvent was removed under reduced pressure to give treprostinil side-chain ibuprofen ester (prodrug XXVI) (2.01 g, 95% yield) (99.23% HPLC purity). This compound was purified by HPLC. 1 H, 13 It was characterized by C NMR, IR and LC-MS.

[0443] Scheme 12: Synthesis of treprostinil side chain naproxen ester (prodrug XXVII)

[0444] [ka]

[0445] Synthesis of Treprostinil Mono-TES Benzyl Ester Side Chain Naproxen Ester (3): Reaction scheme:

[0446] [ka]

[0447] [Table 42]

[0448] Experimental procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with anhydrous DCM (30 ml) and treprostinil mono-TES benzyl ester (1) (2.31 g). To this stirred solution, under argon at room temperature, was added naproxen (2) (1.07 g), triethylamine (1.1 ml), and DMAP (95 mg). After stirring for 10 minutes, EDCI.HCl (0.89 g) was added, and the mixture was stirred overnight under argon at room temperature and checked by TLC (EtOAc / Hex, 1:4). Water (30 ml) was added, and the aqueous layer was extracted with DCM (2 × 15 ml). The combined organic extracts were washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (20 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (4.93 g). Purification by silica gel column chromatography using 1-20% ethyl acetate / hexane gave the desired pure treprostinil mono-TES benzyl ester side chain naproxen ester (3) (2.63 g, 84% yield) (96.89% HPLC purity). 1 It was characterized by 1 H NMR.

[0449] Synthesis of Treprostinil Benzyl Ester Side Chain Naproxen Ester (4): Reaction scheme:

[0450] [ka]

[0451] [Table 43]

[0452] Experimental procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with THF (80 ml), water (16 ml), and treprostinil mono-TES benzyl ester side chain naproxen ester (3) (2.50 g). To this stirred solution, 1.60 ml of 2M HCl solution was added, and the mixture was stirred at room temperature for 1 hour and checked by TLC (1:4 EtOAc / Hex). Triethylamine (1.5 ml) was added, stirred for 10 minutes, and water (50 ml) and EtOAc (50 ml) were added. The aqueous layer was extracted with ethyl acetate (2 x 15 ml). The combined organic layers were washed with water (20 ml), brine (20 ml), and dried over sodium sulfate (20 g). It was filtered, and the solvent was removed under reduced pressure to give the crude product (4.51 g). Purification by silica gel column chromatography using 1-25% ethyl acetate / hexane gave the desired pure treprostinil benzyl ester side chain naproxen ester (4) (2.09 g, 97% yield) (HPLC purity 99.44%). 1 It was characterized by 1 H NMR.

[0453] Synthesis of Treprostinil Side Chain Naproxen Ester (Prodrug XXVII): Reaction scheme:

[0454] [ka]

[0455] [Table 44]

[0456] Experimental procedure: A 100 ml round-bottom flask equipped with a stir bar was charged with ethyl acetate (10 ml) and treprostinil benzyl ester side-chain naproxen ester (4) (0.55 g). To this stirred solution, palladium on carbon (100 mg) was added at room temperature. The system was evacuated and replaced with hydrogen (this process was repeated two more times). The system was connected to a hydrogen balloon, stirred at room temperature for 1 hour, and checked by TLC (EtOAc / Hex, 1:2). The system was evacuated and replaced with air. The mixture was filtered through a pad of Celite (approximately 2 g) and washed with ethyl acetate (2 x 5 ml). The solvent was removed under vacuum to give treprostinil side-chain naproxen ester (prodrug XXVII) (0.44 g, 93% yield) (97.75% HPLC purity). This compound was purified by 1H, 13 It was characterized by C NMR, IR and LC-MS.

[0457] Scheme 13: Synthesis of Treprostinil Cyclic Phenylphosphate (Prodrug XXVIII)

[0458] [ka]

[0459] Synthesis of Treprostinil Benzyl Ester Cyclic Phenylphosphate I(3): Reaction scheme:

[0460] [ka]

[0461] [Table 45]

[0462] Experimental Procedure: A 250 ml round-bottom flask equipped with a stir bar was charged with THF (50 ml) and treprostinil benzyl ester (1) (2.4 g). To this stirred solution, under argon at room temperature, was added DMAP (1.22 g) and dichlorophenyl phosphate (2) (750 μl). The mixture was stirred overnight and checked by TLC (EtOAc / Hex, 1:2). It was filtered and washed with THF (2 × 5 ml). The solvent was removed under reduced pressure to give 3.78 g of crude product. Purification by silica gel column chromatography using 1-30% ethyl acetate / hexane gave treprostinil benzyl ester cyclic phosphate I (3), a white solid (1.02 g, 33% yield) (99.69% HPLC purity), and treprostinil benzyl ester cyclic phosphate II (4), a liquid (0.45 g, 15% yield) (HPLC purity: 96.02%) (I compound). 1 It was characterized by 1 H NMR.

[0463] Synthesis of Treprostinil Cyclic Phenyl Phosphate I (Prodrug XXVIII): Reaction scheme:

[0464] [ka]

[0465] [Table 46]

[0466] Experimental Procedure: A 100 ml round-bottom flask equipped with a stir bar was charged with ethyl acetate (10 ml) and treprostinil benzyl ester cyclic phenyl phosphate I (3) (0.31 g). To this stirred solution, palladium on carbon (50 mg) was added at room temperature. The system was evacuated and replaced with hydrogen (this process was repeated two more times). The flask was then connected to a hydrogen balloon and stirred at room temperature for 4 hours, followed by TLC (EtOAc / Hex, 1:2). The system was evacuated and replaced with air. The mixture was filtered through a pad of Celite (approximately 2 g) and washed with ethyl acetate (3 x 5 ml). The solvent was removed under vacuum to give treprostinil cyclic phenyl phosphate I (prodrug XXVIII) (0.25 g, 96% yield) (99.47% HPLC purity). This compound was purified by filtration. 1 H, 13 C. 31 It was characterized by P NMR, IR and LC-MS.

[0467] Synthesis of Treprostinil Side Chain Acetate (Prodrug XLIII) and Hydroxyacetate (Prodrug XLIV) Treprostinil side chain acetate (prodrug XLIII) and hydroxyacetate (prodrug XLIV) were synthesized as shown in Scheme 14 and Scheme 15, respectively. Treprostinil side chain acetate (prodrug XLIII) (4) was synthesized in three steps from mono-TES treprostinil benzyl ester (1) (Scheme 14). Mono-TES treprostinil benzyl ester (1) was acetylated with acetic anhydride in the presence of 4-(dimethylamino)pyridine (DMAP) to give the acetate derivative (2).

[0468] Scheme 14: Synthesis of Treprostinil Side Chain Acetate (Prodrug XLIII) (4)

[0469] [ka]

[0470] Desilylation of compound (2) with 2N hydrochloric acid in aqueous tetrahydrofuran afforded pure treprostinil benzyl ester side chain acetic acid (3) (purity 98.68%, HPLC) after chromatography. Debenzylation of the pure benzyl ester (3) with 5% palladium on carbon (50% water) in ethyl acetate afforded the product as a foamy solid. Trituration of the foamy solid with heptane afforded treprostinil side chain acetic acid (prodrug XLIII) (4) (purity 99.74%, HPLC) as a white solid, with no free treprostinil.

[0471] Similarly, treprostinil side-chain hydroxyacetic acid (prodrug XLIV) (7) was synthesized in three steps from mono-TES-treprostinil benzyl ester (1) (Scheme 15). Acylation of 1 with benzyloxyacetic acid in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI.HCl), N-ethyldiisopropylamine (N,N-diisopropylethylamine) (DIEA), and 4-(dimethylamino)pyridine (DMAP) afforded TES-treprostinil benzyl ester side-chain benzyloxyacetate (5) after chromatography. Desilylation of compound (5) with 2 N hydrochloric acid in aqueous tetrahydrofuran afforded pure treprostinil benzyl ester side-chain benzyloxyacetic acid (6) (purity 99.33%, HPLC) after chromatography. The pure benzyl ester (6) was debenzylated with 5% palladium on carbon (50% water) in ethyl acetate and water to give the prostinil side-chain hydroxyacetic acid (prodrug XLIV) (7) as a foamy solid (purity, 97.57% by HPLC) and no free treprostinil.

[0472] Scheme 15: Synthesis of Treprostinil Side Chain Hydroxyacetic Acid (Prodrug XLIV) (7)

[0473] [ka]

[0474] experiment: Synthesis of TES-Treprostinil Benzyl Ester Side Chain Acetate (2)

[0475] [Table 47]

[0476] Testing Procedure: To a solution of mono-TES-treprostinil benzyl ester (2.58 g, 4.34 mmol) in anhydrous dichloromethane (20 mL) was added 4-(dimethylamino)pyridine (DMAP) (1.06 g, 8.68 mmol) under argon at room temperature. To this clear solution was added acetic anhydride (0.89 g, 0.82 mL, 8.72 mmol). The reaction mixture was stirred at room temperature for 2 hours and checked by TLC (EtOAc / hexane, 1:4). The reaction was complete. The mixture was treated with hexane (40 mL) and then loaded onto a silica gel (33 g) column. The compound was eluted with ethyl acetate (5-15%) in hexane to obtain pure TES-treprostinil benzyl ester side chain acetic acid (2) as a clear viscous liquid (2.64 g, 95.6%) with a purity of 98.68% by HPLC. Spectroscopic data (IR, 1 It was characterized by H NMR, C NMR, DEPT-NMR and LCMS.

[0477] Synthesis of Treprostinil Benzyl Ester Side Chain Acetate (3): Reaction scheme:

[0478] [ka]

[0479] [Table 48]

[0480] Testing Procedure: To a solution of TES-treprostinil benzyl ester side chain acetic acid (2) (1.40 g, 2.20 mmol) in a mixture of tetrahydrofuran (10 mL) and water (2 mL) (5:1), hydrochloric acid (2N) (1.10 mL, 2.20 mmol) was added, and the reaction mixture (cloudy) was stirred at room temperature for 30 min and checked by TLC (EtOAc / hexane, 1:4). The reaction was complete. The mixture was treated with triethylamine (0.5 mL), then all organic volatiles were removed in vacuo at 30 °C. The residue was treated with water (20 mL) and then extracted with MTBE (2 × 30 mL). The combined MTBE extracts were washed with water (1 × 20 mL), brine (1 × 10 mL), dried (NaSO), filtered, and concentrated in vacuo to give a viscous liquid (1.36 g). The crude product was chromatographed on silica gel (32 g) using ethyl acetate / hexane (5-35%) to give pure prostinyl benzyl ester side chain acetate (3) as a clear viscous liquid (0.18 g) and (0.95 g) (total, 1.13 g, 98.3%), 99.46% purity by HPLC, with spectral data (IR, 1 H NMR, 13 It was characterized by C NMR, DEPT-NMR and LCMS.

[0481] Synthesis of Treprostinil Side Chain Acetate (Prodrug XLIII) (4): Reaction scheme:

[0482] [ka]

[0483] [Table 49]

[0484] Testing Procedure: To a solution of treprostinil benzyl ester side chain acetic acid (3) (0.91 g, 1.74 mmol) in ethyl acetate (30 mL) was added palladium on carbon (5 wt %, -50% water) (0.24 g) at room temperature. The mixture was stirred, evacuated under house vacuum, and then replaced with hydrogen (packed in a balloon). This process was repeated three times. The reaction mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours and checked by TLC (4:6 EtOAc / hexane). The reaction was complete. The mixture was filtered through a pad of Celite (4.19 g) and the solid was washed with ethyl acetate (3 × 15 mL). The filtrate was concentrated under reduced pressure to give a foamy solid (0.74 g). The foamy solid was triturated with heptane (15 mL) and stirred overnight at room temperature. The white solid was collected in a Buchner funnel and washed with hexane (3 × 20 mL). The solid was air-dried under house vacuum, transferred into a vial, and dried under high vacuum to give pure prostinil side chain acetate salt (prodrug XLIII) (4) as a white solid (0.65 g, 86.3% purity, 99.74% by HPLC) with spectral data (IR, 1 H NMR, 13 The compound was characterized by CNMR, DEPT-NMR and LCMS.

[0485] Synthesis of TES-treprostinil benzyl ester side Reaction scheme:

[0486] [ka]

[0487] [Table 50]

[0488] Testing Procedure: A solution of mono-TES-treprostinil benzyl ester (1.83 g, 3.08 mmol) in anhydrous dichloromethane (30 mL) was added with benzyloxyacetic acid (0.49 mL, 3.39 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI.HCl) (1.48 g, 7.70 mmol), N-ethyldiisopropylamine (N,N-diisopropylethylamine) (DIEA) (1.34 mL, 7.70 mmol), and HCl (1.34 mL, 7.70 mmol). The reaction mixture was stirred at room temperature for 4 h and was checked by TLC (EtOAc / hexane, 1:4) to indicate near completion. The mixture was treated with water (10 mL) and extracted with dichloromethane (2 × 10 mL). The combined dichloromethane extracts were washed with brine (1 × 20 mL), dried (NaSO), filtered, and concentrated in vacuo to give the crude product (3.69 g). The crude product was chromatographed on a silica gel (70 g) column using ethyl acetate (1-10%) in hexane to give pure TES-treprostinil benzyl ester side chain benzyloxyacetic acid (5) (1.87 g, 82%), 97.75% purity by HPLC, and spectral data (IR, 1 H NMR, 13 It was characterized by CNMR and LCMS.

[0489] Synthesis of Treprostinil Benzyl Ester Side Chain Benzyloxyacetate (6): Reaction scheme:

[0490] [ka]

[0491] [Table 51]

[0492] Testing Procedure: To a solution of TES-treprostinil benzyl ester side chain benzyloxyacetic acid (5) (1.65 g, 2.22 mmol) in a mixture of tetrahydrofuran (40 mL) and water (8 mL) (5:1), hydrochloric acid (2 N) (1.10 mL, 2.20 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour and checked by TLC (EtOAc / hexane, 1:2). The reaction was complete. The mixture was treated with triethylamine (1 mL), water (20 mL), and ethyl acetate (20 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate (2 × 10 mL). The combined ethyl acetate extracts were washed with brine (1 × 20 mL), dried (NaSO), filtered, and concentrated under reduced pressure to give the crude product (2.30 g). The crude product was chromatographed on a silica gel (50 g) column using ethyl acetate (2-30%) in hexane to give pure prostinyl benzyl ester side chain benzyloxyacetic acid (6) (1.42 g, 100%), 99.33% purity by HPLC, and spectral data (IR, 1 H NMR, 13 It was characterized by CNMR and LCMS.

[0493] Synthesis of Treprostinil Side Chain Hydroxyacetic Acid (Prodrug XLIV) (7): Reaction scheme:

[0494] [ka]

[0495] [Table 52]

[0496] Testing Procedure: To a solution of treprostinil benzyl ester side-chain benzyloxyacetic acid (6) (1.25 g, 1.99 mmol) in ethyl acetate (40 mL) and water (2 mL) was added palladium on carbon (5 wt %, -50% water) (0.30 g) at room temperature. The mixture was stirred, evacuated under house vacuum, and then replaced with hydrogen (packed in a balloon). This process was repeated three times. The reaction mixture was stirred under a hydrogen atmosphere at room temperature for 3 hours and checked by TLC (EtOAc, 100%). The reaction was complete. The mixture was filtered through a pad of Celite (2.0 g), and the solid was washed with ethyl acetate (2 × 10 mL) and a mixture of ethyl acetate (8 mL) and water (2 mL). The filtrate was concentrated in vacuo to give treprostinil side-chain hydroxyacetic acid (prodrug XLIV) (7) (0.81 g, 92%), 97.57% pure by HPLC, and spectral data (IR, 1 H NMR, 13 It was characterized by CNMR and LCMS.

[0497] [Table 53]

[0498] [Table 54]

[0499] Example 3: Pharmacokinetic study Prodrugs IV, XVI, XVII, and VI: Pharmacokinetic evaluation after a single oral gavage or intravenous administration in Sprague Dawley rats summary The objective of this study was to evaluate the pharmacokinetic profiles of prodrugs IV, XVI, XVII, and VI when administered as a single oral (gavage) or intravenous injection (IV bolus) to male Sprague Dawley rats.

[0500] The study design was as follows:

[0501] [Table 55]

[0502] a) Dose calculated from body weight. b) Salt, purity, and water correction. Correction factor for prodrug IV: 1.016 c) Salt, purity, and water correction. Correction factor for prodrug XVI: 1.009 d) Salt, purity, and water correction. Correction factor for prodrug XVII: 1.013 e) Salt, purity, and moisture correction. Correction factor for prodrug VI: 1.002

[0503] Animals were dosed with a single dose by oral gavage or intravenous (bolus) injection. The study evaluated the following parameters and endpoints for clinical signs and pharmacokinetic parameters: One male in the oral 50 mg / kg prodrug XVII and VI groups died on day 2. No macroscopic findings were observed. All other animals survived until scheduled euthanasia.

[0504] All oral 50 mg / kg groups (Groups 3, 6, 9, and 12) and all intravenous bolus 1 mg / kg groups (Groups 13, 14, 15, and 16) showed test article-related clinical findings, including decreased activity, cold sensation, red skin on the skull / forelimbs / hindlimbs, no fur fur grooves, recumbent position, and ocular discharge.

[0505] Based on the results of this study, single oral or intravenous bolus administration of Pro IV, XVI, XVII, and VI at doses of 1, 10, and 50 mg / kg to Crl:CD(SD) rats resulted in lethality at 50 mg / kg oral dose of prodrug XVII and 50 mg / kg oral dose of prodrug Pro VI, and no adverse clinical findings at 1 mg / kg intravenous bolus and 50 mg / kg oral doses for all four test substances.

[0506] Materials and Methods Test substance Prodrug IV (side-chain carbonate ester prodrug of treprostinil). Properties: White powder. Purity: 99.4% Moisture 0.97% Correction factor: 1.016 Storage conditions: Store in a refrigerator set to maintain temperature at 4°C and protect from light. Prodrug XVI (Side Chain Ethyl Carbonate of Treprostinil) Properties: white powder Purity: 99.1% Correction factor: 1.009 Storage conditions: Store in a refrigerator set to maintain 4°C and protected from light. Prodrug XVII (Side Chain Isopropyl Carbonate of Treprostinil) Properties: white powder Purity: 98.7% Correction factor: 1.013 Storage conditions: Store in a refrigerator set to maintain temperature at 4°C and protect from light. Prodrug VI (Treprostinil side chain phosphate ester) Properties: white powder Purity: 99.8% Correction factor: 1.002 Storage conditions: Store in a refrigerator set to maintain temperature at 4°C and protect from light.

[0507] Vehicles for Groups 1-9 and 13-15 20 mM histidine, 125 mM NaCl

[0508] Vehicle Component Sterile water for injection Properties: clear, colorless liquid Stored in a temperature-controlled area maintained at 18-24°C L-Histidine, USP Storage conditions: Store in a temperature-controlled area maintained at 18-24°C. Sodium Chloride, United States Pharmacopoeia Appearance: White crystalline powder Storage conditions: Store in a temperature-controlled area maintained at 18-24°C.

[0509] Groups 10-12 and Group 16 vehicles 20 mM tribasic phosphate, 125 mM NaCl

[0510] Vehicle Component Sterile water for injection Properties: clear, colorless liquid Storage conditions: Store in a temperature-controlled area maintained at 18-24°C. Sterile water for injection Properties: clear, colorless liquid Stored in a temperature-controlled area maintained at 18-24°C Sodium phosphate tribasic anhydrous, FCC Properties: white powder Stored in a temperature-controlled area maintained at 18-24°C Sodium Chloride, United States Pharmacopoeia Storage conditions: Store in a temperature-controlled area maintained at 18-24°C.

[0511] Dosage prescription Solvent preparation Vehicle, 20 mM histidine, 125 mM NaCl (Groups 1-9 and 13-15) and 20 mM tribasic phosphate, 125 mM NaCl (Groups 10-12 and 16) were prepared on July 27 and 31, 2018, respectively, and refrigerated (2°C-8°C).

[0512] Preparation of test substances The test substance dosage formulations were prepared at appropriate concentrations to meet the required dose. The dosage formulations were prepared the day before each administration and stored protected from light (Groups 1-3, 13), at room temperature (18-24°C) (Groups 4-12, 14-16), or refrigerated (2-8°C) until use. The dosage formulations for Groups 1-12 were continuously stirred during the administration period.

[0513] Test system Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC. Animals were 9 weeks old and weighed 296-350 g at the start of dosing.

[0514] Crl:CD(SD) rats were selected as the animal model for this study because they are a rodent species accepted by regulatory agencies for preclinical toxicology testing. The total number of animals used in this study was considered the minimum number necessary to adequately characterize the effects of the test article. The study was designed to avoid requiring an unnecessary number of animals to achieve its objectives.

[0515] Currently, studies using laboratory animals provide the best available basis for extrapolation to humans and are necessary to support regulatory submissions. Currently, no acceptable models that do not use live animals exist.

[0516] Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). After receipt at the testing facility, Crl:CD(SD) rats were acclimated before the start of dosing.

[0517] Animals were assigned to groups by a stratified randomization design designed to achieve similar group mean body weights. Animals were group-housed (2-3 animals per dose group) in solid-bottom cages with appropriate bedding equipped with automatic water valves. Animals were segregated during designated procedures / activities. Each cage was clearly labeled with a color-coded cage card bearing the study number, group number, dose, animal number, and sex. Cages were arranged in group order on racks. Animals were housed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals, Committee for the Update of the Guide for the Care and Use of Laboratory Animals, and Institute for Laboratory Animals. Charles River Ashland's animal facility is accredited by AAALAC International.

[0518] environmental conditions The target temperature was maintained at 68-78°F (20-26°C), and the relative target humidity was maintained at 30-70%. A 12-hour light / 12-hour dark cycle was maintained except when interrupted for a given procedure. A minimum of 10 air changes per hour was maintained in the animal room with 100% fresh air (no recirculation of air).

[0519] food PMI Nutrition International, LLC Certified Rodent Chow LabDiet (登録商標) 5CR 4 The rats were given free access to 100g of corn meal (meal) throughout the study. The supplier analyzed the feed for nutritional content and environmental contaminants. The analysis results were provided by the supplier and are kept at the study facility. There were no known contaminants in the feed that would interfere with the objectives of this study.

[0520] water Municipal tap water, treated with reverse osmosis, was available ad libitum to each animal via an automated water supply system. Water analysis was performed periodically, and the results were kept in the testing facility. No known contaminants that could interfere with the results of the study were considered to be present in the water.

[0521] Animal abundance Animals were socially housed to provide psychological / environmental enrichment and were environmentally enriched as needed to help maintain the animals' oral health.

[0522] Veterinary Care Veterinary care was available throughout the study, but no examinations or treatments were required.

[0523] [Table 56]

[0524] a) Dose calculated from body weight. b) Salt, purity, and water correction. Correction factor for prodrug IV: 1.016 c) Salt, purity, and water correction. Correction factor for prodrug XVI: 1.009 d) Salt, purity, and water correction. Correction factor for prodrug XVII: 1.013 e) Salt, purity, and moisture correction. Correction factor for prodrug VI: 1.002

[0525] Test article formulations were administered as a single dose by oral gavage or intravenous bolus injection. To assess the bioavailability of each test article, the route of administration was oral (gavage) or intravenous injection (IV bolus).

[0526] The dose levels in this study were exploratory. Prodrugs XVI, XVII, and VI were not administered to animals. Intravenous prodrugs were not administered orally or intravenously to rats. These test substances are prodrugs of the active metabolite, treprostinil. Treprostinil is a tricyclic benzidine analog of naturally occurring prostacyclin. Prostacyclin is endogenously produced by the vascular endothelium and possesses potent vasodilatory, antiplatelet, and antiproliferative activity, particularly in the cardiovascular system and smooth muscle. Dose levels were selected based on previous concentrations from treprostinil and provided quantifiable plasma concentrations of the parent compound and active metabolite, treprostinil, in each group without causing animal compulsion to recover.

[0527] During the study, animals were observed twice daily for general health / mortality and moribundity, once in the morning and once in the afternoon. Animals were not to be removed from their cages during the observation period, except as necessary for identification or confirmation of possible findings.

[0528] observation Animals were removed from their cages for detailed clinical observations on the day of animal selection. Cage-side observations were performed 1-2 hours after dosing. Animals were weighed individually upon receipt, on the day of randomization, and on each dosing day (pre-dose). Individual weights are shown below.

[0529] Bioanalysis and Pharmacokinetic Evaluation Blood was collected from the jugular vein into chilled tubes containing NaF / KOx. Samples were taken according to the table below.

[0530] [Table 57]

[0531] Blood samples were kept on wet ice during collection and processing. Plasma was separated in a refrigerated centrifuge and stored in a freezer set to maintain a temperature of -70°C. Plasma samples for analysis were shipped on dry ice via overnight courier.

[0532] Bioanalysis of plasma samples to measure prodrug (parent) and treprostinil (metabolite) concentrations was performed using qualified analytical methods. Data collection and analysis were performed using the Watson Laboratory Information Management System (LIMS) and Microsoft Excel.

[0533] Pharmacokinetic parameters were estimated using Phoenix pharmacokinetic software (Certara, USA) using a non-compartmental approach consistent with the route of administration. All parameters were generated from individual plasma concentrations across all sample days. Parameters were estimated using the nominal sampling times for each dose and nominal dose unless otherwise specified. Whenever possible, plasma concentration values ​​obtained at pre-dose time points were used to estimate concentrations at time 0. Concentration values ​​reported as not quantifiable (BQL) were assigned a value of zero.

[0534] The area under the concentration versus time curve (AUC) was calculated using the linear trapezoidal method with linear interpolation. AUC was not calculated for pharmacokinetic profiles with fewer than three quantifiable concentrations of the test article at separate time points. Where practical, C maxAt least the last three observed concentration values, not including the α, were used to identify the terminal elimination phase of each concentration versus time curve. The slope of the terminal elimination phase was determined using log-linear regression on the unweighted concentration data. If the coefficient of determination was less than 0.800 or the extrapolation of the AUC to infinity was more than 20% of the total area, the parameter on which the determination of the terminal elimination phase depended was not reported.

[0535] [Table 58]

[0536] Terminal Procedure All surviving animals were euthanized and discarded. Animals that died during the study were necropsied and certain tissues were preserved.

[0537] result One male (no. 9004) in the 50 mg / kg prodrug XVI group and one male (no. 12003) in the 50 mg / kg prodrug XVII group died on day 2. No gross findings were observed. All other animals survived until scheduled euthanasia.

[0538] All oral 50 mg / kg groups (Groups 3, 6, 9, and 12) and all intravenous bolus 1 mg / kg groups (Groups 13, 14, 15, and 16) showed test article-related clinical findings, including decreased activity, cold sensation, red skin on the skull / forelimbs / hindlimbs, no fur fur, recumbent position, and ocular discharge. These findings were considered to be exaggerated pharmacological effects and were consistent with the pharmacodynamic response (vasodilation) of the test article.

[0539] Pharmacokinetic evaluation

[0540] [Table 59]

[0541] Prodrug intravenous-oral administration (Groups 1, 2, and 3) Because the plasma concentrations of prodrug IV were all BLQ at 1 mg / kg prodrug IV, the following discussion of prodrug IV is based on data from the 10 and 50 mg / kg dose groups only.

[0542] The variability in mean plasma concentrations of ProIV following single oral administration of the prodrug to animals in Groups 2 and 3 ranged from 40.1% to 200%, as measured by CV values. The higher variability observed was the result of BLQ values, which were converted to zero for parameter estimation and averaged over quantifiable results. The variability in mean plasma concentrations of ProIV following single oral administration of the prodrug to animals in Groups 2 and 3, which do not represent these values, ranged from 40.1% to 51.3%. Intravenous prodrug administration was quantifiable up to 1 hour after administration at 10 mg / kg and up to 1, 2, 8, and 12 hours after administration at 50 mg / kg. Individual peak plasma concentrations of the prodrug were observed by 0.5 hours after administration at 10 mg / kg and by 0.5 or 1 hour after administration at 50 mg / kg.

[0543] After a single oral dose of the prodrug to animals in groups 2 and 3, the mean C of the prodrug IV max The values ​​increased as the dose increased from 10 mg / kg to 50 mg / kg. A five-fold increase in the intravenous prodrug dose (10-50 mg / kg) resulted in a significant increase in the intravenous prodrug C max The mean AUC for intravenous administration of the prodrug increased by approximately 4.6 times. 0-24hr was reported in only one case at 50 mg / kg (animal no. 3003), and was 49.2 hr*ng / mL.

[0544] AUC of intravenous prodrug INF , T 1 / 2 , Cl / F, and V z The values ​​of / F were reported for only one animal at 50 mg / kg (animal no.): 65.4 hr*ng / mL, 4.27 hr, 764000 mL / hr / kg, and 471000 mL / kg.

[0545] Intravenous prodrug - IV bolus injection (Group 13) The variability of mean plasma concentrations of ProIV after a single bolus intravenous injection of the IV prodrug in animals in Group 13 ranged from 53.0% to 120%, as measured by CV values. The IV prodrug was quantifiable up to 1 or 2 hours after administration, and the estimated concentration at time 0 (C0) was measured. However, in two males (animal no.), the IV prodrug concentration increased during the 0.083-0.5 hour sampling interval, resulting in a C0. max Both T°C values ​​were reported: 13002 and 13004. The respective peak plasma concentrations of the prodrugs were observed by 0.083 or 0.5 hours post-dose.

[0546] After a single bolus intravenous injection of prodrug IV in Group 13 animals, the mean C0, C max , and AUC 0-12hr The values ​​were 324 ng / mL, 179 ng / mL, and 94.4 h*ng / mL, respectively. Individual C0 values ​​were 65.6-759 ng / mL, and individual C max Values ​​ranged from 150 to 209 ng / mL, with individual AUC 0-12hr Values ​​ranged from 42.3-155 hr*ng / mL.

[0547] Mean AUC of IV prodrug after a single bolus dose of IV prodrug INF , T 1 / 2 , Cl, V z The individual AUCs were 98.7 h*ng / mL, 0.212 h, 13400 mL / hr / kg, and 3150 mL / kg, respectively. INF Values ​​range from 41.5 to 144 hr*ng / mL, individual T 1 / 2 values ​​were 0.0910-0.276 h, individual Cl values ​​were 6940-24100 mL / hr / kg, and individual V z The values ​​were 2690-3610 mL / kg.

[0548] Bioavailability of prodrug IV Dose-normalized systemic exposure (AUC all / Dose) was lower after a single oral dose of 50 mg / kg of the prodrug intravenously compared with a single bolus dose of 1 mg / kg of the prodrug intravenously. all The oral bioavailability (%F) based on the dose was 1.04% at 50 mg / kg of the prodrug administered intravenously.

[0549] Oral administration of intravenous treprostinil prodrug (Groups 1, 2, and 3) After single oral doses of the prodrug to animals in Groups 1-3, the variability in mean treprostinil plasma concentrations, as measured by CV, ranged from 25.4% to 89.9%. Treprostinil was quantifiable up to 4 or 8 hours after administration of the 1 mg / kg prodrug intravenously and up to 24 hours after administration of the 10 and 50 mg / kg prodrug intravenously. Individual peak plasma concentrations were observed at 0.5 or 2 hours after administration of the 1 mg / kg prodrug intravenously and up to 0.5 hours after administration of the 10 and 50 mg / kg prodrug intravenously.

[0550] Mean C of treprostinil after a single oral dose of the prodrug in animals in Groups 1-3 max and AUC 0-24hr The values ​​increased with increasing dose. A 1:10:50 increase in the intravenous prodrug dose significantly increased the mean treprostinil C max This resulted in an approximately 1:8.4:23.3-fold increase in mean treprostinil AUC 0-24hr This resulted in an approximately 1:6.9:22.0-fold increase in values.

[0551] Systemic exposure to treprostinil (AUC 0-24hr ) was greater than the systemic exposure to intravenous prodrug after a single oral dose of 50 mg / kg of prodrug in animal no. 3003, M:AUC 0-24hr The ratio was 7.60 (AUC of M:P was 0.01 because of limited data available for intravenous prodrug administration). 0-24hr ratio was reported in only one case).

[0552] Secondary parameters of treprostinil (AUC INF , T1 / 2 , Cl / F, and V z / F) has an adjusted R of less than 0.9 2 Insufficient values ​​or plasma concentration-time data could be reported for any of the animals in groups 1 through 3.

[0553] Treprostinil-prodrug IV bolus injection (Group 13) After a single bolus injection of the prodrug IV in animals in Group 13, the variability of mean treprostinil plasma concentrations, as measured by CV values, ranged from 27.4% to 102%. Treprostinil was quantifiable up to 2 hours after administration, and an estimated concentration at time 0 (C0) was measured. However, an increase in treprostinil concentrations was observed during the 0.083-0.5 hour collection interval in two males (animal no. 1), resulting in a C0. max Both T and T°C values ​​were reported: 13002 and 13004. The respective peak plasma concentrations were observed by 0.083 or 0.5 hours post-dose.

[0554] After a single bolus of intravenous prodrug administration to animals in Group 13, the mean C0, C1 of treprostinil max , and AUC 0-12hr The values ​​were 538 ng / mL, 363 ng / mL, and 240-hour* ng / mL, respectively. Individual C0 values ​​ranged from 208-1000 ng / mL, and individual C max Values ​​ranged from 199 to 581 ng / mL, with individual AUC 0-12hr The values ​​were 153-367hr*ng / mL.

[0555] Systemic exposure to treprostinil (AUC 0-12hr ) was greater than the systemic exposure to the IV prodrug after a single bolus injection of 1 mg / kg IV prodrug. Mean M:P AUC 0-12hr The ratio was 2.94, and the individual M:P AUC 0-12hr The ratio was 1.40-3.64.

[0556] Mean AUC of treprostinil after a single bolus intravenous administration of the prodrug INF , T 1 / 2 , Cl, V zThe individual AUCs were 217 h*ng / mL, 0.332 h, 4980 mL / hr / kg, and 2350 mL / kg, respectively. INF Values ​​range from 144-312hr*ng / mL, individual T 1 / 2 values ​​were 0.234-0.408 h, individual Cl values ​​were 3200-6930 mL / hr / kg, and individual V z The values ​​were 1530-3300 mL / kg.

[0557] Bioavailability of treprostinil after intravenous administration of the prodrug. Dose-normalized systemic exposure (AUC all / Dose) was lower after a single oral dose of intravenous prodrug compared with a single bolus injection. all Oral bioavailability (%F) values ​​based on dose / dose were 6.75%, 4.63%, and 2.98% for 1, 10, and 50 mg / kg prodrug intravenously, respectively.

[0558] Oral administration of treprostinil-prodrug XVI (Groups 4, 5, and 6) When animals in Groups 4 through 6 received a single oral dose of prodrug XVI, the variability in mean treprostinil plasma concentrations, as measured by CV values, ranged from 15.7% to 200%. The higher variability observed was the result of BLQ values, which were transformed to zero for parameter estimation and averaged over quantifiable results. When animals in Groups 4 through 6 received a single oral dose of prodrug XVI, the variability in mean plasma treprostinil concentrations ranged from 15.7% to 120% in the absence of these values. Treprostinil was quantifiable up to 1, 8, and 12 hours after dosing with 1 mg / kg prodrug XVI and up to 24 hours after dosing with 10 and 50 mg / kg prodrug XV. The individual peak plasma concentrations were observed at 0.5 or 4 hours after administration for 1 mg / kg prodrug XVI, 0.5, 1, or 4 hours after administration for 10 mg / kg prodrug XV, and 0.5 or 1 hour after administration for 50 mg / kg prodrug XVI.

[0559] Mean C of treprostinil after a single oral dose of prodrug XVI to animals in Groups 4-6 max and AUC 0-24hr The mean treprostinil C values ​​increased with increasing dose. Increasing the dose of prodrug XV by 1:10:50 max The mean treprostinil AUC values ​​increased approximately 1:7.9:25.4-fold. 0-24hr The values ​​increased approximately 1:10.7:34.7-fold. The M:P ratio could not be determined due to insufficient data available for prodrug XVI (all prodrug XVI plasma concentrations were BLQ). The secondary parameters of treprostinil (AUC INF , T 1 / 2 , Cl / F, and V z / F) has an adjusted R of less than 0.9 2 Insufficient values ​​or plasma concentration-time data could be reported for any animals in groups 4 through 6.

[0560] Intravenous bolus injection of treprostinil-prodrug XVI (Group 14) After a single bolus intravenous injection of prodrug XVI in animals from Group 14, the variability of mean treprostinil plasma concentrations, as measured by CV values, ranged from 15.2% to 92.8%. Treprostinil was quantifiable up to 2 hours after administration, and an estimated concentration at time 0 (C0) was measured; however, an increase in treprostinil concentrations was observed in one male (animal no.) between the 0.083- and 0.5-hour sampling intervals, resulting in a C0. max Both T and T°C values ​​were reported. 14003. Individual peak plasma concentrations were observed by 0.083 or 0.5 hours post-dose.

[0561] After a single bolus injection of prodrug XVI in Group 14 animals, the mean C0, C1 of treprostinil max , and AUC 0-12hr The values ​​were 549 ng / mL, 263 ng / mL, and 192 h*ng / mL, respectively. Individual C0 values ​​ranged from 21.4 to 1170 ng / mL, and individual C max Values ​​ranged from 63.7 to 504 ng / mL, with individual AUC 0-12hrThe values ​​were 171 and 214 hr*ng / mL. The M:P ratio could not be determined due to insufficient data available for prodrug XVI (all prodrug XVI plasma concentrations were BLQ). AUC of treprostinil INF , Cl, and V z The mean T values ​​for treprostinil were 183 hr*ng / mL, 5470 mL / hr / kg, and 5700 mL / kg, respectively, after a single oral dose of prodrug XVI (animal no. 1). 1 / 2 The value is the time (individual T 1 / 2 The values ​​were 0.722 and 1.10 hours.

[0562] Treprostinil bioavailability after administration of prodrug XVI Dose-normalized systemic exposure (AUC all / Dose) was lower after a single oral dose of prodrug XVI compared with a single intravenous bolus injection of prodrug XVI. all Oral bioavailability (%F) values ​​based on dose / dose were 5.94%, 6.35%, and 4.13% at 1, 10, and 50 mg / kg, respectively.

[0563] Oral administration of treprostinil-prodrug XVII (Groups 7, 8, and 9) Following single oral doses of prodrug XVII in Groups 7-9, the variability in mean treprostinil plasma concentrations, as measured by CV, ranged from 4.98% to 174%. Treprostinil was quantifiable up to 8 hours after administration of 1 mg / kg prodrug XVI, up to 24 hours after administration of 10 mg / kg prodrug XVI, and up to 12 or 24 hours after administration of 50 mg / kg prodrug XVII. Individual peak plasma concentrations were observed up to 0.5 or 2 hours after administration of 10 mg / kg prodrug XVII, 0.5 or 24 hours after administration of 10 mg / kg prodrug XVII, and 0.5 or 8 hours after administration of 50 mg / kg prodrug XVI.

[0564] Mean C of treprostinil after a single oral dose of prodrug XVII in animals in Groups 7-9 max and AUC 0-24hr The mean treprostinil C values ​​increased with increasing dose. Increasing the dose of prodrug XVII by 1:10:50 fold max The mean treprostinil AUC values ​​increased approximately 1:2.5:21.6-fold. 0-24hr The values ​​increased approximately 1:4.1:24.0-fold. The M:P ratio could not be determined due to insufficient data for prodrug XVII (all prodrug XVII plasma concentrations were BLQ). Secondary parameters of treprostinil (AUC INF , T 1 / 2 , Cl / F, and V z / F) has an adjusted R of less than 0.9 2 Insufficient values ​​or plasma concentration-time data could not be reported for any animals in Groups 7 through 9.

[0565] Bolus intravenous injection of treprostinil-prodrug XVII (Group 15) After a single intravenous bolus injection of prodrug XVII into animals in Group 15, the variability of mean treprostinil plasma concentrations, as measured by CV values, ranged from 42.1% to 200%. The higher variability observed was a result of BLQ values, which were converted to zero for parameter estimation and averaged over quantifiable results. After a single intravenous bolus injection of prodrug XVII into animals in Group 15, the variability of mean treprostinil plasma concentrations in the absence of these values ​​ranged from 42.1% to 81.1%. Treprostinil was quantifiable up to 2 or 8 hours post-dose, and an estimated concentration at time 0 (C0) was measured; however, an increase in treprostinil concentrations was observed during the 0.083-0.5 hour collection interval for one male (animal no.), resulting in a C0. max Both T and T°C values ​​were reported. 15002). Individual peak plasma concentrations were observed by 0.083 or 0.5 hours post-dose.

[0566] After a single intravenous bolus injection of prodrug XVII in Group 15 animals, the mean C0, C1 of treprostinilmax , and AUC 0-12hr The values ​​were 1350 ng / mL, 466 ng / mL, and 274 hr*ng / mL, respectively. The individual C0 values ​​were 227-320 ng / mL, and the individual C max Values ​​ranged from 254 to 917 ng / mL, with individual AUC 0-12hr Values ​​ranged from 167-413 hr*ng / mL. The M:P ratio could not be determined due to insufficient data for prodrug XVII (all prodrug XVII plasma concentrations were BLQ). Secondary parameters of treprostinil (AUC INF , T 1 / 2 , Cl, V z ) has an adjusted R of less than 0.9 2 Due to the values, it was not possible to report on any of the animals in the 15 groups.

[0567] Treprostinil bioavailability after administration of prodrug XVII Dose-normalized systemic exposure (AUC all The AUC (AUC / Dose) was lower after a single oral dose of prodrug XVII compared with a single intravenous bolus injection of prodrug XVII. all The oral bioavailability (%F) values ​​based on the dose / dose were 7.74%, 3.17%, and 3.72% for prodrug XVI at 1, 10, and 50 mg / kg, respectively.

[0568] Prodrug VI - Oral Administration (Groups 10, 11, and 12) All plasma concentrations of prodrug VI were BLQ for 1 and 10 mg / kg prodrug VI. Therefore, the following discussion of prodrug VI is based on data from only the 50 mg / kg prodrug VI group. When animals in Group 12 received a single oral dose of prodrug VI, the variability in mean plasma concentrations of prodrug VI, as measured by CV values, ranged from 38.4% to 173%. The higher variability observed was the result of BLQ values ​​that were converted to zero for parameter estimation and averaged over quantifiable results. When animals in Group 12 received a single oral dose of prodrug XVII, the variability in mean plasma concentrations of prodrug VI, which lacked these values, ranged from 38.4% to 69.8%. Prodrug VI was quantifiable up to 2, 4, 8, or 24 hours post-dose for 50 mg / kg prodrug VI. Peak plasma concentrations of prodrug VI were observed up to 0.5, 8, or 24 hours post-dose for 50 mg / kg prodrug VI.

[0569] Group 12 animals received a single oral dose of 50 mg / kg of prodrug VI, and the mean C max and AUC 0-24hr The C values ​​were 12.4 ng / mL and 242 hr*ng / mL, respectively. max The values ​​ranged from 4.14–32.8 ng / mL for 50 mg / kg of prodrug VI, with individual AUC 0-24hr The values ​​were 26.5 and 457 hr*ng / mL for prodrug VI at 50 mg / kg. AUC INF , T 1 / 2 , Cl / F, and V z The F values ​​were reported for only one animal at 50 mg / kg (animal no.): 28.2 hr ng / mL, 1.76 hr, 1.78 million mL / hr / kg, and 4.52 million mL / kg.

[0570] Oral administration of treprostinil-prodrug VI (Groups 10, 11, and 12) Following single oral doses of prodrug VI to animals in Groups 10-12, the variability in mean treprostinil plasma concentrations, as measured by CV, ranged from 13.8% to 150%. Treprostinil was quantifiable up to 24 hours after dosing for 1 and 10 mg / kg prodrug VI and up to 8 or 24 hours after dosing for 50 mg / kg prodrug VI. Individual peak plasma concentrations were observed at 1, 2, or 4 hours after dosing for 1 mg / kg prodrug VI, 1 or 2 hours after dosing for 10 mg / kg prodrug XVII, and 2, 8, or 12 hours after dosing for 50 mg / kg prodrug VI.

[0571] Mean C of treprostinil after a single oral dose of prodrug VI in animals in Groups 10-12 max and AUC 0-24hr The values ​​increased with increasing dose. A 1:10:50 increase in the dose of prodrug VI resulted in a mean treprostinil C max The mean treprostinil AUC 0-24hr The values ​​increased by approximately 1:8.5:60.8 times.

[0572] Systemic exposure to treprostinil (AUC 0-24hr ) was greater than the systemic exposure to prodrug VI after a single oral dose of 50 mg / kg of prodrug VI to animal numbers 12002 and 12004, with M:P ratios of 12.1 and 30.0, respectively (M:AUC 0-24hr (Only two ratios could be reported.) The mean M:P ratio was 21.0.

[0573] Secondary parameters of treprostinil (AUC INF , T 1 / 2 , Cl / F, V z / F) has an adjusted R of less than 0.9 2 value, insufficient plasma concentration-time data, or AUC > 25% INF %AUC of ExtrapDue to the values, the mean AUC of treprostinil could not be reported for prodrug VI at 10 mg / kg or 50 mg / kg. INF , T 1 / 2 , Cl / F, and V z The AUC values ​​were 38.4 hr*ng / mL, 14.0 hr, 26900 mL / hr / kg, and 243000 mL / kg, respectively, for 1 mg / kg of prodrug VI. INF Values ​​range from 31.7 to 48.4 h*ng / mL, individual T 1 / 2 The values ​​ranged from 4.71 to 36.9 hours (T 1 / 2 The value of 36.9 hours is estimated to be less than the half-life of 3 hours, and should be observed with caution. 1 / 2 values ​​4.71-7.35 hours, otherwise individual Cl / F values ​​20700-31600 mL / hr / kg, individual V z The F value was 194,000-316,000 mL / kg.

[0574] Bolus intravenous injection of treprostinil-prodrug VI (Group 16) The variability of mean treprostinil plasma concentrations, as measured by CV values, ranged from 17.6% to 71.2% after a single bolus intravenous injection of prodrug VI to animals in Group 16. The higher variability observed was the result of BLQ values, which were converted to zero for parameter estimation and averaged over quantifiable outcomes. In the absence of these values, the variability of mean plasma treprostinil concentrations ranged from 17.6% to 48.4% after a single bolus intravenous injection of prodrug VI to animals in Group 16. Treprostinil was quantifiable for up to 8 or 12 hours after administration, and an estimated concentration at time 0 (C0) was determined.

[0575] Mean C0 and AUC of treprostinil after a single bolus injection of prodrug VI in Group 16 animals 0-12hr The values ​​were 277 ng / mL and 129 hr*ng / mL, respectively. The individual C0 values ​​ranged from 207 to 454 ng / mL, and the individual AUC 0-12hrValues ​​ranged from 97.4-158 hr*ng / mL. M:P ratios could not be determined due to insufficient data available (plasma concentrations of all prodrugs VI were at BLQ).

[0576] Mean AUC of treprostinil after a single intravenous bolus dose of prodrug VI INF , T 1 / 2 , Cl and V z The values ​​were 130 h*ng / mL, 1.90 h, 7920 mL / hr / kg, and 22000 mL / kg, respectively. INF Values ​​range from 99.0 to 159 hr*ng / mL, individual T 1 / 2 values ​​were 1.60-2.28 hours, individual Cl values ​​were 6290-10100 mL / hr / kg, and individual V z The values ​​were 16,800-33,200 mL / kg.

[0577] Treprostinil bioavailability after administration of prodrug VI Dose-normalized systemic exposure to treprostinil (AUC ) was significantly greater after a single oral dose of prodrug VI compared to a single intravenous bolus injection of prodrug VI. all / Dose) was low. AUC all Based on the dose / dose, oral bioavailability (%F) values ​​were 28.4%, 24.3% and 34.7% for prodrug XVII at 1, 10 and 50 mg / kg, respectively.

[0578] conclusion Based on the results of this study, single oral or intravenous bolus administration of Pro IV, XVI, XVII, and VI to Crl:CD(SD) rats at doses of 1, 10, and 50 mg / kg resulted in lethality with oral 50 mg / kg of prodrug XVII and oral 50 mg / kg of prodrug Pro VI, and adverse clinical findings with all four test substances at 1 mg / kg intravenous bolus and 50 mg / kg oral.

[0579] Example 4: Prodrugs IV, XVI, XVII and VI: Pharmacokinetic evaluation after a single oral gavage or intravenous administration in Sprague Dawley rats List of Abbreviations Adjusted goodness-of-fit statistics for the terminal elimination phase (R 2 ) T last AUC extrapolated to 12 and / or 24 hours 0-24hr The proportion of and / or T last AUC extrapolated from to infinity INF Percentage of %AUC Extrap Area under the plasma concentration-time curve from 0 to 12 hours (bolus intravenous administration only) or area under the plasma concentration-time curve up to 24 hours (oral administration only) AUC all Dose-normalized area under the plasma concentration-time curve from 0 to 12 hours (bolus intravenous administration only) or up to 24 hours (oral administration only) AUC all / Dose Area under the plasma concentration-time curve from 0 to 12 hours AUC 0-12hr Area under the plasma concentration-time curve from 0 to 24 hours AUC 0-24hr Area under the plasma concentration-time curve from zero to infinity AUC INF AUC INF / Dose Area under the plasma concentration-time curve from zero to infinity based on the final predicted concentration normalized to the dose Area under the plasma concentration-time curve (from time 0 to the last quantifiable sample time) Tlast BLQ, BQL below the limit of quantification C0 Estimated concentration at time 0 C0 / Dose Estimated concentration at time 0 normalized to dose C max Maximum plasma concentration C max C max / Dose Maximum plasma concentration normalized by dose Cl total body clearance Cl / F Total body clearance divided by the fraction of absorbed dose CV Coefficient of variation in percent F Bioavailability, percentage of absorbed dose for intravenous administration Time Sampling interval, time after administration IV LLOQ Lower limit of quantification M:P metabolite / parent exposure ratio N The number of values ​​used to calculate the statistic NA Not Applicable PO Oral SD standard deviation T 1 / 2 Terminal half-life = ln(2) / λ z T last Time of last quantifiable sample T max Time to reach maximum plasma concentration V z Distribution volume by disappearing phase V z / F Distribution volume based on the final elimination phase divided by the absorbed dose rate λ z Terminal elimination rate constant

[0580] [Table 60]

[0581] [Table 61-1]

[0582] [Table 61-2]

[0583] Test Method Plasma concentration-time profiles of prodrugs IV, XVI, XVII, and VI, and individual prodrugs from animals treated with prodrugs IV, XVI, XVII, or VI were analyzed using a model-independent method. An intravenous bolus model was used to analyze the pharmacokinetic data for both the prodrug and treprostinil intravenous bolus groups due to the rapid conversion of the prodrugs to treprostinil. Pharmacokinetic parameters were obtained for each animal after a single oral or intravenous bolus administration of ProIV, XVI, XVII, or VI below the lower limit of quantitation (LLOQ < 2 ng / mL for prodrugs XVI and XVII, LLOQ < 1.00 ng / mL for prodrugs XVI and XVII, and < 0.2 ng / mL or < 1.00 ng / mL for treprostinil).

[0584] For each animal, the following pharmacokinetic parameters were measured: estimated concentration at time 0; (C0, IV bolus group only), maximum plasma concentration (C max ), time of maximum plasma concentration (T℃), area under the plasma concentration-time curve (AUC). AUC (AUC 0-12hr , IV bolus group only), time 0-24 hours AUC (AUC 0-24hr , PO group only), AUC from time 0 to the time of the last quantifiable sample (AUC Tlast ), AUC from time 0 to infinity (AUC INF ) was calculated by the linear trapezoidal method for all animals with at least three consecutive quantifiable concentrations. On Day 1, 0 was used as an estimate of the 0-hour concentration for the oral dose group. The terminal elimination phase (T max (at least 3 samples not containing α-glucan) had sufficient plasma concentrations and the adjusted R 2 For each plasma concentration-time profile of ≥ 0.9, the half-life value (T 1 / 2 ) were reported. Additional pharmacokinetic parameters were clearance (Cl, IV bolus group only), clearance divided by fraction of absorbed dose (Cl / F, PO group only), and volume of distribution (V z , IV bolus group only), and the volume of distribution divided by the fraction of absorbed dose (V zOral administration group (V z The secondary parameters of HCl / F or Cl / F were not normalized for the fraction of the dose absorbed.

[0585] The metabolite to parent ratio (M:P), where appropriate, was calculated for each animal using the following formula: Intravenous bolus group: M:P = AUC 0-12hr Treprostinil ÷ AUC 0-12hr Intravenous prodrug, XVI, XVII, or VI Oral administration group: M:P = AUC 0-24hr Treprostinil ÷ AUC 0-24hr Prodrug IV, XVI, XVII or VI T last If is not equal to the last sampling interval, AUC 0-12hr (IV bolus administration group) or AUC 0-24hr (Oral administration group) Extrapolated AUC% (%AUC Extrap ) was calculated as follows: AUC 0-12hr =[(AUC 0-12hr -AUC Tlast ) / AUC 0-12hr ]×AUC 0-24hr =[(AUC 0-24hr -AUC Tlast ) / AUC Tlast ]×100% AUC INF Extrapolation rate (%AUC Extrap ) was calculated as follows: %AUC Extrap =[(AUC INF -AUC Tlast ) / AUC INF ] x 100 AUC values ​​and %AUC calculated with an extrapolation rate exceeding 25% Extrap The value is not reported but is stored in the test file.

[0586] Data exclusion No data exclusion was performed for pharmacokinetic data analysis.

[0587] information After a single bolus injection of ProVI, XVII, and VI. Because the plasma concentrations of prodrugs XVI, XVII, and VI were all at the base quotient (BQL) (<1.00 ng / mL), the mean plasma concentrations and pharmacokinetic parameters of prodrugs XVI, XVII, and VI were not reported or discussed for groups 14, 15, and 16.

[0588] After a single oral dose of prodrug XVI or XVII, the plasma concentrations of all prodrugs XVI or XVII were at the BQL (<1.00 ng / mL), and therefore, the mean plasma concentrations and pharmacokinetic parameters of prodrugs XVI and XVII were not reported or discussed in groups 4 to 9.

[0589] After a single intravenous bolus injection or oral administration of prodrug IV or VI, the majority of the plasma concentrations of prodrug IV or VI were at the BQL (<2.00 ng / mL); therefore, for informational purposes, mean plasma concentrations and pharmacokinetic parameters are reported, as appropriate, as follows for Groups 1-3 (prodrug IV), 10-12 (prodrug VI), and 13 (prodrug IV):

[0590] The plasma concentration-time profiles of the intravenous prodrug and treprostinil were observed in two males receiving 1 mg / kg of the intravenous prodrug (animal no. 13002 and 13004). The plasma concentration-time profile of treprostinil in one male receiving 1 mg / kg of the prodrug XV (animal no. 13002). In one male receiving 1 mg / kg of the prodrug XVII (animal no. 13002), and one male receiving 1 mg / kg of the prodrug XVII (animal no. 13002), the plasma concentrations of the intravenous prodrug or treprostinil increased over the 0.083-0.5 hour sampling interval. The increases observed in the aforementioned animals may be due to the C0C max Both T°C and T°C were reported.

[0591] One male animal receiving 50 mg / kg of prodrug XVII (animal no. 9003) was confirmed dead before the 24-hour collection period, and therefore no samples were collected.

[0592] No samples were obtained from one male at 50 mg / kg prodrug XVII (animal no. 12003). The dose was administered 4 hours after dosing due to the animal's struggle during blood collection. Animal no. 12003 died before the 12-hour collection, so no samples were collected at 12 or 24 hours post-dose.

[0593] Due to sample clotting, the following samples taken 12 hours after administration were not analyzed. Group 3 (50 mg / kg prodrug intravenous injection): Male animal number 3001 Group 5 (10 mg / kg of prodrug XVI): Male animal number 5002 Group 10 (1 mg / kg of prodrug XVII): Male animal number 10003 Group 11 (10 mg / kg of prodrug VI): Male animal number 11004

[0594] result The mean prodrug IV and treprostinil plasma concentration-time profiles are shown in Figures 2 and 5A-B, and the mean treprostinil plasma concentration-time profiles are shown in Figures 6 (prodrug XVI) and 3 / 7 (prodrug XVII). The mean prodrug VI and treprostinil plasma concentration-time profiles are shown in Figures 8A-B and 4.

[0595] Consideration Prodrug intravenous-oral administration (Groups 1, 2, and 3) Because the plasma concentrations of prodrug IV were all BLQ at 1 mg / kg prodrug IV, the following discussion of prodrug IV is based on data from the 10 and 50 mg / kg dose groups only.

[0596] The variability in mean plasma concentrations of ProIV following single oral administration of the prodrug to animals in Groups 2 and 3 ranged from 40.1% to 200%, as measured by CV values. The higher variability observed was the result of BLQ values, which were converted to zero for parameter estimation and averaged over quantifiable results. The variability in mean plasma concentrations of ProIV following single oral administration of the prodrug to animals in Groups 2 and 3, which do not represent these values, ranged from 40.1% to 51.3%. Intravenous prodrug administration was quantifiable up to 1 hour after administration at 10 mg / kg and up to 1, 2, 8, and 12 hours after administration at 50 mg / kg. Individual peak plasma concentrations of the prodrug were observed by 0.5 hours after administration at 10 mg / kg and by 0.5 or 1 hour after administration at 50 mg / kg.

[0597] After a single oral dose of the prodrug to animals in groups 2 and 3, the mean C of the prodrug IV max The values ​​increased as the dose increased from 10 mg / kg to 50 mg / kg. A five-fold increase in the intravenous prodrug dose (10-50 mg / kg) resulted in a significant increase in the intravenous prodrug C max The mean AUC for intravenous administration of the prodrug increased by approximately 4.6 times. 0-24hr The values ​​were reported in only one case at 50 mg / kg (animal no.), and were 3003 and 49.2 hr*ng / mL.

[0598] AUC of intravenous prodrug INF , T 1 / 2 , Cl / F, and V z The values ​​of / F were reported for only one animal at 50 mg / kg (animal no.): 65.4 hr*ng / mL, 4.27 hr, 764000 mL / hr / kg, and 471000 mL / kg.

[0599] Intravenous prodrug - IV bolus injection (Group 13) The variability of mean plasma concentrations of ProIV after a single bolus intravenous injection of the IV prodrug in animals in Group 13 ranged from 53.0% to 120%, as measured by CV values. The IV prodrug was quantifiable up to 1 or 2 hours after administration, and the estimated concentration at time 0 (C0) was measured. However, in two males (animal no.), the IV prodrug concentration increased during the 0.083-0.5 hour sampling interval, resulting in a C0. max Both T°C values ​​were reported: 13002 and 13004. The respective peak plasma concentrations of the prodrugs were observed by 0.083 or 0.5 hours post-dose.

[0600] After a single bolus intravenous injection of prodrug IV in Group 13 animals, the mean C0, C max , and AUC 0-12hr The values ​​were 324 ng / mL, 179 ng / mL, and 94.4 h*ng / mL, respectively. Individual C0 values ​​were 65.6-759 ng / mL, and individual C max Values ​​ranged from 150 to 209 ng / mL, with individual AUC 0-12hr Values ​​ranged from 42.3-155 hr*ng / mL.

[0601] Mean AUC of IV prodrug after a single bolus dose of IV prodrug INF , T 1 / 2 , Cl, V z The individual AUCs were 98.7 h*ng / mL, 0.212 h, 13400 mL / hr / kg, and 3150 mL / kg, respectively. INF Values ​​range from 41.5 to 144 hr*ng / mL, individual T 1 / 2 values ​​were 0.0910-0.276 h, individual Cl values ​​were 6940-24100 mL / hr / kg, and individual V z The values ​​were 2690-3610 mL / kg.

[0602] Bioavailability of prodrug IV Dose-normalized systemic exposure (AUC all / Dose) was lower after a single oral dose of 50 mg / kg of the prodrug intravenously compared with a single bolus dose of 1 mg / kg of the prodrug intravenously. all The oral bioavailability (%F) based on the dose was 1.04% at 50 mg / kg of the prodrug administered intravenously.

[0603] Oral administration of intravenous treprostinil prodrug (Groups 1, 2, and 3) After single oral doses of the prodrug to animals in Groups 1-3, the variability in mean treprostinil plasma concentrations, as measured by CV, ranged from 25.4% to 89.9%. Treprostinil was quantifiable up to 4 or 8 hours after administration of the 1 mg / kg prodrug intravenously and up to 24 hours after administration of the 10 and 50 mg / kg prodrug intravenously. Individual peak plasma concentrations were observed at 0.5 or 2 hours after administration of the 1 mg / kg prodrug intravenously and up to 0.5 hours after administration of the 10 and 50 mg / kg prodrug intravenously.

[0604] Mean C of treprostinil after a single oral dose of the prodrug in animals in Groups 1-3 max and AUC 0-24hr The values ​​increased with increasing dose. A 1:10:50 increase in the intravenous prodrug dose significantly increased the mean treprostinil C max This resulted in an approximately 1:8.4:23.3-fold increase in mean treprostinil AUC 0-24hr This resulted in an approximately 1:6.9:22.0-fold increase in values.

[0605] Systemic exposure to treprostinil (AUC 0-24hr ) was greater than the systemic exposure to intravenous prodrug after a single oral dose of 50 mg / kg of prodrug in animal no. 3003, M:AUC 0-24hr The ratio was 7.60 (AUC of M:P was 0.01 because of limited data available for intravenous prodrug administration). 0-24hr ratio was reported in only one case).

[0606] Secondary parameters of treprostinil (AUC INF , T1 / 2 , Cl / F, and V z / F) has an adjusted R of less than 0.9 2 Insufficient values ​​or plasma concentration-time data could be reported for any of the animals in groups 1 through 3.

[0607] Treprostinil-lodrug IV bolus injection (Group 13) After a single bolus injection of the prodrug IV in animals in Group 13, the variability of mean treprostinil plasma concentrations, as measured by CV values, ranged from 27.4% to 102%. Treprostinil was quantifiable up to 2 hours after administration, and an estimated concentration at time 0 (C0) was measured. However, an increase in treprostinil concentrations was observed during the 0.083-0.5 hour collection interval in two males (animal no. 1), resulting in a C0. max Both T and T°C values ​​were reported: 13002 and 13004. The respective peak plasma concentrations were observed by 0.083 or 0.5 hours post-dose.

[0608] After a single bolus of intravenous prodrug administration to animals in Group 13, the mean C0, C1 of treprostinil max , and AUC 0-12hr The values ​​were 538 ng / mL, 363 ng / mL, and 240-hour* ng / mL, respectively. Individual C0 values ​​ranged from 208-1000 ng / mL, and individual C max Values ​​ranged from 199 to 581 ng / mL, with individual AUC 0-12hr The values ​​were 153-367hr*ng / mL.

[0609] Systemic exposure to treprostinil (AUC 0-12hr ) was greater than the systemic exposure to the IV prodrug after a single bolus injection of 1 mg / kg IV prodrug. Mean M:P AUC 0-12hr The ratio was 2.94, and the individual M:P AUC 0-12hr The ratio was 1.40-3.64.

[0610] Mean AUC of treprostinil after a single bolus intravenous administration of the prodrug INF , T 1 / 2 , Cl, V zThe individual AUCs were 217 h*ng / mL, 0.332 h, 4980 mL / hr / kg, and 2350 mL / kg, respectively. INF Values ​​range from 144-312hr*ng / mL, individual T 1 / 2 values ​​were 0.234-0.408 h, individual Cl values ​​were 3200-6930 mL / hr / kg, and individual V z The values ​​were 1530-3300 mL / kg.

[0611] Bioavailability of treprostinil after intravenous administration of the prodrug. Dose-normalized systemic exposure (AUC all / Dose) was lower after a single oral dose of the IV prodrug compared with a single bolus injection of the IV prodrug. all Oral bioavailability (%F) values ​​based on dose / dose were 6.75%, 4.63%, and 2.98% for 1, 10, and 50 mg / kg prodrug intravenously, respectively.

[0612] Oral administration of treprostinil-prodrug XVI (Groups 4, 5, and 6) When animals in Groups 4 through 6 received a single oral dose of prodrug XVI, the variability in mean treprostinil plasma concentrations, as measured by CV values, ranged from 15.7% to 200%. The higher variability observed was the result of BLQ values, which were transformed to zero for parameter estimation and averaged over quantifiable results. When animals in Groups 4 through 6 received a single oral dose of prodrug XVI, the variability in mean plasma treprostinil concentrations ranged from 15.7% to 120% in the absence of these values. Treprostinil was quantifiable up to 1, 8, and 12 hours after dosing for 1 mg / kg prodrug XVI and up to 24 hours after dosing for 10 and 50 mg / kg prodrug XVI. The individual peak plasma concentrations were observed at 0.5 or 4 hours after administration for 1 mg / kg prodrug VI, 0.5, 1, or 4 hours after administration for 10 mg / kg prodrug XVI, and 0.5 or 1 hour after administration for 50 mg / kg prodrug XVI.

[0613] Mean C of treprostinil after a single oral dose of prodrug XVI to animals in Groups 4-6 max and AUC 0-24hr The values ​​increased with increasing dose. A 1:10:50 increase in the dose of prodrug XVI resulted in a mean treprostinil C max The mean treprostinil AUC values ​​increased approximately 1:7.9:25.4-fold. 0-24hr The values ​​increased by approximately 1:10.7:34.7 times.

[0614] The M:P ratio could not be determined due to insufficient data available for prodrug XVI (plasma concentrations of all prodrug XVI were at BLQ).

[0615] Secondary parameters of treprostinil (AUC INF , T 1 / 2 , Cl / F, and V z / F) has an adjusted R of less than 0.9 2 Insufficient values ​​or plasma concentration-time data could be reported for any animals in groups 4 through 6.

[0616] Intravenous bolus injection of treprostinil-prodrug XVI (Group 14) After a single bolus intravenous injection of prodrug XVI in animals from Group 14, the variability of mean treprostinil plasma concentrations, as measured by CV values, ranged from 15.2% to 92.8%. Treprostinil was quantifiable up to 2 hours after administration, and an estimated concentration at time 0 (C0) was measured; however, an increase in treprostinil concentrations was observed in one male (animal no.) between the 0.083- and 0.5-hour sampling intervals, resulting in a C0. max Both T and T°C values ​​were reported. 14003. Individual peak plasma concentrations were observed by 0.083 or 0.5 hours post-dose.

[0617] After a single bolus injection of prodrug XVI in Group 14 animals, the mean C0, C1 of treprostinil max , and AUC 0-12hrThe values ​​were 549 ng / mL, 263 ng / mL, and 192 h*ng / mL, respectively. Individual C0 values ​​ranged from 21.4 to 1170 ng / mL, and individual C max Values ​​ranged from 63.7 to 504 ng / mL, with individual AUC 0-12hr The values ​​were 171 and 214 hr*ng / mL.

[0618] There was insufficient data for prodrug XV (all plasma concentrations of prodrug XVI were BLQ) to determine the M:P ratio.

[0619] AUC of treprostinil INF , Cl, and V z The values ​​could only be estimated after a single oral dose of prodrug XVI (animal no.) and were 183 hr*ng / mL, 5470 mL / hr / kg, and 5700 mL / kg, respectively. 1 / 2 The mean value of the individual T 1 / 2 The values ​​were 0.722 and 1.10 hours.

[0620] Treprostinil bioavailability after administration of prodrug XVI Dose-normalized systemic exposure (AUC all / Dose) was lower after a single oral dose of prodrug XVI compared with a single intravenous bolus injection of prodrug XVI. all The oral bioavailability (%F) values ​​based on / Dose were 5.94%, 6.35%, and 4.13% for prodrug XVI at 1, 10, and 50 mg / kg, respectively.

[0621] Oral administration of treprostinil-prodrug XVII (Groups 7, 8, and 9) Following single oral doses of prodrug XVII to animals in Groups 7-9, the variability in mean treprostinil plasma concentrations, as measured by CV, ranged from 4.98% to 174%. Treprostinil was quantifiable up to 8 hours after administration for 1 mg / kg prodrug XVII, up to 24 hours after administration for 10 mg / kg prodrug XVII, and up to 12 or 24 hours after administration for 50 mg / kg prodrug XVII. Individual peak plasma concentrations were observed up to 0.5 or 2 hours after administration for 1 mg / kg prodrug XVII, up to 0.5 or 24 hours after administration for 10 mg / kg prodrug XVII, and up to 0.5 or 8 hours after administration for 50 mg prodrug XVII.

[0622] Mean C of treprostinil after a single oral dose of prodrug XVII in animals in Groups 7-9 max and AUC 0-24hr The mean treprostinil C values ​​increased with increasing dose. Increasing the dose of prodrug XVII by 1:10:50 fold max The mean treprostinil AUC values ​​increased approximately 1:2.5:21.6-fold. 0-24hr The values ​​increased by approximately 1:4.1:24.0 times.

[0623] The M:P ratio could not be determined due to insufficient data for prodrug XVII (all prodrug XVII plasma concentrations were at BLQ).

[0624] Secondary parameters of treprostinil (AUC INF , T 1 / 2 , Cl / F, and V z / F) has an adjusted R of less than 0.9 2 Insufficient values ​​or plasma concentration-time data could not be reported for any animals in Groups 7 through 9.

[0625] Bolus intravenous injection of treprostinil-prodrug XVII (Group 15) After a single intravenous bolus injection of prodrug XVII into animals in Group 15, the variability of mean treprostinil plasma concentrations, as measured by CV values, ranged from 42.1% to 200%. The higher variability observed was a result of BLQ values, which were converted to zero for parameter estimation and averaged over quantifiable results. After a single intravenous bolus injection of prodrug XVII into animals in Group 15, the variability of mean treprostinil plasma concentrations in the absence of these values ​​ranged from 42.1% to 81.1%. Treprostinil was quantifiable up to 2 or 8 hours post-dose, and an estimated concentration at time 0 (C0) was measured; however, an increase in treprostinil concentrations was observed during the 0.083-0.5 hour collection interval for one male (animal no.), resulting in a C0. max Both T and T°C values ​​were reported. 15002. Individual peak plasma concentrations were observed by 0.083 or 0.5 hours post-dose.

[0626] After a single intravenous bolus injection of prodrug XVII in Group 15 animals, the mean C0, C1 of treprostinil max , and AUC 0-12hr The values ​​were 1350 ng / mL, 466 ng / mL, and 274 hr*ng / mL, respectively. The individual C0 values ​​were 227-320 ng / mL, and the individual C max Values ​​ranged from 254 to 917 ng / mL, with individual AUC 0-12hr Values ​​ranged from 167-413 hr*ng / mL.

[0627] The M:P ratio could not be determined due to insufficient data for prodrug XVII (all prodrug XVII plasma concentrations were at BLQ).

[0628] Secondary parameters of treprostinil (AUC INF , T 1 / 2 , Cl, V z ) has an adjusted R of less than 0.9 2 Due to the values, it was not possible to report on any of the animals in the 15 groups.

[0629] Treprostinil bioavailability after administration of prodrug XVII Dose-normalized systemic exposure to treprostinil (AUC ) was significantly greater after a single oral dose of prodrug XVII compared with a single intravenous bolus injection of prodrug XVI. all / Dose) was low. AUC all The oral bioavailability (%F) values ​​based on the dose / dose were 7.74%, 3.17%, and 3.72% for prodrug XVII at 1, 10, and 50 mg / kg, respectively.

[0630] Prodrug VI - Oral Administration (Groups 10, 11, and 12) Because all plasma concentrations of prodrug VI were BLQ at 1 and 10 mg / kg of prodrug XVII, the following discussion of prodrug VI is based on data from only the 50 mg / kg prodrug VI dose group.

[0631] After a single oral dose of prodrug VI to animals in Group 12, the variability in mean plasma concentrations of prodrug VI, as measured by CV values, ranged from 38.4% to 173%. The higher variability observed was the result of BLQ values, which were converted to zero for parameter estimation and averaged over quantifiable results. The variability in mean plasma concentrations of prodrug VI, absent these values, ranged from 38.4% to 69.8% after a single oral dose of prodrug VI to animals in Group 12. Prodrug VI was quantifiable up to 2, 4, 8, or 24 hours post-dose for 50 mg / kg prodrug VI. Peak plasma concentrations of prodrug VI were observed up to 0.5, 8, or 24 hours post-dose for 50 mg / kg prodrug VI.

[0632] Group 12 animals received a single oral dose of 50 mg / kg of prodrug VI, and the mean C max and AUC 0-24hr The C values ​​were 12.4 ng / mL and 242 hr*ng / mL, respectively. max The values ​​ranged from 4.14–32.8 ng / mL for 50 mg / kg of prodrug VI, with individual AUC 0-24hrThe values ​​were 26.5 and 457 hr*ng / mL for 50 mg / kg of prodrug VI.

[0633] AUC of prodrug VI INF , T 1 / 2 , Cl / F, and V z The F values ​​were reported for only one animal at 50 mg / kg (animal no.): 28.2 hr ng / mL, 1.76 hr, 1.78 million mL / hr / kg, and 4.52 million mL / kg.

[0634] Oral administration of treprostinil-prodrug VI (Groups 10, 11, and 12) Following single oral doses of prodrug VI to animals in Groups 10-12, the variability in mean treprostinil plasma concentrations, as measured by CV, ranged from 13.8% to 150%. Treprostinil was quantifiable up to 24 hours after dosing for 1 and 10 mg / kg prodrug XVII and up to 8 or 24 hours after dosing for 50 mg / kg prodrug VI. Individual peak plasma concentrations were observed at 1, 2, or 4 hours after dosing for 1 mg / kg prodrug XVII, 1 or 2 hours after dosing for 10 mg / kg prodrug VI, and 2, 8, or 12 hours after dosing for 50 mg / kg prodrug VI.

[0635] Mean C of treprostinil after a single oral dose of prodrug VI in animals in Groups 10-12 max and AUC 0-24hr The values ​​increased with increasing dose. A 1:10:50 increase in the dose of prodrug VI resulted in a mean treprostinil C max The mean treprostinil AUC 0-24hr The values ​​increased by approximately 1:8.5:60.8 times.

[0636] Systemic exposure to treprostinil (AUC 0-24hr) was greater than the systemic exposure to prodrug VI after a single oral dose of 50 mg / kg of prodrug VI to animal numbers 12002 and 12004, with M:P ratios of 12.1 and 30.0, respectively (M:AUC 0-24hr (Only two ratios could be reported.) The mean M:P ratio was 21.0.

[0637] Secondary parameters of treprostinil (AUC INF , T 1 / 2 , Cl / F, V z / F) has an adjusted R of less than 0.9 2 values, insufficient plasma concentration-time data, or AUC > 25% INF %AUC of Extrap Due to the values, the mean AUC of treprostinil could not be reported for prodrug VI at 10 mg / kg or 50 mg / kg. INF , T 1 / 2 , Cl / F, and V z The AUC values ​​were 38.4 hr*ng / mL, 14.0 hr, 26900 mL / hr / kg, and 243000 mL / kg, respectively, for 1 mg / kg of prodrug VI. INF Values ​​range from 31.7 to 48.4 h*ng / mL, individual T 1 / 2 The values ​​ranged from 4.71 to 36.9 hours (T 1 / 2 The value of 36.9 hours is estimated to be less than the half-life of 3 hours, and should be observed with caution. 1 / 2 values ​​4.71-7.35 hours, otherwise individual Cl / F values ​​20700-31600 mL / hr / kg, individual V z The F value was 194,000-316,000 mL / kg.

[0638] Bolus intravenous injection of treprostinil-prodrug VI (Group 16) The variability of mean treprostinil plasma concentrations, as measured by CV values, ranged from 17.6% to 71.2% after a single bolus intravenous injection of prodrug VI in animals in Group 16. The higher variability observed was a result of BLQ values ​​that were transformed to zero for parameter estimation and averaged across quantifiable outcomes.

[0639] In the absence of these values, the variability of mean plasma treprostinil concentrations ranged from 17.6% to 48.4% after a single bolus intravenous injection of prodrug VI in animals in Group 16. Treprostinil was quantifiable up to 8 or 12 hours after administration, and an estimated concentration at time 0 (C0) was determined.

[0640] Mean C0 and AUC of treprostinil after a single bolus injection of prodrug VI in Group 16 animals 0-12hr The values ​​were 277 ng / mL and 129 hr*ng / mL, respectively. The individual C0 values ​​ranged from 207 to 454 ng / mL, and the individual AUC 0-12hr Values ​​ranged from 97.4-158 hr*ng / mL.

[0641] The M:P ratio could not be determined due to insufficient data available for prodrug VI (plasma concentrations of all prodrug VI were at BLQ).

[0642] Mean AUC of treprostinil after a single intravenous bolus dose of prodrug VI INF , T 1 / 2 , Cl and V z The values ​​were 130 h*ng / mL, 1.90 h, 7920 mL / hr / kg, and 22000 mL / kg, respectively. INF Values ​​range from 99.0 to 159 hr*ng / mL, individual T 1 / 2 values ​​were 1.60-2.28 hours, individual Cl values ​​were 6290-10100 mL / hr / kg, and individual V z The values ​​were 16,800-33,200 mL / kg.

[0643] Treprostinil bioavailability after administration of prodrug VI Dose-normalized systemic exposure to treprostinil (AUC ) was significantly greater after a single oral dose of prodrug VI compared to a single intravenous bolus injection of prodrug VI. all / Dose) was low. AUC allOral bioavailability (%F) values ​​based on / Dose were 28.4%, 24.3%, and 34.7% for prodrug VI at 1, 10, and 50 mg / kg, respectively.

[0644] conclusion Prodrug IV (Groups 1, 2, 3, and 13) After a single oral dose of prodrug IV, the mean C of prodrug IV max Values ​​appeared to increase with increasing dose in an approximately dose-proportional manner from 10 to 50 mg / kg. Oral bioavailability of the prodrug IV was 1.04% at 50 mg / kg prodrug IV.

[0645] Treprostinil (Groups 1, 2, 3, and 13) Mean C of treprostinil after a single oral dose of the prodrug in animals in Groups 1-3 max and AUC 0-24hr The values ​​increased with increasing dose less than dose-proportional between 1 and 50 mg / kg. The systemic exposure of treprostinil (AUC 0-24hr ) was approximately 8 times the systemic exposure of the intravenous prodrug after a single oral dose of 50 mg / kg of the prodrug in animal number 3003. 0-12hr ) was approximately three times the systemic exposure to the intravenous prodrug after a single bolus injection of the prodrug. The oral bioavailability of treprostinil ranged from 2.98% to 6.75% after intravenous prodrug administration.

[0646] Prodrug XVI (Groups 4, 5, 6, and 14) After a single oral or intravenous bolus injection of prodrug XVI, the plasma concentrations of all prodrugs XVI were at the BQL (<1.00 ng / mL).

[0647] Treprostinil (Groups 4, 5, 6, and 14) Mean C of treprostinil after a single oral dose of prodrug XVI to animals in Groups 4-6 max and AUC0-24hr The values ​​increased non-proportionally with increasing doses of prodrug XVI from 1 to 50 mg / kg. The oral bioavailability of treprostinil ranged from 4.13% to 6.35% after administration of prodrug XVI.

[0648] Prodrug XVII (Groups 7, 8, 9, 15) After a single oral or intravenous bolus injection of prodrug XVII, the plasma concentrations of all prodrugs XVII were at the BQL (<1.00 ng / mL).

[0649] Treprostinil (Groups 7, 8, 9, and 15) Mean C of treprostinil after a single oral dose of prodrug XVII max and AUC 0-24hr The values ​​increased with increasing dose in a less than dose-proportional manner.The oral bioavailability of treprostinil ranged from 3.17%-7.74% after administration of prodrug XVII.

[0650] Prodrug VI (Groups 10, 11, 12, 16) After a single bolus injection of prodrug VI, the plasma concentrations of all prodrugs VI were at the BQL (<1.00 ng / mL).

[0651] Treprostinil (Groups 10, 11, 12, and 16) Mean C of treprostinil after a single oral dose of prodrug VI max and AUC 0-24hr The values ​​increased with increasing dose in an approximately dose-proportional manner.

[0652] Systemic exposure to treprostinil (AUC 0-24hr ) was approximately 21-fold greater than the systemic exposure to prodrug VI after a single oral dose of 50 mg / kg of prodrug XVII.

[0653] The oral bioavailability of treprostinil ranged from 24.3% to 34.7% after a single dose of prodrug VI.

[0654] Example 5: Calculation of Compound Solubility and pKa

[0655] [Table 62-1]

[0656] [Table 62-2]

[0657] [Table 62-3]

[0658] [Table 62-4]

[0659] [Table 62-5]

[0660] 6.1. Stability in plasma 6.1.1 Experimental Procedure Studies were conducted using mixed human plasma, male Sprague-Dawley rat plasma, and male beagle dog plasma. All plasma was obtained from bioreclamation and collected on K2EDTA. Plasma was adjusted to pH 7.4 before the start of the experiment. DMSO stocks were first prepared for the test article. Aliquots of DMSO solution were administered to 1.5 mL of plasma pre-warmed to 37°C for a final test article concentration of 1 μM. Vials were stored in a benchtop Thermomixer® for the duration of the study. Aliquots (200 μL) were withdrawn at each time point (0, 15, 30, 60, and 120 minutes) and added to a 96-well plate pre-filled with 400 μL of acetonitrile (ACN). Samples were stored at 4°C until the end of the experiment. After sampling at the final time point, the plate was mixed and then centrifuged at 3000 rpm for 10 minutes. An aliquot of the supernatant was removed, diluted 1:1 in distilled water containing an internal standard, and analyzed by LC-MS / MS against calibration standards prepared in a matching matrix. All samples were analyzed for the administered prodrug as well as the drug, treprostinil. Test article concentrations were compared to the zero-point concentration to determine the percentage of test article remaining at each time point. Half-lives were calculated using GraphPad software, fitting a monophasic exponential decay equation.

[0661] 6.1.2.Experimental Results

[0662] [Table 63]

[0663] Calculated half-life is longer than the test period a When calculated, the half-life is expressed as > the longest incubation period. If the calculated half-life is less than twice the experimental period, the calculated half-life is listed in parentheses. Similarly, if the calculated half-life is shorter than the first non-zero time point, the half-life is listed as < 15, and the calculated half-life, if applicable, is also listed in parentheses.

[0664] [Table 64]

[0665] 6.2. Stability in Liver Microsomes 6.2.1 Experimental Procedure Combined human liver microsomes, male Sprague-Dawley rat liver microsomes, and male beagle dog liver microsomes were purchased from XenoTech. Reaction mixtures excluding NADPH were prepared as follows: Test substances were added to the reaction mixtures in duplicate at a final concentration of 1 μM. The control compound, testosterone, was tested simultaneously with the test substances in separate reactions. Aliquots of the reaction mixture (without cofactors) were equilibrated in a shaking water bath at 37°C for 5 minutes. The reaction was initiated by the addition of cofactors, and the mixture was incubated in a shaking water bath at 37°C. Aliquots (200 μL) were withdrawn at 0, 10, 20, 30, and 60 minutes. Test substance samples were immediately mixed with 400 μL of ice-cold acetonitrile (ACN) to terminate the reaction. Testosterone samples were immediately combined with 400 μL of ice-cold 50 / 50 ACN / H2O containing 0.1% formic acid and the internal standard to terminate the reaction. The samples were then mixed and centrifuged to precipitate proteins. An aliquot of the supernatant was removed, diluted 1:1 in distilled water containing an internal standard, and analyzed by LC-MS / MS against calibration standards prepared in a matching matrix. All samples were analyzed for the administered prodrug and the drug, treprostinil. Test article concentrations were compared to the zero-point concentration to determine the percentage of test article remaining at each time point. Half-lives and clearance were calculated using GraphPad software, fitting a monophasic exponential decay equation.

[0666] 6.2.2 Reaction composition Liver microsomes 0.5mg / mL NADPH (cofactor) 1mM Potassium phosphate, pH 7.4 100mM Magnesium chloride 5mM Test substance 1 μM

[0667] 6.2.3.Experimental Results

[0668] [Table 65]

[0669] Calculated half-life is longer than the test period a When calculating half-lives, the half-life is expressed as > the longest incubation period. If the calculated half-life is less than twice the experimental period, the calculated half-life is listed in parentheses. Similarly, if the calculated half-life is shorter than the first non-zero time point, the half-life is listed as < 10 and the calculated half-life is also listed in parentheses. b Intrinsic Clearance (CL int ) is CL int = k / P, where k is the elimination rate constant and P is the protein concentration during incubation. * There was little prodrug present in the time zero sample. It is likely that the test article underwent non-CYP-mediated degradation during the 5-minute preincubation period. Stability results should be interpreted with caution for these experiments.

[0670] [Table 66]

[0671] [Table 67]

[0672] 6.3. Stability in Hepatocytes 6.3.1 Experimental Procedure Cryopreserved mixed human hepatocytes, male Sprague-Dawley rat hepatocytes, and male beagle dog hepatocytes were purchased from XenoTech. Hepatocytes were thawed and prepared according to the vendor's instructions, pooled in Krebs-Henseleit buffer (KHB, pH 7.4), and placed on ice before the experiment. The hepatocyte suspension was equilibrated at 37°C in a shaking water bath for 3 minutes, after which the test substance was added to the hepatocyte suspension (1.5 x 10 cells) at a final test substance concentration of 1 μM. 6The reaction was initiated by spiking (in duplicate) 100 μg of 1000 μM of 7-hydroxycoumarin (7-HC) (100 μM) into 100 μM cells / mL. The final DMSO content in the incubation mixture was ≤0.1%. The reaction mixture was incubated at 37°C in a shaking water bath. Positive controls, testosterone (1 μM) and 7-hydroxycoumarin (7-HC) (100 μM), were run in parallel to confirm hepatocyte activity. Aliquots of the test substances were collected at 0, 15, 30, 60, and 120 min (n = 1). Aliquots of testosterone (n = 1) were removed at 0, 5, 15, 30, 60, and 120 min. Aliquots of 7-HC were collected at 0 and 15 min (n = 1). The reaction was immediately terminated by adding 2 volumes of ice-cold acetonitrile (ACN) and 3 volumes of ACN containing the internal standard to the positive control sample. The samples were then mixed and centrifuged to precipitate proteins. Aliquots of the supernatant were then diluted with water and analyzed by LC-MS / MS against calibration standards prepared in a matching matrix. Testosterone samples were analyzed without calibration standards. All test article samples were analyzed for the administered prodrug and the drug, treprostinil. Test article concentrations were compared to the zero-point concentration to determine the percentage of test article remaining at each time point. Half-life and clearance values ​​were calculated using GraphPad software, fitting a one-phase exponential decay equation.

[0673] 6.3.2. Experimental Results

[0674] [Table 68]

[0675] Calculated half-life is longer than the test period a When calculating half-life, the half-life is expressed as > the longest incubation period. Then, if the calculated half-life is less than twice the experimental period, the calculated half-life is listed in parentheses. Similarly, if the calculated half-life is shorter than the first non-zero time point, the half-life is listed as < 15, and the calculated half-life, if applicable, is also listed in parentheses. b Intrinsic Clearance (CL int ) is CL int= k / P, where k is the elimination rate constant and P is the cell concentration during incubation.

[0676] [Table 69]

[0677] [Table 70]

[0678] [Table 71]

[0679] 6.4. Stability in simulated intestinal fluid 6.4.1 Experimental Procedure Studies were conducted using simulated intestinal fluid (SIF). SIF was prepared by dissolving 6.8 g of KH2PO4 in 250 mL of water, mixing, and then adding 77 mL of 0.2 N NaOH and 750 mL of water. Pancreas (10 g) was added, mixed, and the pH was adjusted to 6.8 with 10 N NaOH. DMSO stocks were first prepared for the test article. Aliquots of the DMSO solution were administered to 0.4 mL of matrix pre-warmed to 37°C for a final test article concentration of 1 μM. Vials were stored in a benchtop Thermomixer® for the duration of the study. Separate tubes were administered for each time point in each matrix. At the appropriate times (0, 15, 30, 60, and 120 min), 0.8 mL of acetonitrile (ACN) containing 1% formic acid and internal standard was added directly to a single tube. Samples were mixed and immediately stored at 4°C until the end of the experiment. After the final time point was sampled, the plate was mixed and then centrifuged at 3000 rpm for 10 minutes. An aliquot of the supernatant was removed, diluted 1:1 in distilled water containing an internal standard, and analyzed by LC-MS / MS against calibration standards prepared in a matching matrix. All samples were analyzed for the administered prodrug as well as the drug, treprostinil. Test article concentrations were compared to the concentration at time 0 to determine the percentage of test article remaining at each time point. Half-lives were calculated using GraphPad software, fitting a monophasic exponential decay equation.

[0680] [Table 72]

[0681] Calculated half-life is longer than the test period a When calculated, the half-life is expressed as the longest incubation period. If the calculated half-life is less than twice the experimental period, the calculated half-life is listed in parentheses.

[0682] [Table 73]

[0683] 6.5. Stability of Human Subcutaneous Skin Hormogenates The objective of this study was to determine the stability of seven test articles in human subcutaneous skin homogenates.

[0684] 6.5.1 Experimental Procedure The stability of calcein-AM and test articles (prodrugs, LXIV, LXVII, LXXX, LXXI, LXXII, and LXXIII) was evaluated in subcutaneous skin homogenates (BioIVT, lot information in Table 14). A pool of skin homogenates was prepared by combining equal volumes of the three lots tested in the initial skin homogenate lot evaluation study.

[0685] A stock solution of calcein-AM was first prepared at 5 mM in DMSO and then serially diluted in methanol to a concentration of 100 μM. 495 μL of skin homogenate was thawed and warmed to 37°C, and 5 μL aliquots of the 100 μM calcein-AM solution were spiked into the skin homogenate for a final calcein-AM dosing concentration of 1 μM. After brief mixing, 150 μL aliquots were removed three times and transferred to a 96-well Falcon plate. The plate was placed on a Thermomixer and maintained at 37°C with gentle shaking for the duration of the experiment. At each time point (0, 15, 30, 60, and 120 minutes), the plate was removed from the Thermomixer and placed in a FLUOstar® tube. (登録商標) The plate was transferred to a plate reader and the production of calcein (Sigma) was monitored by fluorescence (490 / 515 nm). Calibration standards were prepared in distilled water by serial dilution of the calcein DMSO stock at final concentrations ranging from 1 μM to 1 nM. All samples and standards were read simultaneously (Table 15).

[0686] For each test article, 495 μL of skin homogenate was added to triplicate centrifuge tubes. 5 μL of a 100 μM solution of test article was added to each tube, resulting in a final test article dose concentration of 1 μM. The tubes were placed on a thermomixer and maintained at 37°C with gentle shaking for the duration of the experiment. At each time point (0, 30, 60, and 120 minutes), a 100 μL aliquot was removed from each tube and combined with 200 μL of acetonitrile to terminate the stability reaction. The tubes were mixed and centrifuged at 3000 rpm for 10 minutes. An aliquot of the supernatant was removed and diluted 1:1 with distilled water containing the internal standard (2 μM treprostinil-d4). Calibration standards for treprostinil analysis were prepared for each test article in a surrogate matrix (supernatant from a human plasma:acetonitrile mixture) at concentrations ranging from 1 μM to 1 nM. Calibration standards were also diluted 1:1 with distilled water containing an internal standard. Analytical conditions are outlined in Appendix 1. The disappearance of each test article and the production of treprostinil were monitored (Tables 16 and 17).

[0687] [Table 74]

[0688] 6.5.2. Experimental Results

[0689] [Table 75]

[0690] [Table 76]

[0691] [Table 77]

[0692] 6.6. In Vitro Pharmacological Evaluation of Disubstituted Treprostinil Prodrugs LXX-LXXIII 6.6.1. Summary of findings Comparison of disubstituted prodrugs with treprostinil: (A) Prodrugs LXX, LXXI, and LXXII are inactive, and prodrug LXXIII is approximately 200-fold less active at the prostaglandin I2 (PGI2) receptor (IP); (B) Prodrugs LXX, LXXI, and LXXIII are inactive, and prodrug LXXII exhibits a non-traditional dose-response curve at prostaglandin E2 (PGE2) receptor 2 (EP2). (C) Each of the prodrugs LXX-LXXIII exhibits a non-traditional dose-response curve, with prodrugs LXX, LXXII, and LXXIII being several hundred times less active and prodrug LXXI being approximately 2000 times less active at the prostaglandin D2 (PGD2) receptor 1 (DP1); (D) Each of the prodrugs LXX-LXXIII is inactive at the PGE2 receptor 1 (EP1).

[0693] 6.6.2.Materials Cells and control agonists: The cells and control agonists used in this study are summarized in the table below.

[0694] [Table 78]

[0695] [Table 79]

[0696] Cyclic AMP assay kit: Multiscreen™ TR-FRET cAMP 1.0 No Wash Assay Kit (Multispan, Inc.) Calcium assay kit: MultiScreen™ Calcium 1.0 No Wash Kit (Multispan, Inc., Cat# MSCA01-1) Assay Buffer: EP1 calcium and DP1 cAMP assays: HBSS + 20mM HEPES EP2 and IP1 cAMP assay: 1 mM IBMX + 20 mM HEPES in HBSS Equipment: FlexStation III (Molecular Devices) and FLIPR 384 (Molecular Devices)

[0697] 6.6.3 Method Cells were thawed from frozen cells and resuspended in assay buffer at the desired concentration. cAMP or calcium assays were performed using the Multiscreen™ TR-FRET cAMP 1.0 No Wash Assay Kit or the Multiscreen™ Calcium 1.0 No Wash Kit according to the manufacturer's protocol.

[0698] Gas Cyclic AMP (cAMP) Assay: For agonist-mode testing, cells were seeded at the appropriate density into 384-well plates, then treated with compounds and incubated at 37°C for 20 minutes. The reaction was stopped by the sequential addition of trFluor™ Eu-labeled cAMP and trFluor™ 650-labeled anti-cAMP antibody in lysis buffer. The plate was then incubated at room temperature for 30 minutes before reading fluorescence at 620 nm and 665 nm on a FlexStation III (Molecular Devices). All test wells contained a final concentration of 0.1% DMSO.

[0699] Calcium assay: Cells were seeded at an appropriate density in 384-well plates. Calcium assays were performed according to the manufacturer's protocol (Multiscreen Calcium 1.0 No Wash Assay Kit). Calcium dye loading buffer was added to the cells and incubated at 37°C for 1 hour. For agonist mode, iloprost control agonist or test compound was injected into the cells using a FLIPR, calcium mobilization was monitored for 180 seconds, and compound was injected into the well at 19 seconds. Fluorescence emission was read at 525 nm with excitation at 490 nm in a FLIPR 384 instrument (Molecular Devices).

[0700] Data Analysis Cyclic AMP (cAMP) assay: Results of the cyclic AMP assay are shown as the ratio 665 / 620 × 10,000 (ratio of fluorescence at 665 nm and 620 nm × 10,000). Graphed data are presented as mean ± SD. Sigmoid colon dose-response curves were fitted to the dose-dependent responses using GraphPad Prism version 6 (Graphpad Prism), allowing for variable slopes.

[0701] Calcium Assay: Calcium assay results are expressed as "RFU" as defined in the figure below. Data are expressed as Mean ± SEM. Dose-response curves were fitted using the "Sigmoidal dose-response (variable slope)" function in GraphPad Prism 6. EC 50 Values ​​were calculated based on curve fitting.

[0702] Results of 6.6.5. Prostaglandin receptor activity assessments using treprostinil and treprostinil analogs have been conducted, including the historical positive control agonists for these receptors, iloprost (IP, EP2, and EP1) or PGD2 (DP1). These studies used cell lines overexpressing either the human IP, EP2, or DP1 receptor, or the EP1 receptor, and measured cAMP levels using fluorescence resonance energy transfer (FRET) after incubation with various concentrations of the compounds. The 665 / 620 (acceptor / donor emission signal) ratio is inversely proportional to cAMP concentration.

[0703] The relative differences between treprostinil and its positive controls (iloprost and PGD2) were consistent with previous evaluations. As shown in Tables 20 and 24-25 below, and in Figures 11 and 12 compared to treprostinil, the disubstituted analogs have lower activity and very different pharmacological profiles compared to treprostinil.

[0704] [Table 80]

[0705] DP1, PGD2 receptor 1; EC50, concentration giving half-maximal response; EP2, PGE2 receptor 2; EP1, PGE2 receptor 1; IP, PGI2 receptor *Partial agonist or odd-numbered dose-response curve; NC = too inactive to realistically calculate. Data on prodrug conversion in hepatocytes are also presented in Table 21 and FIG.

[0706] [Table 81]

[0707] Data on prodrug conversion in liver microsomes are also presented in Table 22 and FIG.

[0708] [Table 82]

[0709] Table 23 presents data on the conversion of selected treprostinil prodrugs in skin homogenates.

[0710] [Table 83]

[0711] Tables 24-25 and Figures 11-12 present data on the activity of selected treprostinil prodrugs at IP, EP1, EP2 and DP receptors compared to that of treprostinil.

[0712] [Table 84]

[0713] EC of treprostinil 50 Compare to a value * Partial agonists or equivocal dose-response curves

[0714] "Emoji" = Too expensive to calculate realistically

[0715] [Table 85]

[0716] EC of treprostinil 50 Compare to a value * Partial agonists or equivocal dose-response curves "Emoji" = Too expensive to calculate realistically Tables 26, 27, and 28 present data on the hydrolysis of selected treprostinil prodrugs at pH 6, 7, and 8, and at 40° C., respectively.

[0717] [Table 86]

[0718] [Table 87]

[0719] [Table 88]

[0720] Example 7 7.1. Synthesis of Triprostil Monosubstituted Prodrugs Scheme 16: Synthesis of Prodrugs XXXXII-XXXVIII, XLIII-XLIV, XLVIII, XLVIII, XLIX, XLVIII, XLIX, LIV, and LV

[0721] [ka]

[0722] In Scheme 16, R 72 may be triethylsilyl (TES) or another silyl ester, such as trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, phenyldimethylsilyl, substituted benzyl, while R 71 is benzyl or substituted benzyl, i.e., a benzyl group substituted with one or more substituents which may be independently selected from the group consisting of -NO, -CN, -halogen (e.g., -F, -Cl, -Br, or -I), (C1-C3)alkyl, halo(C1-C3)alkoxy, and halo(C1-C3)alkoxy;

[0723] Experiments: General procedure for the synthesis of treprostinil side chain substituted mono-TES benzyl esters (73) (coupling reaction): To a stirred solution of treprostinil mono-TES benzyl ester (71) (1.0 equiv.) in dichloromethane (DCM) (10 v / wt), acid (72) (1.2 equiv.), diisopropylethylamine (DIPEA) (2.5 equiv.) or triethylamine (2.5 equiv.) and 4-N,N-dimethylaminopyridine (EDCI.HCl) were added. After stirring for 10 min, EDCI.HCl (2.5 equiv.) was added and the mixture was stirred at room temperature under argon for 3 h. Water was added and the aqueous layer was extracted with DCM. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography to give treprostinil side chain substituted mono-TES benzyl ester (73). Compound 73 was synthesized by 1 It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0724] General procedure for the synthesis of treprostinil side chain substituted benzyl esters (74) (first deprotected or desilylated): To a stirred solution of treprostinil side chain substituted mono-TES benzyl ester (74) (1.0 equiv.) in THF (15 v / wt) and water (3 v / wt), HCl solution (2N) (1.0 equiv.) was added, and the mixture was stirred at room temperature for 1 hour. Water and EtOAc were added. The aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography to give treprostinil side chain substituted benzyl ester (74). Compound 74 was synthesized as follows: 1 It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0725] General procedure for the synthesis of treprostinil side chain ester (75) (second deprotection or debenzylation): To a stirred solution of treprostinil side-chain substituted benzyl ester (74) (1.0 equiv.) 5% palladium on carbon (25 wt%) in ethyl acetate (20 v / wt) was added. The system was evacuated under house vacuum and replaced with hydrogen (this process was repeated two more times). The system was connected to a hydrogen balloon and stirred at room temperature for 1 hour. The system was filtered through a pad of Celite and washed with ethyl acetate. The filtrate was concentrated under reduced pressure to give treprostinil side-chain ester (75). Compound 75 was characterized by infrared and NMR spectroscopy. 1 H NMR, 13 It was characterized by CNMR and LCMS, and the purity was determined by HPLC.

[0726] Similarly, prodrugs XXXXXII, XXXIII, XXIV, XXXV, XXXVI, XXVI, XXVII, XLIII, XLIV, XLVIII, XLVIII, XLIX, LIV and LV were synthesized following the general procedure above. XXXXII, treprostinil side chain succinate ester dimethylamide. XXXXIII, treprostinil side chain succinate ester morpholine amide. XXXIV, treprostinil side chain succinate ester N-methylpiperazine. XXXXV, treprostinil side chain lysine ester. XXXXVI, treprostinil side chain proline ester. XXXXVII, treprostinil side chain β-alanine ester. XLIII, treprostinil side chain acetate. XLIV, treprostinil side chain hydroxyacetate ester. XLVIII, treprostinil side chain p-toluyl ester. XLIX, treprostinil side chain trimethyl acetate ester. LIV, treprostinil side chain (4-hydroxyphenoxy) acetate ester. Left ventricle, treprostinil side-chain propionate.

[0727] Scheme 17: Synthesis of Treprostinil Side Chain Monosubstituted Carbamates (Prodrugs XXXXXIII and XLII)

[0728] [ka]

[0729] In Scheme 17, R 71 and R 72 is the same as Scheme 16.

[0730] Synthetic Method for Treprostinil Mono-TES Benzyl Ester Side Chain (4-Nitrophenol) Carbonate (77) To a stirred solution of triprostinil mono-TES benzyl ester (71) (1.0 equiv.) in THF (15 v / wt) at room temperature, pyridine (5.0 equiv.) was added. The solution was cooled to 0°C, and then a solution of 4-nitrophenol chloroformate (76) (1.5 equiv.) in THF (7 v / wt) was added dropwise and stirred at room temperature for 2 hours. Water was added, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. It was purified by silica gel column chromatography to give treprostinil mono-TES benzyl ester side chain (4-nitrophenol) carbonate (77). Compound 77 was prepared as follows: 1 It was characterized by 1 HNMR and LCMS.

[0731] General procedure for the synthesis of treprostinil mono-TES benzyl ester side chain carbamates (79a-b) 79a: To a stirred solution of treprostinil mono-TES benzyl ester side chain (4-nitrophenol) carbonate (7) (1.0 equiv.) in THF (20 v / wt) and water (1 v / wt), benzylglycine hydrochloride (78a) (1.1 equiv.) and potassium carbonate (1.2 equiv.) were added and the mixture was stirred at room temperature overnight. 79b: To a stirred solution of treprostinil mono-TES benzyl ester side chain (4-nitrophenol) carbonate (7) (1.0 equiv.) in THF (20 v / wt) was added ammonia solution (7N / methanol) (78b) (10 equiv.), and the mixture was stirred at room temperature for 4 hours.

[0732] Water and ethyl acetate were added, and the layers were separated. The aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography to give treprostinil mono-TES benzyl ester side chain carbamate (79a-b). Compounds 79a-b were obtained. 1 It was characterized by 1 HNMR and LCMS.

[0733] General procedure for the synthesis of treprostinil benzyl ester side chain carbamates (80a-b) (first deprotection or desilylation): Treprostinil benzyl ester side chain carbamates of compound 74 (80a-b) were prepared and characterized using the general procedure described above.

[0734] General procedure for the synthesis of treprostinil side chain carbamates (81a-b) (second deprotection or debenzylation): Similarly, treprostinil benzyl ester side chain carbamates (81a-b) were prepared and characterized following the general procedure described for compound 75. Prodrugs XXXVIII and XLII were prepared accordingly.

[0735] XXXXVIII, treprostinil side chain glycine carbamate (81a). XLII, treprostinil side chain carbamate (81b).

[0736] Scheme 18: Synthesis of treprostinil side chain monosubstituted carbonates (prodrug XXXIX) [ka]

[0737] In Scheme 18, R 71 and R 72 is the same as in Schemes 16-17.

[0738] Synthetic Method for Treprostinil Mono-TES Benzyl Ester Side Chain Benzyl Glycolic Acid Carbonate (84) To a stirred solution of triphosgene (82) (1.0 equiv.) in toluene (10 v / wt.) at 0° C., a solution of treprostinil mono-TES benzyl ester (71) (1.0 equiv.) and pyridine (1.1 equiv.) in toluene (10 v / wt.) was added via an addition funnel. The mixture was stirred for 3 hours. To this was added a solution of benzyl glycolic acid (83) (10 equiv.) and pyridine (10 equiv.) in toluene (5 v / wt.). The mixture was stirred overnight. Saturated sodium bicarbonate solution and ethyl acetate were added, stirred for 10 minutes, and the layers were separated. The aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography to give triprostinil mono-TES benzyl ester side-chain benzyl glycolate carbonate (84). Compound 84 was synthesized as follows: 1 It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0739] Method for the synthesis of treprostinil benzyl ester side chain benzyl glycolic acid carbonate (85) (first deperonation or desilylation): Using the general procedure described for compound 74, treprostinil benzyl ester side chain benzyl glycolic acid carbonate (85) was prepared and characterized.

[0740] Synthetic Procedure for Treprostinil Side Chain Glycolic Acid (16) (Prodrug XXXIX) (Second Deprotection or Debenzylation): Using the general procedure described for compound 75, treprostinil side chain glycolate carbonate (86) (prodrug XXXIX) was prepared and characterized accordingly.

[0741] Scheme 19: Synthesis of Treprostinil Side Chain Ethers (Prodrugs XLV, LII, LVI, and LVII) [ka]

[0742] The first reaction in Scheme 19 may be an alkylation or a Mitsunobu or diazoacetic acid coupling carried out in the presence of a base. 71 may be the same as in Schemes 16-18. SiR 74 may be a silyl ester such as triethylsilyl, trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, phenyldimethylsilyl, etc. In some embodiments, R 74 may be Et3 or t-BuMe2.

[0743] Treprostinil mono-TBDMS benzyl ester side chain methyl ether (88a, R 74 Synthesis of t-BuMe2: Treprostinil mono-TBDMS benzyl ester (87, R 74 A stirred solution of 4-methyl-bis(2,6-tert-butyl)pyridine (15 equiv.) and methyl triflate (10 equiv.) was stirred in an oil bath at 35°C for 6 hours. Water was added and the layers were separated. The aqueous layer was extracted with DCM. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. Purification by column chromatography afforded the treprostinil mono-TBDMS benzyl ester side chain methyl ether (88a, R 74 Compound (88a, R 74 =t-BuMe2) 1 It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0744] Treprostinil mono-TES benzyl ester side chain methyl pivalate ether (88b, R 74 =Et3) synthesis method Treprostinil mono-TES benzyl ester (71) (=87) in anhydrous DMF (6.5 v / wt) at room temperature, R 74To a stirred solution of 1.0 equiv. of Et was added cesium carbonate (6.0 equiv.), sodium iodide (5.0 equiv.), and chloromethyl pivalate (5.0 equiv.). The resulting mixture was stirred at room temperature for 3 days. After filtration, the filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography to give treprostinil mono-TES benzyl ester side-chain methyl pivalate ether (88b, R 74 =Et3) was obtained. 74 =Et3) 1 It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0745] Treprostinil mono-TBDMS benzyl ester side chain benzoic acid ether (88c, R 74 =t-BuMe2) synthesis method 3'AU90 mono-TBDMS (87', R 74 To a stirred solution of 3'AU90 mono-TBDMS benzyl ester (3.0 eq.) and 3'AU90 tetrahydrofuran (2.0 eq.) was added potassium carbonate (3.0 eq.) and sodium iodide (3 mol%). The solution was stirred at room temperature overnight. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography to give the pure product 3'AU90 mono-TBDMS benzyl ester, which was 1 It was characterized by HNMR and LCMS, and the purity was confirmed by HPLC.

[0746] To a stirred solution of 3'AU90 mono-TBDMS benzyl ester (1.0 equiv.), 2-benzyl salicylate (5.0 equiv.), and triphenylphosphine (2.0 equiv.) in THF (25 v / wt.) at 0 °C under argon was added DIAD (2.0 equiv.) in THF (8 v / wt.) via an addition funnel. The reaction mixture was stirred overnight at that temperature (slowly warming to room temperature) under argon. Water and EtOAc were added, and the layers were separated. The aqueous layer was extracted with EtOAc. The combined EtOAc layers were washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. This was purified by silica gel column chromatography to give the treprostinil mono-TBDMS benzyl ester side-chain benzoate ether (88c, R 74 =t-BuMe2), and 1 Characterized by H NMR and LCMS. Purity was determined by HPLC.

[0747] Treprostinil mono-TBDMS benzyl ester side chain ethyl acetate ether (88d, R 74 Synthesis of t-BuMe2: Treprostinil mono-TBDMS benzyl ester (87,R 74 A suspension of treprostinil mono-TBDMS benzyl ester (1.0 equiv.) and rhodium(II) acetate dimer (10 mol%) in anhydrous toluene (10 v / wt) at 80 °C was prepared by dissolving 15 wt% of ethyl diazoacetate (4.0 equiv.) in toluene (1 v / v) (1 v / v). The reaction mixture was concentrated in vacuo. This was purified by column chromatography on silica gel to give treprostinil mono-TBDMS benzyl ester side-chain ethyl acetate ether (88d, R 74 This compound (88d, R 74 =t-BuMe2) 1 It was characterized by HNMR.

[0748] General procedure for the synthesis of treprostinil benzyl ester side chain ethers (89a, 89c, 89d) Treprostinil mono-TMS benzyl ester side chain ether (88a or 88c or 88d, R 74 To a stirred solution of HF.Py (89a, 89c, 89d) (1.0 equiv.) was added HF.Py (10 equiv.). The solution was stirred at room temperature for 4 hours. Saturated aqueous sodium bicarbonate solution was slowly added to pH-7, followed by ethyl acetate, and stirred for 10 minutes to obtain separated layers. The aqueous layer was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography to obtain the treprostinil benzyl ester side chain ethers (89a, 89c, 89d). These compounds 89a, 89c, and 89d were 1 It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0749] Method for the synthesis of treprostinil benzyl ester side chain methyl pivalate ether (89b) (first deprotection or desilylation): Using the general procedure described for compound 74, treprostinil benzyl ester side chain methyl pivalate ether (89b) was prepared and characterized.

[0750] Method for the synthesis of treprostinil side chain ethers (90a-d) (second deprotection or debenzylation): Similarly, using the general procedure described for compound 75, treprostinil side chain ethers (90a-d) were prepared and characterized accordingly.

[0751] Scheme 20: Synthesis of treprostinil side chain ethers (prodrugs XLVI and L)

[0752] [ka]

[0753] In Scheme 20, R 71 may be the same as in Schemes 16-19. 76may be a silyl ester such as triethylsilyl, trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, phenyldimethylsilyl, etc. In some embodiments, R 76 R may be t-butyldimethylsilyl. 77 R may be a hydroxyl protecting group such as acetyl or benzoyl. 78 can be a sulfonic acid group such as methyl sulfonate (mesylate) or methyl p-toluenesulfonate (tosylate).

[0754] Synthesis of treprostinil benzyl ester acetate side chain TBDMS ether (92): To a solution of treprostinil benzyl ester side chain TBDMS ether (91) (1.0 equivalent) and dimethylaminopyridine (DMAP) (2.6 equivalents) in anhydrous dichloromethane (10 v / wt) was added acetic anhydride (2.0 equivalents) at ambient temperature under argon. After 1 hour, the reaction was complete. The reaction mixture was evaporated in vacuo to give the crude product. The crude compound was purified by column chromatography using silica gel to give treprostinil benzyl ester acetate side chain TBDMS ether (92). Compound 92 was synthesized as follows: 1 It was characterized by HNMR.

[0755] General procedure for the synthesis of treprostinil benzyl ester acetate side chain ethers (94a-b) To a solution of treprostinil benzyl ester side-chain TBDMS acetate (92) (1.0 equiv.) in acetonitrile (20 v / wt.) was added triethylsilane (1.5 equiv.). To this mixture was added a solution of bismuth bromide (7 mol%) in acetonitrile (2 v / wt.) under argon. Propionaldehyde (1.5 equiv.) was then slowly added over 5 min. The reaction mixture was stirred at room temperature under argon for 20 min. The reaction mixture was quenched at saturation. The water and sodium bicarbonate solution were extracted with ethyl acetate. The precipitate was filtered. The filtrate was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain pure treprostinil benzyl ester acetate side-chain ethers (94a-b). These compounds 94a-b were synthesized. 1 Characterized by 1 H NMR and LCMS.

[0756] General procedure for the synthesis of treprostinil acetate side chain ethers (95a-b) (first deprotection or debenzylation): Similarly, using the general procedure described for compound 75, treprostinil acetate side chain ether acetates (95a-b) were prepared and characterized accordingly.

[0757] Method for the synthesis of treprostinil side chain propyl ether (96) (prodrug XLVI): To a solution of treprostinil acetate side chain propyl ether (95a) (1.0 equiv.) in methanol (25 v / wt.) was added an aqueous solution (6 v / wt.) of potassium hydroxide (4.0 equiv.). This was stirred overnight at room temperature. The reaction mixture was evaporated under vacuum, and the residue was dissolved in water. The pH of this solution was adjusted to pH 2-3 with 1N HCl, and then extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and evaporated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography to give treprostinil side chain propyl ether (96) (prodrug XLVI). Compound 96 was characterized by infrared, 1 H NMR, 13It was characterized by CNMR and LCMS, and the purity was determined by HPLC.

[0758] Synthetic method for treprostinil benzyl ester side chain mesyloxyethyl ether (97) To a stirred solution of treprostinil hydroxyethyl ether (95b) (1.0 equiv.) and benzyl bromide (1.1 equiv.) in acetone (40 v / wt) was added K2CO3 (2.0 equiv.). The mixture was stirred overnight under argon and the reaction was found to be incomplete. NaI (25 mol %) and additional benzyl bromide (1.1 equiv.) were added and stirring continued for another 5 h. The mixture was passed through a pad of Celite and washed with acetone. The filtrate was concentrated under reduced pressure to give the crude product. This crude product was purified by silica gel column chromatography to give the treprostinil benzyl ester side chain hydroxyethyl ether, which was 1 H NMR, 13 Characterization was performed by CNMR and LCMS. Purity was determined by HPLC.

[0759] Under argon 0-5 0 To a stirred solution of treprostinil benzyl ester side chain hydroxyethyl ether (1.0 equiv.) and triethylamine (6.0 equiv.) in DCM (40 v / wt.) of C was added dropwise methylsulfonyl chloride (6.0 equiv.) in DCM (2 v / wt.). The mixture was stirred under argon for 1 hour, and the reaction was found to be complete. The mixture was concentrated in vacuo, and the crude product was purified by silica gel column chromatography to give treprostinil benzyl ester side chain mesyloxyethyl ether (97). Compound 97 was synthesized as follows: 1 H NMR, 13 It was characterized by C NMR and LCMS, and the purity was determined by HPLC.

[0760] Synthetic Method for Treprostinil Benzyl Ester Side Chain Morpholine Ethyl Ether (28) To a stirred solution of treprostinil benzyl ester side-chain mesyloxyethyl ether (97) (1.0 equiv.) and DIPEA (10 equiv.) in anhydrous CH3CN (20 v / wt) was added morpholine (10 equiv.). The mixture was heated to 60-70 °C under argon for 11 h, then cooled to room temperature, concentrated in vacuo, and the crude product purified by silica gel column chromatography to give the desired treprostinil benzyl ester side-chain morpholine ethyl ether (98). Compound 98 was identified by infrared, 1 H NMR, 13 It was characterized by CNMR and LCMS, and the purity was determined by HPLC.

[0761] Method for the synthesis of treprostinil side chain morphine ethyl ether (99) (prodrug L): To a stirred solution of treprostinil benzyl ester side-chain morpholine ethyl ether (98) (1.0 equiv.) in methanol (25 v / wt) was added 5% palladium on carbon (50% water) (25 wt%). The mixture was evacuated and flushed with hydrogen (from a hydrogen balloon) three times and stirred under an H atmosphere for 3 h. The mixture was passed through a Celite pad and washed with methanol. The solvent was removed under reduced pressure to give a white solid, which was dissolved in methanol (25 v / wt). NaOH (5.0 equiv.) in H2O (5 v / wt) was then added. The mixture was stirred at room temperature under argon for 4 h until the reaction was complete. Water was added to the mixture, and the mixture was extracted with MTBE. The aqueous solution was then adjusted to pH 1-2 with 2N HCl and extracted with EtOAc. The extract was washed with brine and dried over Na2SO4. The solvent was removed under vacuum to give pure treprostinil side chain morpholine ethyl ether (99) (prodrug L). 1 H NMR, 13 It was characterized by CNMR and LCMS, and the purity was determined by HPLC.

[0762] Scheme 21: Synthesis of treprostinil cyclopentyl methyl ether (prodrug LIII)

[0763] [ka]

[0764] Method for the synthesis of treprostinil methyl ester cyclopentyl methyl ether (101) To a stirred solution of treprostinil (100) (1.0 equiv.) in anhydrous DMSO (20 v / wt.) at room temperature, potassium hydroxide (9.0 equiv.) and methyl iodide (20 equiv.) were added. The resulting mixture was stirred at room temperature overnight. After filtration, the filtrate was concentrated under reduced pressure to give the crude product. Water and DCM were added, and the layers were separated. The aqueous layer was extracted with DCM. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography to give treprostinil methyl ester cyclopentyl methyl ether (101). Compound 101 was synthesized as follows: 1 It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0765] Synthesis of treprostinil cyclopentyl methyl ether (102) (prodrug LXIII): To a stirred solution of treprostinil methyl ester cyclopentyl methyl ether (101) (1.0 equiv.) in methanol (20 v / wt.) was added sodium hydroxide (10 equiv.) in water (2 v / wt.). The resulting mixture was stirred at room temperature for 6 hours. The solvent was evaporated under reduced pressure. Water was added to the residue, and the basic solution was extracted with MTBE. The aqueous layer was cooled to 0°C and adjusted to pH -2 with 2N HCl. The acidic solution was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product. It was dissolved in EtOAc and slowly added to hexane to form a solid. It was filtered and then dried in air overnight to give treprostinil cyclopentyl methyl ether (102) (prodrug LIII). Compound 102 was characterized by infrared and NMR spectroscopy. 1 H NMR, 13 It was characterized by CNMR and LCMS, and the purity was determined by HPLC.

[0766] Example 8 Synthesis of Treprostinil Disubstituted Prodrugs LXX-LXXIII

[0767] [ka]

[0768] Scheme 22: Synthesis of Treprostinil Disubstituted Prodrugs LXX-LXXIII [ka]

[0769] In Scheme 22, R 71 may be the same as in Schemes 16-20. The functionalization reaction may be, for example, acylation, carbonylation or phosphorylation.

[0770] experiment: General procedure for the synthesis of treprostinil benzyl ester diacylate (104a, 104c) (acylation): To a stirred solution of treprostinil benzyl ester (103) (1.0 equivalent) and DMAP (4.0 equivalents) in dichloromethane (DCM) (20 v / wt), propionic anhydride (2.5 equivalents) (for 104a) or acetic anhydride (2.5 equivalents) (for 104c) was added. The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure to give the crude product, which was purified by silica gel column chromatography to give the streptostinil benzyl ester diacylate (104a or 104c). These compounds (104a, 104c) were 1 It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0771] Method for synthesizing treprostinil benzyl ester dicarbonate (104b) To a solution of treprostinil benzyl ester (103) (1.0 equiv.) in anhydrous pyridine (5 v / wt.) at 0-5°C under argon, a solution of methyl chloroformate (6.0 equiv.) in anhydrous dichloromethane (5 v / wt.) was added dropwise over 5 min. After complete addition, the reaction mixture was stirred at room temperature for 2 h at 0°C. The mixture was treated with water and then extracted with dichloromethane. The dichloromethane extract was washed with water, brine, dried over Na2SO4, filtered, and concentrated in vacuo to give a crude oil. The crude product was purified by silica gel column chromatography to give pure treprostinil benzyl ester dicarbonate (104b) as a white solid. The pure product was characterized by IR, 1 H NMR, 13 It was characterized by CNMR, DEPT-135 and LC-MS, and purity was determined by HPLC.

[0772] Method for the synthesis of treprostinil benzyl ester di(dibenzyl)phosphate (104d) To a stirred solution of treprostinil benzyl ester (103) (1.0 equiv.), 1H-tetrazole (4.0 equiv.) (0.45 M in acetonitrile) was added via an addition funnel under argon. The resulting mixture was stirred at room temperature for 10 minutes, and dibenzyl-N,N-diisopropylphosphoramidite (3.0 equiv.) in DCM (7 v / wt) was added dropwise. The mixture was stirred at room temperature for 2 hours. The reaction was complete at this stage, and the system was cooled to -78 °C (dry ice-acetone). 3-Chloroperoxybenzoic acid (mCPBA) (70-75%) (4.2 equiv.) was added in one portion and stirred at that temperature for 2 hours. The reaction was complete, and sodium sulfite solution (10%) was added and stirred overnight (slowly warming to room temperature). The DCM layer was checked with a Peroxide 100 test tip to ensure no peroxide was present in the solution (if peroxide was present, it was washed with more sodium sulfite solution (10%)). The DCM layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to give the crude product, which was purified by silica gel column chromatography to give treprostinil benzyl ester di(dibenzyl)phosphate (104d). Compound 104d was 1It was characterized by HNMR and LCMS, and the purity was determined by HPLC.

[0773] General procedure for the synthesis of treprostinil disubstituted prodrugs (105a-d) (deprotected or debenzylated): To a solution of treprostinil benzyl ester disubstituted prodrugs (104a-d) (1.0 equiv.) in ethyl acetate (20 v / wt) (1 v / wt water for 104d), 5% palladium on carbon (-50% water) (25 wt%) was added under argon. The mixture was evacuated under house vacuum and replaced with hydrogen (packed in a balloon) at room temperature, and this process was repeated three times. The reaction mixture was stirred under an atmosphere of hydrogen at room temperature for 2.5 hours. The mixture was filtered through a pad of Celite and washed with EtOAc. The filtrate was evaporated in vacuo to give pure treprostinil disubstituted prodrugs (105a-d). The pure products were identified by IR, 1 H NMR, 13 CNMR, DEPT-135 (105d 31 The product was characterized by PNMR and LC-MS. The purity was determined by HPLC.

[0774] Similarly, following the general procedure described above, treprostinil disubstituted prodrugs LXX-LXXIII were synthesized: LXX, treprostinil dipropionate (105a); LXXI, treprostinil dicarbonate (105b); LXXII, treprostinil diacetate (105c); and LXXIII, treprostinil diphosphate (105d).

[0775] Additional Embodiments 1.The following formula:

[0776] [ka]

[0777] or a pharmaceutically acceptable salt of a compound having the formula: 3の Compounds where each is not H. 2. The compound of embodiment 1, wherein R9 is H, one of R2 and R3 is H, and the other of R2 and R3 is a second drug moiety. 3. The compound of embodiment 2, wherein R2 is H and R3 is a second drug moiety. 4. The compound of embodiment 2, wherein R2 is a second drug moiety and R3 is H. 5. The compound of any one of paragraphs 1-4, wherein the second drug moiety is a pain-relieving drug moiety. 6. The compound of embodiment 5, wherein the second drug moiety is a nonsteroidal anti-inflammatory drug moiety. 7. The compound of embodiment 6, wherein the nonsteroidal anti-inflammatory drug moiety is selected from the group consisting of aspirin, naproxen, and ibuprofen. 8.Following formula:

[0778] [ka]

[0779] wherein X is OH and R2 and R3 together form a carbonyl-containing group or a phosphorus-containing group. 9. R2 and R3 together

[0780] [ka]

[0781] The compound of embodiment 8, which forms: 10. R2 and R3 together

[0782] [ka] ,

[0783] Form R 23 is H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted aryl. 11.The following formula:

[0784] [ka]

[0785] or a pharmaceutical salt thereof, wherein X is OR9 and R9 is a compound of the formula:

[0786] [ka]

[0787] where R 11 does not exist, or R 11 is a substituted or unsubstituted alkyl, and R 12 and R 13 are each independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted aryl, R and R, where OR or OR are independently selected from H, a phosphate group, an alkyl, or a group to form an ester-containing group. 12.R 12、 R 13 , R2, and R3 are each H, and R 11 is C 1-4 The compound of embodiment 11, wherein the aryl is alkyl. 13.The following formula:

[0788] [ka]

[0789] wherein X is OH, and R2 and R3 are each independently H or

[0790] [ka]

[0791] wherein Y is independently selected from a) NR4R5, where R4 and R5 form a substituted or unsubstituted C3-C8 cycloalkyl group; b) OR 4 where R 4 is optionally substituted with a carboxy or hydroxy group 1-6 is an alkyl group, provided that R 2 and R 3 is not H. 14.R 3 is H. 15.R 2 is H. 16.R 3 is below

[0792] [ka]

[0793] and Y is NR4R5, where R4 and R5 form a substituted or unsubstituted C3-C8 cycloalkyl group. 17. Compounds according to embodiment 16, wherein R4 and R5 form a C3-C8 cycloalkyl group substituted with one or more substituents selected from substituted or unsubstituted alkyl groups and substituted or unsubstituted cycloalkyl groups. 18.R3 is:

[0794] [ka]

[0795] wherein Y is OR4, and R4 is C optionally substituted with a carboxy or hydroxy group. 1-6 The compound of embodiment 15, wherein the alkyl group is an alkyl group. 19. A compound having a formula selected from the group consisting of:

[0796] [ka]

[0797] [ka]

[0798] [ka]

[0799] 20. A pharmaceutical composition comprising: (A) an effective amount of a compound of any one of embodiments 1-19; and (B) a pharmaceutically acceptable carrier. 21. The pharmaceutical composition of embodiment 20, which is an oral pharmaceutical composition. 22. The pharmaceutical composition of embodiment 20, which is a subcutaneous pharmaceutical composition. 23. A method of treating a disease or condition, comprising administering the composition of embodiment 20 to a subject. 24. The method of embodiment 23, wherein the disease or condition is one or more selected from the group consisting of pulmonary hypertension, congestive heart failure, peripheral vascular disease, Raynaud's phenomenon, scleroderma, renal failure, peripheral neuropathy, digital ulcers, intermittent claudication, ischemic limb disease, peripheral ischemic lesions, pulmonary fibrosis, and asthma. 25. The method of embodiment 23, wherein the disease is pulmonary hypertension. 26. The method of any one of embodiments 23-25, wherein the composition is administered orally. 27. The method of embodiment 26, wherein the subject has detectable treprostinil plasma levels for at least 24 hours after administration. 28. The method of any one of embodiments 23-25, wherein the composition is administered by injection. 29. The method of embodiment 28, wherein the administration is subcutaneous. 30. The method of embodiment 29, wherein the administration is continuous subcutaneous administration. 31. The method of any one of claims 28-30, wherein no or reduced pain occurs at the injection site compared to administering treprostinil. 32. The method of any one of 23-31, wherein the subject is a human. 33. A method of treating a disease or condition comprising administering to a subject a prodrug of treprostinil, wherein said prodrug, upon administration, is converted to a metabolite consisting essentially of treprostinil. 34. The method of embodiment 33, wherein the metabolite consists of treprostinil. 35. The method of embodiment 33 or 34, wherein said administering is performed orally. 36. The method of embodiment 35, wherein the subject has detectable treprostinil plasma levels for at least 24 hours after administration. 37. A prodrug has the following formula:

[0800] [ka]

[0801] or a pharmaceutically acceptable salt of the compound, wherein X is OR9, R9 and R2 are H, and R3 is a non-hydrogen group. 38. The method of embodiment 37, wherein R3 is a phosphorus-containing group or OR3 is an ester group. 39.R 3 But the following formula:

[0802] [ka]

[0803] where R 31 does not exist, or R 31 is a substituted or unsubstituted alkyloxy, and R 32 and dR 33 wherein each of is independently selected from H, substituted or unsubstituted alkyloxy, substituted or unsubstituted alkenyloxy, substituted or unsubstituted cycloalkyloxy, and substituted or unsubstituted aryloxy. 40.R 31 C 1-5 alkyl, and R 32and R 33 40. The method of embodiment 39, wherein each of is H. 41. The method of embodiment 38, wherein OR3 is an ester of an amino acid. 42. The method of embodiment 41, wherein the amino acid is alanine, valine, or glycine. 43. The method of embodiment 38, wherein OR3 is an ester of the second drug moiety. 44. The method of embodiment 43, wherein the second drug moiety is a pain-relieving drug moiety. 45. The method of embodiment 44, wherein the second drug moiety is a nonsteroidal anti-inflammatory drug moiety. 46. ​​The method of embodiment 45, wherein the second drug moiety is selected from the group consisting of aspirin, naproxen, and ibuprofen. 47.R 3 but

[0804] [ka]

[0805] wherein Y is OR4 or NR4R5, where each of R4 and R5 is H and C 1-4 38. The method of embodiment 37, wherein said alkyl is independently selected from alkyl. 48.R3 is,

[0806] [ka]

[0807] and Y is NR 4 R 5 wherein R4 and R5 form a substituted or unsubstituted C3-C8 cycloalkyl group. 49. The method of embodiment 48, wherein R4 and R5 form a C3-C8 cycloalkyl group substituted with one or more substituents selected from substituted or unsubstituted alkyl groups and substituted or unsubstituted cycloalkyl groups. 50.R 3 but

[0808] [ka]

[0809] wherein Y is OR4, and R4 is C optionally substituted with a carboxy group or an alkoxy group. 1-6 38. The method of embodiment 37, wherein the group is an alkyl group. 51. The method according to one of embodiments 33-37, wherein the prodrug is selected from the group consisting of:

[0810] [ka]

[0811] [ka]

[0812] [ka]

[0813] [ka]

[0814] 52. The method of any one of embodiments 33-51, wherein the disease or condition is one or more selected from the group consisting of pulmonary hypertension, congestive heart failure, peripheral vascular disease, Raynaud's phenomenon, scleroderma, renal failure, peripheral neuropathy, digital ulcers, intermittent claudication, ischemic limb disease, peripheral ischemic lesions, pulmonary fibrosis, and asthma. 53. The method of embodiment 53, wherein the disease is pulmonary hypertension. 54. The method of any one of embodiments 33-53, wherein the subject is a human.

[0815] While the foregoing refers to certain preferred embodiments, it will be understood that the present invention is not limited thereto. Those skilled in the art will recognize that various modifications can be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.

[0816] All publications, patent applications and patents cited herein are hereby incorporated by reference in their entirety.

Claims

1. The following formula: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein X is OR １４ , -NR １ SO ２ R １ , -NR １ CO ２ H. 【Chemistry 2】 and Each R １ are independently H or C １ -C ４ alkyl, and R ８ is an optionally substituted C １ -C ６ alkyl or amino acid side groups, or R １ and R ８ together with to form a 4-7 membered heterocycle; R １４ is H, optionally substituted C １ -C ６ alkyl, a first drug moiety, or 【Transformation 3】 and R １１ is absent or optionally substituted C １ -C ６ Alkylene or -Q １ -O and Q １ is an optionally substituted C １ -C ６ alkylene; R １２ and R １３ each of which is selected from H, OH, optionally substituted C １ -C ６ Alkoxy, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ Alkenyl, optionally substituted C １ -C ８ Cycloalkyl, optionally substituted C １ -C １０ is aryl, R ２ and R ３ each independently represents a second drug moiety, a third drug moiety, H, a phosphorus-containing group, —C(O)R ６ or an -ABC substituent, A is an optionally substituted C １ -C ６ Alkylene, —NR ６ -, -C(O)-, -C(O)O- or -C(O)NR ６ - and B is a bond, optionally substituted C １ -C ６ alkylene, —C(O)—, —O—, —S—, or optionally substituted heterocyclyl; C is optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted cycloalkyl, -(°CH ２ CH ２ ) ｑ -OR ６ , -C(O)N(R ６ ) ２ , -C(O)N(R １８ ) ２、 -C(O)R ６ , -CO ２ H, -OR ６ -N(R １８ ) ２ , -N(R ６ ) ２ , or 【Chemistry 4】 Both R １８ together form an optionally substituted 3-8 membered heterocyclyl; Each R ６ are independently H, optionally substituted C １ -C ６ alkyl, optionally substituted heteroaryl, optionally substituted aryl, or R ６ and both taken together form a 4-8 membered optionally substituted heterocyclyl or a 5 membered optionally substituted heteroaryl; Or, R ２ and R ３ are bonded to each other to form -C(O)-, -SO ２ -, 8-12 members, 【Transformation 5】 Forming Each R １０ is H, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ alkenyl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted aryl; q is 0, 1, 2, 3, 4, 5 or 6; however, When A is —C(O)—, B is not a bond and C is —N(R ６ ) ２ Not; When A is —C(O)—, B is not a bond and C is —OR ６ Not; R １４ , R ２ , R ３ is not H; When X is OH, R ２ and R ３ are not both H; and R ８ is H, then R ２ and R ３ or a pharmaceutically acceptable salt thereof, wherein at least one of

2. X is OH, —CH ２ OPO ３ H ２ , 【Transformation 6】 -NHSO ２ CH ３ where R ８ is OH or —CO ２ C optionally substituted with H １ -C ２ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

3. R ３ But -C(O)R １ ７ , -OPO ３ H ２ or -ABC, A is -C(O)-, -C(O)O-, CH ２ or —C(O)NR ６ - and B is -CHR １６ - or - (CH ２ ) ｑ - and C is C １ -C ３ Alkoxy, heterocyclyl, OR ６ , O.P.O. ３ H ２ , CO ２ H, OH, NH ２ , -C(O)R ６ , -C(O)N(R １８ ) ２ , or -C(O)N(R ６ ) ２ and Where: R １６ is H or C １ -C ３ is alkyl, R １ ７ is C １ -C ３ alkyl, optionally substituted aryl, or optionally substituted heteroaryl; 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, or 2.

4. R ２ or R ３ is the ABC portion, A and B are CH ２ and C is CO ２ H, amino, C(O)N(R １８ ) ２ , or -C(O)N(R ６ ) ２ The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein:

5. R ２ or R ３ is the ABC moiety of formula -C(O)-C, C is optionally substituted phenyl, C １ -C ４ The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, which is alkyl, optionally substituted piperidinyl, optionally substituted morpholino, optionally substituted azepanyl, optionally substituted aziridinyl, optionally substituted azetidinyl, optionally substituted pyrrolidinyl, or optionally substituted piperazinyl.

6. R ２ or R ３ is the formula -C(O)-CHCH ３ 6. The compound according to any one of claims 1 to 5, wherein the ABC moiety of -C is optionally substituted aryl or optionally substituted heteroaryl, or a pharmaceutically acceptable salt thereof.

7. R ２ or R ３ -C(O)-X-CH ２ CO ２ H, and X is O or NR １ The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein:

8. R ２ or R ３ -C(O)-(℃H ２ CH ２ ) ｑ -OR ６ and R ６ is C １ -C ６ The compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

9. R ２ -C(O)-(CH ２ ) ２ CO ２ H or C(O)-(CHCH ３ 20. The compound of claim 18, wherein C is optionally substituted aryl or optionally substituted heteroaryl, or a pharmaceutically acceptable salt thereof.

10. Phosphorus-containing groups. 【Transformation 7】 where R ３１ is absent, and optionally substituted C １ -C ６ alkylene, or -Q-OA, where Q is an optionally substituted C １ -C ６ alkylene; R ３２ andR ３３ Each of １ -C ６ Alkoxy, optionally substituted C １ -C ６ Alkenyloxy, optionally substituted C １－ C ６ The compound of any one of claims 1-9, independently selected from cycloalkoxy and optionally substituted aryloxy.

11. formula: 【Transformation 8】 3. The compound of claim 1 or 2, wherein: 【Request Item 12】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition comprising: (A) an effective amount of a compound according to any one of claims 1 to 12; and (B) a pharmaceutically acceptable carrier.

14. 14. The pharmaceutical composition of claim 13, which is an oral pharmaceutical composition.

15. 14. The pharmaceutical composition of claim 13, which is a subcutaneous pharmaceutical composition.

16. 14. A method of treating a disease or condition, comprising administering to a subject in need thereof the composition of claim 13.

17. 17. The method of claim 16, wherein the disease or condition is one or more selected from the group consisting of pulmonary hypertension, congestive heart failure, peripheral vascular disease, Raynaud's phenomenon, scleroderma, renal failure, peripheral neuropathy, digital ulcers, intermittent claudication, ischemic limb disease, peripheral ischemic lesions, pulmonary fibrosis, and asthma.

18. 18. The method of claim 16 or 17, wherein the disease is pulmonary hypertension.

19. The method of any one of claims 16 to 18, wherein the composition is administered orally.

20. 20. The method of any one of claims 16 to 19, wherein the subject has detectable treprostinil plasma levels for at least 24 hours after administration.

21. The method of any one of claims 16 to 18 and 20, wherein the composition is administered by injection.

22. The method of any one of claims 16 to 18, 20 and 21, wherein the administration is subcutaneous.

23. 23. The method of claim 22, wherein the administration is continuous subcutaneous administration.

24. 24. The method of any one of claims 16 to 18 and 20 to 23, wherein said administration results in no or less pain at the injection site compared to administration of treprostinil.

25. The method of any one of claims 16 to 24, wherein the subject is a human.

26. A method of treating a disease or condition comprising administering to a subject a prodrug of treprostinil, wherein said prodrug, upon administration, is converted to a metabolite consisting essentially of treprostinil.

27. 27. The method of claim 26, wherein the metabolite consists of treprostinil.

28. 28. The method of claim 26 or 27, wherein the administration is orally.

29. 29. The method of any one of claims 26 to 28, wherein the subject has detectable treprostinil plasma levels for at least 24 hours after administration.

30. The prodrug may have the formula: 【Chemistry 15】 or a pharmaceutically acceptable salt thereof, X is OR １４ , -NR １ SO ２ R １ , -NR １ CO ２ H. 【Chemistry 16】 and Each R １ are independently H or C １ -C ４ alkyl, and R ８ is an optionally substituted C １ -C ６ alkyl or amino acid side groups, or R １ and R ８ together with R １４ is H, optionally substituted C １ -C ６ alkyl, a first drug moiety, or 【Chemistry 17】 Either R １１ is absent and optionally substituted C １ -C ６ Alkylene or -Q １ -O, Q １ is an optionally substituted C １ -C ６ alkylene; R １２ and R １３ each of which is selected from H, OH, optionally substituted C １ -C ６ Alkoxy, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ Alkenyl, optionally substituted C １ -C ８ Cycloalkyl, optionally substituted C １ -C １０ and R ２ and R ３ each independently represents a second drug moiety, a third drug moiety, H, a phosphorus-containing group, —C(O)R ６ or an -ABC substituent, A is an optionally substituted C １ -C ６ Alkylene, —NR ６ -, -C(O)-, -C(O)O- or -C(O)NR ６ - and B is a bond, optionally substituted C １ -C ６ alkylene, —C(O)—, —O—, —S—, or heterocyclyl; C is optionally substituted heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, optionally substituted cycloalkyl, -(°CH ２ CH ２ ) ｑ -OR ６ , -C(O)N(R ６ ) ２ , -C(O)N(R １８ ) ２ , -C(O)R ６ , -CO ２ H, -OR ６ , -N(R １８ ) ２ , -N(R ６ ) ２ ,or [Chemistry 18] and Both R １８ together form an optionally substituted 3-8 membered heterocyclyl; Each R ６ are independently H, optionally substituted C １ -C ６ alkyl, optionally substituted heteroaryl, optionally substituted aryl, or R ６ together form a 4-8 membered optionally substituted heterocyclyl or a 5 membered optionally substituted heteroaryl; Or, R ２ and R ３ are bonded to each other to form -C(O)-, -SO ２ -, 8-12 membered heterocyclyl 【Chemistry 19】 and Each R １０ is H, optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ alkenyl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted aryl; q is 0, 1, 2, 3, 4, 5 or 6; however, When A is —C(O)—, B is not a bond and C is —N(R ６ ) ２ Not; When A is —C(O)—, B is not a bond and C is —OR ６ Not ; R １４ , R ２ , R ３ is not H; When X is OH, R ２ and R ３ is not H; and R ８ is H, then R ２ and R ３ At least one of is not H.

31. R ３ but, 【Chemistry 20】 or -C(O)R ６ 31. The method of claim 30, wherein:

32. R ３ But the formula: 【Chemistry 21】 is a phosphorus-containing group of R ３１ is absent, and optionally substituted C １ -C ６ alkylene, or -Q-O-, where Q is an optionally substituted C １ -C ６ alkylene; R ３２ and ３３ Each of １ -C ６ Alkoxy, optionally substituted C １ -C ６ Alkenyloxy, optionally substituted C １ -C ６ is independently selected from cycloalkoxy, and optionally substituted aryloxy.

31. The method of claim 30

33. R ３１ is C １ -C ６ alkylene, and R ３２ and R ３３ 33. The method of claim 32, wherein each is H.

34. R ３ is -C(O)-CHR １９ -N(R ６ ) ２ and R １９ The method of claim 30, wherein is a side group of an amino acid.

35. 35. The method of claim 34, wherein the amino acid is alanine, valine, or glycine.

36. R ３ 31. The method of claim 30, wherein is a third drug moiety linked to the compound via an ester.

37. 37. The method of any one of claims 30-36, wherein the second drug moiety is a pain-relieving drug moiety.

38. 37. The method of any one of claims 30-36, wherein the second drug moiety is a nonsteroidal anti-inflammatory drug moiety.

39. 39. The method of claim 38, wherein the second drug moiety is selected from the group consisting of aspirin, naproxen, and ibuprofen.

40. R ３ is the -ABC portion, A is —C(O)—, B is a bond, and C is -N(R １８ ) ２ 31. The method of claim 30, wherein:

41. Both R １８ is an optionally substituted C １ -C ６ alkyl, optionally substituted heterocyclyl, and optionally substituted C １ -C ８ 41. The method of claim 40, wherein together they form a 3-8 membered heterocyclyl optionally substituted with one or more substituents selected from cycloalkyl.

42. R ３ is the -ABC portion, A is —C(O)—, B is an optionally substituted C １ -C ６ is alkylene, C is CO ２ H, -OR ６ where R ６ is an optionally substituted C １ -C ６ 31. The method of claim 30, wherein the alkyl is alkyl.

43. The prodrug may be: 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemistry 27】 The method of any one of claims 26 to 42, selected from the group consisting of:

44. 44. The method of any one of claims 26 to 43, wherein the disease or condition is one or more selected from the group consisting of pulmonary hypertension, congestive heart failure, peripheral vascular disease, Raynaud's phenomenon, scleroderma, renal failure, peripheral neuropathy, digital ulcers, intermittent claudication, ischemic limb disease, peripheral ischemic lesions, pulmonary fibrosis, and asthma.

45. 45. The method of claim 44, wherein the disease is pulmonary hypertension.

46. The method of any one of claims 26 to 45, wherein the subject is a human.

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