Methods of Making and Using PDE9 Inhibitors

Abstract

The present invention relates to PDE9 inhibitors, their synthesis, and their use for the treatment of benign prostatic hyperplasia, beta-thalassemia, and sickle cell disease. [Selection diagram] None

Description

[Technical field]

[0001] (Reference to Related Application) This application claims priority to U.S. Provisional Patent Application No. 62 / 511,367, entitled Methods of Making and Using PDE9 Inhibitors, filed May 26, 2017, the contents of which are incorporated by reference herein in their entirety.

[0002] FIELD OF THE DISCLOSURE The present invention relates to cyclic guanylate monophosphate (cGMP)-specific phosphodiesterase 9 inhibitors (hereinafter referred to as PDE9 inhibitors). [Background technology]

[0003] Phosphodiesterases (PDEs) are a family of enzymes that degrade cyclic nucleotides, thereby controlling the cellular levels of second messengers throughout the body. PDEs represent attractive drug targets, as evidenced by the large number of compounds introduced into clinical testing and on the market, respectively. PDEs are encoded by 21 genes that are functionally separated into 11 families that differ with respect to their mechanical properties, substrate selectivity, expression, localization patterns, activation, regulators and inhibitor sensitivity. The function of PDEs is the degradation of the cyclic nucleotide monophosphates cyclic adenosine monophosphate (cAMP) and / or guanosine monophosphate (cGMP), which are important intracellular mediators involved in numerous biological processes, including neurotransmission and the control of smooth muscle contraction and relaxation.

[0004] PDE9 is hypothesized to be a key player in controlling cGMP levels because it is cGMP specific (Km for cAMP is 1000-fold higher than Km for cGMP) and has the lowest Km of any PDE for this nucleotide. PDE9 is expressed throughout the brain at low levels, potentially controlling basal cGMP.

[0005] WO 2012 / 040230 discloses PDE9 inhibitors having an "imidazopyrazinone" scaffold for use as drugs in the treatment of PDE9-related diseases, including CNS and neurodegenerative disorders.

[0006] WO 2008 / 139293 and WO 2010 / 084438 both disclose aminoheterocyclic compounds that are PDE9 inhibitors and their use in the treatment of neurodegenerative and cognitive disorders.

[0007] In the periphery, PDE9 expression is highest in the prostate, intestine, kidney and hematopoietic cells, allowing therapeutic potential in a variety of non-CNS indications. Summary of the Invention

[0008] The present invention provides the method of making and using PDE9 inhibitors, which have been shown to have low blood-brain barrier penetration, and are therefore particularly useful for treating peripheral diseases such as benign prostatic hyperplasia (BPH), urinary tract dysfunction epithelial disease, erectile dysfunction, type 2 diabetes and sickle cell disease (SCD).Furthermore, the PDE9 inhibitors of the present invention are significantly more potent PDE9 inhibitors than PDE1 inhibitors.This PDE inhibition selectivity is important because PDE1 is expressed in heart and testis, and the inhibition of these PDE1 isoforms is believed to be a potential cause of cardiovascular and reproductive side effects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] I. Compounds of the Invention

[0010] One aspect of the present invention provides PDE9-inhibiting compounds or PDE9 inhibitors that can be used to treat sickle cell disease (SCD).The PDE9 inhibitors of the present invention have been shown to have low blood-brain barrier penetration, and therefore can be particularly useful for treating peripheral diseases, such as benign prostatic hyperplasia (BPH), urinary tract dysfunction epithelial disease, erectile dysfunction, type 2 diabetes and sickle cell disease (SCD).In addition, the PDE9 inhibitors of the present invention are significantly more potent PDE9 inhibitors than PDE1 inhibitors.This PDE inhibition selectivity is important because PDE1 is expressed in the heart and testis, and the inhibition of these PDE1 isoforms is believed to be a potential cause of cardiovascular and reproductive side effects. (PDE9 inhibitors)

[0011] In the context of the present invention, the IC of any of the three PDE9 isoforms 50 Compound is considered to be PDE9 inhibitor when the amount required to reach the level is 10 micromolar or less, preferably less than 9 micromolar, for example 8 micromolar or less, for example 7 micromolar or less, for example 6 micromolar or less, for example 5 micromolar or less, for example 4 micromolar or less, for example 3 micromolar or less, more preferably 2 micromolar or less, for example 1 micromolar or less, particularly 500 nanomolar or less.In a preferred embodiment, the amount required to reach the IC50 level of PDE9 is 400 nanomolar or less, for example 300 nanomolar or less, 200 nanomolar or less, 100 nanomolar or less, or 80 nanomolar or less, for example 50 nanomolar or less, for example 25 nanomolar.

[0012] Throughout this application, the notation IC 50 and IC50 are used interchangeably.

[0013] In some embodiments, the PDE9 inhibitor of the present invention has low or no blood-brain barrier penetration.For example, the ratio of the concentration of the PDE9 inhibitor of the present invention in the brain to the concentration of the PDE9 inhibitor of the present invention in plasma (brain / plasma ratio) can be less than about 0.50, less than about 0.40, less than about 0.30, less than about 0.20, less than about 0.10, less than about 0.05, less than about 0.04, less than about 0.03, less than about 0.02, or less than about 0.01.The brain / plasma ratio can be measured 30 minutes or 120 minutes after administration of the PDE9 inhibitor. (isomeric type)

[0014] Where the compounds of the invention contain one or more chiral centers, reference to any of the compounds will include enantiomerically or diastereomerically pure compounds as well as mixtures of the enantiomers or diastereomers in any ratio, unless otherwise specified.

[0015] In one embodiment, the PDE9 inhibitor compounds of the present invention used to treat sickle cell disease contain an imidazopyrazinones structure, which may have the structure (I) (also referred to as compounds of formula (I)) and tautomers and pharma- ceutically acceptable acid addition salts thereof, and polymorphs thereof:

[0016] [ka] wherein R2 is cyclized with either R1 or R3; R1, R2 and R3 are R1 and when cyclized with R2,

[0017] [ka] and R7 is selected from the group consisting of H, -CH3, -C2H5 and -C3H7; * indicates the cyclization point. If no cyclization occurs, R1 is H and

[0018] [ka] is selected from the group consisting of R7 is selected from the group consisting of H, -CH3, -C2H5 and -C3H7; R2 is

[0019] [ka] is selected from the group consisting of R8 and R12 are independently selected from the group consisting of H, -CH3, -C2H5 and -C3H7; * indicates the cyclization point; When cyclized with R2, R3 is

[0020] [ka] and * indicates the cyclization point; R9 is H, C1-C6 alkyl, substituted C1-C6 alkyl, branched C3-C6 alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, C6-C 10 Aryl, Substituted C6-C 10 Aryl, C3-C9 heteroaryl, substituted C3-C9 heteroaryl, C1-C6 alkoxy, substituted C1-C6 alkoxy, branched C3-C6 alkoxy, C3-C6 cycloalkoxy, substituted C3-C6 cycloalkoxy, C6-C 10 Aryloxy, substituted C6-C 10 aryloxy, C3-C9 heteroaryloxy, substituted C3-C9 heteroaryloxy; When not cyclized, R3 is

[0021] [ka] and R10 is selected from the group consisting of H, -CH3 and -C2H5; R11 is C6-C 10 Aryl, Substituted C6-C 10selected from the group consisting of aryl, C3-C9 heteroaryl, and substituted C3-C9 heteroaryl; R4 is selected from the group consisting of hydrogen, -CH3, -C2H5, -C3H7, -CF3, -CN, F and Cl; R5 is C6-C 10 Aryl, Substituted C6-C 10 selected from the group consisting of aryl, C3-C9 heteroaryl, substituted C3-C9 heteroaryl, C3-C6 heterocyclyl, substituted C3-C6 heterocyclyl, C3-C6 cycloalkyl, and substituted C3-C6 cycloalkyl; R6 is selected from the group consisting of hydrogen, F, Cl, CN, -CH3, -C2H5, -C3H7 and -CF3; A is absent or -CH2-.

[0022] Non-limiting examples of PDE9-inhibiting compounds of formula (I) are disclosed in WO 2013 / 053690, the contents of which are incorporated herein by reference in their entirety.

[0023] For example, the PDE9 inhibitor having an imidazopyrazinone scaffold is selected from the group consisting of (compound P1), (compound P2), and (compound P3) (racemic and enantiomerically enriched or pure forms).

[0024] [ka]

[0025] [ka]

[0026] and,

[0027] [ka]

[0028] In another embodiment, the PDE9 inhibitor compounds of the present invention used to treat sickle cell disease contain an imidazopyrazinones scaffold. They may have the structure (II) (also referred to as compounds of formula (II)) and its tautomers and pharmaceutically acceptable acid addition salts thereof, and its polymorphs:

[0029] [ka] wherein R2 is cyclized with either R1 or R3; R1, R2 and R3 are R1 and when cyclized with R2,

[0030] [ka] and R6 is selected from the group consisting of H, -CH3, -C2H5 and -C3H7; * indicates the cyclization point; When not cyclized, R1 is H and

[0031] [ka] is selected from the group consisting of R6 is selected from the group consisting of H, -CH3, -C2H5 and -C3H7; R2 is

[0032] [ka] is a compound selected from the group consisting of R7 and R11 are independently selected from the group consisting of H, -CH3, -C2H5, and -C3H7; * indicates the cyclization point, and when cyclization is performed at R2, R3 is

[0033] [ka] and * indicates the cyclization point; R8 is H, C1-C6 alkyl, substituted C1-C6 alkyl, branched C3-C6 alkyl, C3-C6 cycloalkyl, substituted C3-C6 cycloalkyl, C6-C 10 Aryl, Substituted C6-C 10 Aryl, C3-C9 heteroaryl, substituted C3-C9 heteroaryl, C1-C6 alkoxy, substituted C1-C6 alkoxy, branched C3-C6 alkoxy, C3-C6 cycloalkoxy, substituted C3-C6 cycloalkoxy, C6-C 10 Aryloxy, substituted C6-C 10 aryloxy, C3-C9 heteroaryloxy, substituted C3-C9 heteroaryloxy; When not cyclized, R3 is

[0034] [ka] and R9 is selected from the group consisting of H, -CH3 and -C2H5; R10 is C6-C 10 Aryl, Substituted C6-C 10 aryl, C3-C9 heteroaryl, substituted C3-C9 heteroaryl; R4 is selected from the group consisting of C6-C 10 Aryl, Substituted C6-C 10 selected from the group consisting of aryl, C3-C9 heteroaryl, substituted C3-C9 heteroaryl, C3-C6 heterocyclyl, substituted C3-C6 heterocyclyl, C3-C6 cycloalkyl, and substituted C3-C6 cycloalkyl; R5 is selected from the group consisting of hydrogen, F, Cl, CN, -CH3, -C2H5, -C3H7, and -CF3; A is absent or -CH2-.

[0035] Non-limiting examples of PDE9 inhibitors of formula (II) are disclosed in WO 2013 / 110768, the contents of which are incorporated herein by reference in their entirety.

[0036] For example, a PDE9 inhibitor having an imidazopyrazinone backbone can be (compound P4).

[0037] [ka]

[0038] Non-Limiting Embodiments of the Invention

[0039] Table 1 lists exemplary compounds of the present invention and the corresponding IC50 values ​​(nM) that were determined. Additionally, the concentrations of the compounds in plasma and brain, as determined as described in the "Blood-Brain Barrier Penetration" section, are listed. Each of the compounds constitutes an individual embodiment of the present invention.

[0040] Table 1: Examples of compounds of the invention, IC50 values ​​and plasma / brain concentrations

[0041] [Table 1] II. Pharmaceutical Compositions

[0042] The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of any of the compounds of the present invention and a pharma- ceutically acceptable carrier or diluent.The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one of the specific compounds disclosed herein and a pharma- ceutically acceptable carrier or diluent. (Pharmaceutically acceptable salts)

[0043] The present invention also includes salts of the compounds, typically pharma- ceutically acceptable salts. Such salts include pharma- ceutically acceptable acid addition salts. Acid addition salts include salts of inorganic and organic acids.

[0044] Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, sulfamic, nitric, etc. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, itaconic, lactic, methanesulfonic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene, salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic. p-toluenesulfonic acid, theophylline acetate, 8-halotheophylline, e.g., 8-bromotheophylline, etc. Further examples of pharma- ceutically acceptable inorganic or organic acid addition salts include the pharma- ceutically acceptable salts listed in Berge, SM et al., J. Pharm. Sci. 1977, 66, 2., the contents of which are incorporated by reference.

[0045] Additionally, the compounds of the present invention can exist in unsolvated as well as solvated forms with pharma- ceutically acceptable solvents such as water, ethanol, etc. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.

[0046] The compounds of the present invention can be administered alone or in combination with pharma- ceutical acceptable carriers, diluents or excipients, either in single or multiple doses.The pharmaceutical compositions of the present invention can be formulated with pharma- ceutical acceptable carriers or diluents and any other known adjuvants and excipients according to the prior art, such as those disclosed in Remington: The Sciene and Practice of Pharmacy, 22nd Edition, Gennato, Ed., Mack Publishing Co., Easton, PA, 2013.

[0047] The pharmaceutical compositions may be specifically formulated for administration by any suitable route, for example, oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) routes. It will be understood that the route will depend on the general health and age of the subject being treated, the nature of the condition being treated and the active ingredient.

[0048] Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, the compositions may be prepared with coatings such as enteric coatings, or may be formulated to provide controlled release of active ingredients, such as sustained or extended release, according to methods well known in the art. Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs.

[0049] Pharmaceutical compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions, as well as sterile powders to be reconstituted in a sterile injectable solution or dispersion prior to use. Other suitable administration forms include, but are not limited to, suppositories, sprays, ointments, creams, gels, inhalants, skin patches and implants.

[0050] A typical oral dosage is in the range of about 0.001 to about 100 mg / kg body weight per day. A typical oral dosage is also in the range of about 0.01 to about 50 mg / kg body weight per day. A typical oral dosage is further in the range of about 0.05 to about 10 mg / kg body weight per day. Oral dosages are usually administered in one or more doses, typically 1 to 3 doses per day. The exact dosage will depend on the frequency and mode of administration, the sex, age, weight and general health of the subject, the nature and severity of the condition being treated, and any co-morbidities being treated and other factors evident to one skilled in the art.

[0051] The formulations may also be presented in unit dosage form by methods known to those of skill in the art. For purposes of illustration, a typical unit dosage form for oral administration may contain from about 0.01 to about 1000 mg, from about 0.05 to about 500 mg, or from about 0.5 mg to about 200 mg.

[0052] For parenteral routes, such as intravenous, intrathecal, intramuscular, and the like, typical dosages are about half of the dosage used for oral administration.

[0053] The present invention also provides a method for preparing a pharmaceutical composition comprising mixing a therapeutically effective amount of a compound of the present invention with at least one pharma- ceutically acceptable carrier or diluent. In one embodiment of the present invention, the compound used in the aforementioned process is one of the specific compounds disclosed in the experimental section of this specification.

[0054] The compound of the present invention is usually utilized as free substance or as its pharmaceutically acceptable salt.This salt is prepared by treating the solution or suspension of the compound of the present invention with one molar equivalent of pharmaceutically acceptable acid in a conventional manner.The representative examples of suitable organic and inorganic acids are described above.

[0055] For parenteral administration, the solution of the compound of the present invention in sterile aqueous solution, aqueous propylene glycol, aqueous vitamin E or sesame or peanut oil can be used.Such aqueous solution should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose.Aqueous solution is particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.The compound of the present invention can be easily incorporated into known sterile aqueous media using standard techniques known to those skilled in the art.

[0056] Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers include lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Similarly, carriers or diluents can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with wax. The pharmaceutical composition formed by combining the compound of the present invention with a pharmaceutically acceptable carrier is then easily administered in a variety of dosage forms suitable for the disclosed administration route. The formulation can be conveniently provided in unit dosage form by methods known in the art of pharmacy.

[0057] Formulations of the present invention suitable for oral administration may be presented as discrete units, such as capsules or tablets, each containing a predetermined amount of the active ingredient and, optionally, suitable excipients. Further, an orally available formulation may be in the form of a powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion.

[0058] When a solid carrier is used for oral administration, the preparation may be tableted, placed in a hard gelatin capsule in the form of a powder or pellet, or the preparation may be in the form of a troche or lozenge. The amount of solid carrier varies widely, but may range from about 25 mg to about 1 g per dosage unit. When a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, or a sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

[0059] The pharmaceutical composition of the present invention can be prepared by conventional methods in the art.For example, tablets can be prepared by mixing the active ingredient with common adjuvants and / or diluents, and then compressing the mixture in a conventional tablet press to prepare tablets.Examples of adjuvants or diluents include corn starch, potato starch, talcum, magnesium stearate, gelatin, lactose, gum, etc. Any other adjuvants or additives that are usually used for such purposes, such as colorants, flavorings, preservatives, etc., can be used as long as they are compatible with the active ingredient.

[0060] The pharmaceutical compositions may comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% by weight of a PDE9 inhibitor of the invention.

[0061] In one embodiment, a pharmaceutical composition comprising a compound of the invention is used in combination with an additional activating agent, such as HU.

[0062] III. Methods of Using the Compounds of the Invention

[0063] PDE9 is specifically expressed in the human hematopoietic system, including neutrophils, reticulocytes, and erythroleukemia cells. Furthermore, sickle cell disease (SCD) patients show a marked and significant increase in PDE9 expression in reticulocytes and neutrophils compared to healthy individuals (Almeida et al., Br J Haematol. 2008 Sep;142(5):836-44). Evidence further substantiates the link between PDE9 and cell adhesion, as pharmacological PDE9 inhibition ameliorates the increased adhesiveness of SCD neutrophils (Miguel et al., Inflamm Res. 2011 Jul;60(7):633-42). The mechanism by which PDE9 inhibition reduces cell adhesion has been shown to be mediated by increased cGMP and reduced endothelial adhesion molecule expression. Importantly, in animal models of SCD, PDE9 inhibitor-mediated reduction in cell adhesion had the functional effect of increased cell survival. In addition to demonstrating reduced cell adhesion comparable to hydroxyurea (HU), PDE9 inhibition increased fetal non-sickle hemoglobin (HBF) production, which reduced the cellular concentration of hemoglobin (Hb S) in red blood cells (RBCs) and reduced polymerization of hemoglobin and its associated sequelae. The importance of increasing HbF in treating SCD is evidenced by the results of large studies such as the Sickle Cell Disease Collaborative and studies of various patient cohorts outside the United States (Alsultan et al. Am J Hematol. 88(6):531-2 (2013)), which show that HbF is one of the most important modifiers of the disease, as well as data showing that modifiers of HbF improve other hematological parameters (Akinsheye, Blood, 118(1):19-27 (2011)). Finally, Almeida and colleagues demonstrated that treatment with HU combined with PDE9 inhibition in a mouse model of SCD leads to a further beneficial amplification of the cGMP elevating effect of HU (Almeida et al., Blood. 2012 Oct 4, 120(14):2879-88). In conclusion, PDE9 inhibition can modulate both the expression of fetal hemoglobin production and the reduction of cell adhesion, both mechanisms being important for the treatment of SCD.

[0064] One aspect of the present invention provides methods of using the PDE9 inhibitors of the present invention and pharmaceutical compositions comprising the PDE9 inhibitors of the present invention.

[0065] The PDE9 inhibitors of the present invention may be used to treat sickle cell disease or any disease and / or condition associated with sickle cell disease, such as anemia, sickle hemoglobin C disease (SC), beta thalassemia (beta plus thalassemia and beta zero thalassemia), vaso-occlusive crisis, pain crisis (sickle cell crisis), splenic retention crisis, acute chest syndrome, bone marrow aplastic crisis, hemolytic crisis, prolonged pain, bacterial infection, and stroke.

[0066] In one embodiment, the PDE9 inhibitors of the present invention are used to treat beta thalassemia in a subject and / or to increase hemoglobin levels in a subject.

[0067] In another embodiment, the PDE9 inhibitor of the present invention is used to increase cGMP level in cells or in the plasma of a subject, and the subject suffers from sickle cell disease.Cells can be, but are not limited to, red blood cells and / or white blood cells.cGMP level can be increased by at least 50%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or 25-fold.

[0068] In another embodiment, the PDE9 inhibitors of the present invention are used to increase the number of fetal hemoglobin (HbF) positive red blood cells in a subject suffering from sickle cell disease, and the number of HbF positive red blood cells is increased by at least 50%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or 25-fold.

[0069] In another embodiment, the PDE9 inhibitors of the present invention are used to reduce the percentage of sickle red blood cells (% sickle cells), the percentage of stasis (% stasis), total bilirubin or total white blood cell count in a subject suffering from sickle cell disease, and the % sickle red blood cells, % stasis, total bilirubin, total white blood cell count or spleen weight is reduced by at least 10%, 20%, 30%, 40%, 50%, 60% or 70%.

[0070] cGMP levels can be measured using any suitable method in the art, such as enzyme immunoassay.

[0071] As used herein, HbF positive cells refer to red blood cells that contain HbF. HbF positive cells can be measured from blood samples using any suitable method in the art, such as electrophoresis and / or colorimetry.

[0072] Sickle cells, sickled red blood cells, as used herein, refer to crescent-shaped or sickle-shaped red blood cells. % sickle cells can be measured from a blood sample using any suitable method in the art.

[0073] Hemostasis or capillary hemostasis, as used herein, is the severe slowing or complete cessation of blood or lymph flow through a vessel. % hemostasis is the number of static (no flow) venules divided by the number of flowing venules times 100. % hemostasis can be measured using any suitable method in the art.

[0074] Total bilirubin, as used herein, refers to both unconjugated and conjugated bilirubin. Total bilirubin levels can be measured from a blood sample using any suitable method in the art.

[0075] A total white blood cell count or total leukocyte count, as used herein, is a blood test that measures the number of white blood cells in the body. It can be measured from a blood sample using any suitable method in the art.

[0076] Another aspect of the present invention provides a method of using the PDE9 inhibitor of the present invention in combination with at least one other active agent. They can be administered simultaneously or sequentially. They can be present as a mixture for simultaneous administration or each can be present in a separate container for sequential administration.

[0077] The term "co-administration," as used herein, is not particularly limited and means that the PDE9 inhibitor of the present invention and at least one other active agent are administered substantially simultaneously, for example, as a mixture or in immediate succession.

[0078] The term "sequential administration" as used herein is not particularly limited, and refers to the PDE9 inhibitor of the present invention and at least one other active agent being administered not simultaneously, but one after the other, or in groups, with a certain time interval between administrations. The time intervals can be the same or different between each administration of the PDE9 inhibitor of the present invention and the at least one other active agent, and can be selected, for example, from the range of 2 minutes to 96 hours, 1 to 7 days, or 1, 2, or 3 weeks. Usually, the time interval between administrations can be in the range of several minutes to several hours, for example, in the range of 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Further examples include time intervals in the range of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.

[0079] The molar ratio of the PDE9 inhibitor of the present invention and at least one other active agent is not particularly limited.For example, when the PDE9 inhibitor of the present invention and one other active agent are combined in a composition, their molar ratio can be in the range of 1:500-500:1, or in the range of 1:100-100:1, or in the range of 1:50-50:1, or in the range of 1:20-20:1, or in the range of 1:5-5:1, or in the range of 1:1.When the PDE9 inhibitor of the present invention and two or more other active agents are combined in a composition, the same molar ratio applies. The PDE9 inhibitors of the present invention may comprise a given molar weight percentage of the composition, between about 1% and 10%, or between about 10% and about 20%, or between about 20% and about 30%, or between about 30% and 40%, or between about 40% and 50%, or between about 50% and 60%, or between about 60% and 70%, or between about 70% and 80%, or between about 80% and 90%, or between about 90% and 99%.

[0080] The other active agent may be a different PDE9 inhibitor of the present invention or HU. The other active agent may also be an antibiotic such as penicillin, a nonsteroidal anti-inflammatory drug (NSAIDS) such as diclofenac or naproxen, an analgesic such as an opioid, or folic acid.

[0081] Yet another aspect of the present invention provides methods of using the PDE9 inhibitors of the present invention in combination with at least one other therapy, such as, but not limited to, blood transfusion, bone marrow transplant, or gene therapy.

[0082] IV. KITS AND DEVICES

[0083] The present invention provides various kits and devices for conveniently and effectively carrying out the methods of the present invention. Typically, the kits will contain sufficient amounts and / or numbers of components to allow a user to perform multiple treatments of subjects and / or perform multiple experiments.

[0084] In one embodiment, the present invention provides a kit for treating sickle cell disease comprising a PDE9 inhibitor compound of the present invention or a combination of PDE9 inhibitor compounds of the present invention, optionally in combination with any other active agent such as HU, an antibiotic such as penicillin, a nonsteroidal anti-inflammatory drug (NSAIDS) such as diclofenac or naproxen, an analgesic such as an opioid, or folic acid.

[0085] The kit may further include packaging and instructions and / or a delivery agent for forming a formulation composition. The delivery agent may include saline, buffer, or any delivery agent disclosed herein. The amount of each component may be varied to allow for consistent, reproducible, more concentrated saline or simple buffer formulations. The components may also be varied to increase the stability of the PDE9 inhibitor compound in the buffer over a period of time and / or under various conditions.

[0086] The present invention provides devices that can incorporate the PDE9 inhibitor compounds of the present invention. These devices contain stable formulations that are available for immediate delivery to a subject in need thereof, such as a human patient with sickle cell disease or beta thalassemia.

[0087] Non-limiting examples of device include pump, catheter, needle, transdermal patch, pressurized olfactory delivery device, iontophoretic device, multi-layer microfluidic device.Device can be used to deliver the PDE9 inhibitor compound of the present invention according to single, multiple or divided administration regime.Device can be used to deliver the PDE9 inhibitor compound of the present invention to whole body tissue, intradermally, subcutaneously or intramuscularly. More examples of devices suitable for delivering PDE9 inhibitor compounds include medical devices for intravesical drug delivery as disclosed in International Publication WO 2014036555, vials made of type I glass as disclosed in US Patent Publication No. 20080108697, drug eluting devices comprising a film made of a degradable polymer and an active agent as disclosed in US Patent Publication No. 20140308336, infusion devices with an infusion micropump as disclosed in US Patent Publication No. 5716988, or containers containing a pharma- ceutical stable formulation of an active agent, implantable devices comprising a reservoir and a channeled member in fluid communication with the reservoir as disclosed in International Publication WO 2015023557, hollow fiber-based biocompatible drug delivery devices with one or more layers as disclosed in US Patent Publication No. 20090220612, and the like. No. 2013170069, and the bioabsorbable implant device disclosed in U.S. Pat. No. 7,326,421, the contents of each of which are incorporated herein by reference in their entirety.

[0088] V. Definition

[0089] The articles "a" and "an," as used herein, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0090] The phrase "and / or," as used herein, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements, other than those specifically identified by the "and / or" clause, may be present as appropriate, whether or not related to those elements specifically identified, unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," refers in one embodiment to A without B (optionally including elements other than B), in another embodiment to B without A (optionally including elements other than A), and in yet another embodiment to both A and B (optionally including other elements).

[0091] As used herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed inclusively, i.e., including at least one of a number or list of elements, but also including a plurality, and including additional unlisted items, as appropriate. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of one element of a number or list of elements b.

[0092] In general, the term "or" as used herein shall only be construed as indicating exclusive alternatives (i.e., either one or the other, but not both) when preceded by terms of exclusivity such as "either," "one," "only one," or "exactly one." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0093] As used herein, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition allows for elements to optionally be present other than the elements specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0094] Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer to, in one embodiment, at least one, optionally including more than one A, and no B (and optionally including elements other than B), in another embodiment, at least one, optionally including more than one B, and no A (and optionally including elements other than A), in yet another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements), etc.

[0095] As used herein, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like, should be understood to be open-ended, i.e., meaning including but not limited to.

[0096] As set forth in the United States Patent Office Manual of Patent Examining Procedure, only the transitional phrases "consisting of" and "consisting essentially of" must be closed or semi-closed transitional phrases, respectively.

[0097] As used herein, "subject" or "patient" refers to any mammal, such as a mammal susceptible to a disease or disorder, e.g., tumor formation or cancer (e.g., a human). Examples include humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents, such as mice, rats, hamsters, or guinea pigs. In various embodiments, a subject refers to one that has been or is the subject of a treatment, observation, or experiment. For example, a subject may be a subject diagnosed with or known to have cancer, or a subject selected for treatment, observation, or experiment based on a known cancer in the subject.

[0098] As used herein, "treatment" or "treating" refers to the amelioration of a disease or disorder, or at least one sign or symptom thereof. "Treatment" or "treating" can refer to a reduction in the progression of a disease or disorder, e.g., as determined by the stabilization of at least one sign or symptom, or a reduction in the rate of progression, e.g., as determined by a reduction in the rate of progression of at least one sign or symptom. In another embodiment, "treatment" or "treating" refers to delaying the onset of a disease or disorder.

[0099] As used herein, "prevention" or "preventing" refers to the reduction in the risk of acquiring or having the signs or symptoms of a given disease or disorder, i.e., prophylactic treatment.

[0100] As used herein, the phrase "therapeutically effective amount" refers to an amount of a compound, material, or composition, including a compound of the present teachings, effective to produce a desired therapeutic effect. Accordingly, a therapeutically effective amount treats or prevents a disease or disorder, e.g., ameliorates at least one sign or symptom of the disorder. In some embodiments, the disease or condition is cancer.

[0101] A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment of the substituent. For example, -CONH2 is attached through a carbon atom (C).

[0102] By "optional" or "optionally," it is meant that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur. For example, "optionally substituted aryl" encompasses both "aryl" and "substituted aryl" as defined herein. With respect to any functional group that contains one or more substituents, it will be understood by those of skill in the art that such functional groups are not intended to introduce any substitutions or substitution patterns that are sterically impractical, synthetically impractical, and / or inherently unstable.

[0103] The term "alkyl" as used herein refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-22, 1-8, 1-6, or 1-4 carbon atoms, respectively, (C1-C 22)alkyl, (C1-C8)alkyl, (C1-C6)alkyl, and (C1-C4)alkyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

[0104] The term "alkenyl," as used herein, refers to an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond (e.g., represented as "="), such as a straight-chain or branched group of 2-22, 2-8, 2-6, or 2-4 carbon atoms, respectively (C2-C 22 )alkenyl, (C2-C8)alkenyl, (C2-C6)alkenyl, and (C2-C4)alkenyl. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl.

[0105] The term "alkynyl," as used herein, refers to an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon triple bond (e.g., depicted as ""), such as a straight-chain or branched group of 2-22, 2-8, 2-6, or 2-4 carbon atoms, respectively, (C2-C 22)alkynyl, (C2-C8)alkynyl, (C2-C6)alkynyl, and (C2-C4)alkynyl. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl.

[0106] The term "cycloalkyl" as used herein refers to a saturated or unsaturated monocyclic, bicyclic, other polycyclic, or bridged ring hydrocarbon group. Cycloalkyl groups have 3-22, 3-12, or 3-8 ring carbons, respectively (C3-C4). 22 )cycloalkyl, (C3-C 12 )cycloalkyl, or (C3-C8)cycloalkyl. Cycloalkyl groups can also have one or more carbon-carbon double bonds or carbon-carbon triple bonds.

[0107] Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopentane (cyclopentyl), cyclopentene (cyclopentenyl), cyclohexane (cyclohexyl), cyclohexene (cyclohexenyl), cycloheptane (cycloheptyl), cycloheptene (cycloheptenyl), cyclooctane (cyclooctyl), cyclooctene (cyclooctenyl), cyclononane (cyclononyl), cyclononene (cyclononenyl), cyclodecane (cyclodecyl), cyclodecene (cyclodecenyl), cycloundecane (cycloundecyl), cycloundecene (cycloundecenyl), cyclododecane (cyclododecyl), and cyclododecene (cyclododecenyl). Other exemplary cycloalkyl groups, including bicyclic, polycyclic, and bridged ring groups, include, but are not limited to, bicyclobutane (bicyclobutyl), bicyclopentane (bicyclopentyl), bicyclohexane (bicyclohexyl), bicycloheptane (bicycloheptyl, including bicyclo[2,2,1]heptane) (bicyclo[2,2,1]heptyl) and bicyclo[3,2,0]heptane (bicyclo[3,2,0]heptyl), bicyclooctane (bicyclooctyl, including octahydropentalene (octahydropentalenyl), bicyclo[3,2,1]octane (bicyclo[3,2,1]octyl), and bicyclo[2,2,2]octane (bicyclo[2,2,2]octyl)), and adamantane (adamantyl). The cycloalkyl group can be fused to another cycloalkyl saturated or unsaturated, aryl, or heterocyclyl group.

[0108] The term "aryl" as used herein refers to a monocyclic, bicyclic, or other polycarbocyclic aromatic ring system. Aryl has 6-22, 6-18, 6-14, or 6-10 carbons, respectively, (C6-C 22 )Aryl, (C6-C 18 )Aryl, (C6-C 14 ) aryl, or (C6-C 10 )Aryl(C6-C 14), which are referred to herein as aryl, cycloalkyl, and heterocyclyl. The term "bicyclic aryl" as used herein refers to an aryl group fused to another aromatic or non-aromatic carbocyclic or heterocyclic ring. Exemplary aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. Exemplary aryl groups also include, but are not limited to, monocyclic aromatic ring systems, in which the ring contains six carbon atoms, referred to herein as "(C6)aryl" or phenyl. A phenyl group can also be fused to a cyclohexane or cyclopentane ring to form another aryl.

[0109] The term "arylalkyl" as used herein refers to an alkyl group having at least one aryl substituent (e.g., -aryl-alkyl-). Exemplary arylalkyl groups include, but are not limited to, arylalkyl groups having a monocyclic aromatic ring system, the ring containing 6 carbon atoms, referred to herein as "(C6)arylalkyl". The term "benzyl" as used herein refers to a -CH2-phenyl group.

[0110] The term "heteroalkyl" refers to an alkyl group as described herein in which one or more carbon atoms are replaced with a heteroatom. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and the like.

[0111] The terms "heteroalkenyl" and "heteroalkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the heteroalkyls described above, but that contain at least one double or triple bond respectively.

[0112] The term "heterocycle" refers to a cyclic group containing at least one heteroatom as a ring atom, in some cases 1-3 heteroatoms as ring atoms, with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and the like. In some cases, the heterocycle can be a 3- to 10-membered ring structure or a 3- to 7-membered ring structure, the ring structure containing 1-4 heteroatoms. The term "heterocycle" can include a heteroaryl group, a saturated heterocycle (e.g., cycloheteroalkyl) group, or a combination thereof. The heterocycle can be a saturated molecule or can contain one or more double bonds. In some cases, the heterocycle is a nitrogen heterocycle, with at least one ring containing at least one nitrogen ring atom. The heterocycle can be fused to other rings to form a polycyclic heterocycle. Thus, the heterocycle can also be any of the above heterocycles fused to one or two rings independently selected from aryl, cycloalkyl, and heterocycle. Heterocyclic rings can also be fused to spirocyclic groups.

[0113] Examples of heterocycles include thiophene, benzothiophene, thianthrene, furan, tetrahydrofuran, pyran, isobenzofuran, chromene, xanthene, phenoxate, pyrrole, dihydropyrrole, pyrrolidine, imidazole, pyrazole, pyrazine, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, thiazole ... Examples of suitable aryl groups include riazoles, tetrazoles, oxazoles, isoxazoles, thiazoles, isothiazoles, phenanthridines, acridines, pyrimidines, phenanthrolines, phenazines, phenarsazines, phenothiazines, furazans, phenoxazines, pyrrolidines, oxolanes, thiolanes, oxazoles, oxazines, piperidine, homopiperidine (hexamethyleneimine), piperazines (e.g., N-methylpiperazine), morpholines, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and other saturated and / or unsaturated derivatives.

[0114] In some cases, the heterocycle may be bonded to the compound through a heteroatom ring atom (e.g., nitrogen). In some cases, the heterocycle may be bonded to the compound through a carbon ring atom. In some cases, the heterocycle is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridine-9-amine, bipyridine, naphthyridine, quinoline, isoquinoline, benzoquinoline, benzoisoquinoline, phenanthridine-1,9-diamine, etc.

[0115] The term "heteroaromatic" or "heteroaryl" as used herein refers to a monocyclic, bicyclic, or polycyclic aromatic ring system containing one or more heteroatoms, e.g., 1-3 heteroatoms such as nitrogen, oxygen, and sulfur. Heteroaryls can also be fused to non-aromatic rings. In various embodiments, the term "heteroaromatic" or "heteroaryl" as used herein refers to a stable 5- to 7-membered monocyclic, stable 9- to 10-membered fused bicyclic, or stable 12- to 14-membered fused tricyclic heterocycle containing an aromatic ring containing at least one heteroatom selected from the group consisting of N, O, and S, unless otherwise specified. In some embodiments, at least one nitrogen is in an aromatic ring.

[0116] Heteroaromatics or heteroaryls can include, but are not limited to, monocyclic aromatic rings, where the ring contains 2 to 5 carbon atoms and 1 to 3 heteroatoms, referred to herein as "(C2-C5)heteroaryl. Illustrative examples of monocyclic heteroaromatics (or heteroaryls) include, but are not limited to, pyridine (pyridinyl), pyridazine (pyridazinyl), pyrimidine (pyrimidinyl), pyrazine (pyrazyl), triazine (triazinyl), pyrrole (pyrrolyl), pyrazo, pyrazolyl ... Examples of aromatic aryl groups include pyrazole (pyrazolyl), imidazole (imidazolyl), (1,2,3)- and (1,2,4)-triazole ((1,2,3)- and (1,2,4)-triazolyl), pyrazine (pyrazinyl), pyrimidine (pyrimidinyl), tetrazole (tetrazolyl), furan (furyl), thiophene (thienyl), isoxazole (isoxazolyl), thiazole (thiazolyl), isoxazole (isoxazolyl), and oxazole (oxazolyl).

[0117] The term "bicyclic heteroaromatic" or "bicyclic heteroaryl" as used herein refers to a heteroaryl group fused to another aromatic or non-aromatic carbocyclic or heterocyclic ring. Exemplary bicyclic heteroaromatics or heteroaryls include, but are not limited to, 5,6- or 6,6-fused systems in which one or both rings contain heteroatoms. The term "bicyclic heteroaromatic" or "bicyclic heteroaryl" also includes reduced or partially reduced forms of fused aromatic systems in which one or both rings contain ring heteroatoms. The ring system may contain up to three heteroatoms independently selected from oxygen, nitrogen, and sulfur.

[0118] Exemplary bicyclic heteroaromatics (or heteroaryls) include, but are not limited to, quinazoline (quinazolinyl), benzoxazole (benzoxazolyl), benzothiophene (benzothiophenyl), benzoxazole (benzoxazolyl), benzisoxazole (benzisoxazolyl), benzimidazole (benzimidazolyl), benzothiazole (benzothiazolyl), benzfuran (benzofuranylin), benzisothiazole (benzisothiazolyl), indole (indolyl), indazole (indazolyl), indolizine (indolizinyl), quinoline (quinolinyl), isoquinoline (isoquinolinyl), naphthyridine (naphthyridyl), phthalazine (phthalazinyl), phthalazine (phthalazinyl) purine (purinyl), benzotriazole (benzotriazolyl), and benzofuran (benzofuranyl). In some embodiments, the bicyclic heteroaromatic (or bicyclic heteroaryl) is selected from quinazoline (quinazolinyl), benzimidazole (benzimidazolyl), benzothiazole (benzothiazolyl), indole (indolyl), quinoline (quinolinyl), isoquinoline (isoquinolinyl), and phthalazine (phthalazinyl). In certain embodiments, the bicyclic heteroaromatic (or bicyclic heteroaryl) is quinoline (quinolinyl) or isoquinoline (isoquinolinyl).

[0119] The term "tricyclic heteroaromatic" or "tricyclic heteroaryl" as used herein refers to a bicyclic heteroaryl group fused to another aromatic or non-aromatic carbocyclic or heterocyclic ring. The term "tricyclic heteroaromatic" or "tricyclic heteroaryl" also encompasses reduced or partially reduced forms of fused aromatic systems in which one or both rings contain ring heteroatoms. Each of the rings in a tricyclic heteroaromatic (tricyclic heteroaryl) can contain up to three heteroatoms independently selected from oxygen, nitrogen, and sulfur.

[0120] Exemplary tricyclic heteroaromatics (or heteroaryls) include, but are not limited to, acridine (acridinyl), 9H-pyrido[3,4-b]indole (9H-pyrido[3,4-b]indolyl), phenanthridine (phenanthridinyl), pyrido[1,2-a]benzimidazole (pyrido[1,2-a]benzimidazolyl), and pyrido[1,2-b]indazole (pyrido[1,2-b]indazolyl).

[0121] The term "alkoxy" as used herein refers to an alkyl group bonded to oxygen (-O-alkyl-). An "alkoxy" group also includes an alkenyl group bonded to oxygen ("alkenyloxy") or an alkynyl group bonded to oxygen ("alkynyloxy"). Exemplary alkoxy groups include, but are not limited to, groups having alkyl, alkenyl or alkynyl groups of 1 to 22, 1 to 8, or 1 to 6 carbon atoms, each of which are defined herein as (C1-C 22 )alkoxy, (C1-C8)alkoxy, or (C1-C6)alkoxy. Exemplary alkoxy groups include, but are not limited to, methoxy and ethoxy.

[0122] The term "cycloalkoxy" as used herein refers to a cycloalkyl group attached to an oxygen.

[0123] The term "aryloxy" or "aroxy" as used herein refers to an aryl group bonded to an oxygen atom. Exemplary aryloxy groups include, but are not limited to, aryloxy having a monocyclic aromatic ring system, the ring containing six carbon atoms, referred to herein as "(C6)aryloxy". The term "arylalkoxy" as used herein refers to an arylalkyl group bonded to an oxygen atom. An exemplary aralkyl group is the benzyloxy group.

[0124] As used herein, the term "amine" or "amino" refers to both unsubstituted and substituted amines, e.g., NR a R b R b’R a , R b , and R b’ is independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, carbamate, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen; R a , R b , and R b’ At least one of R is not hydrogen. The amine or amino may be attached to the parent molecular group through the nitrogen. The amine or amino may also be cyclic, e.g., R a , R b , and R b’ Any two of may be joined together and / or with N to form a 3-12 membered ring (e.g., morpholino or piperidinyl). The term amino also includes the corresponding quaternary ammonium salts of any amino group. Exemplary amines include R a , R b or R b’ an alkylamine, in which at least one of R is an alkyl group; a , R b or R b’ At least one of the groups is a cycloalkyl group.

[0125] The term "ammonia" as used herein refers to NH3.

[0126] The term "aldehyde" or "formyl" as used herein refers to --CHO.

[0127] The term "acyl" as used herein refers to a carbonyl radical bound to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. Exemplary acyl groups include, but are not limited to, acetyl, formyl, propionyl, benzoyl, and the like.

[0128] As used herein, the term "amide" refers to -NR c C(O)(R d )- or -C(O)NRc R e Refers to the format of R c , R d , and R e are each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. Amides are formed from carbon, nitrogen, R c , R d , R e The amide may also be cyclic, for example, R c and R e can be linked to form a 3-12 membered ring, such as a 3-10 membered ring or a 5- or 6-membered ring. The term "amide" includes groups such as sulfonamide, urea, ureido, carbamate, carbamic acid, and cyclic versions thereof. The term "amide" also includes salts of an amide group attached to a carboxy group, such as -amide-COOH or -amide-COONa.

[0129] The term "arylthio" as used herein refers to an aryl group attached to a sulfur atom. Exemplary arylthio groups include, but are not limited to, arylthio groups having a monocyclic aromatic ring system, the ring containing 6 carbon atoms, referred to herein as "(C6)arylthio".

[0130] The term "arylsulfonyl" as used herein refers to an aryl group bonded to a sulfonyl group, e.g., -S(O)2-aryl-. Exemplary arylsulfonyl groups include, but are not limited to, arylsulfonyl having a monocyclic aromatic ring system, the ring containing 6 carbon atoms, referred to herein as "(C6)arylsulfonyl."

[0131] As used herein, the term "carbamate" refers to a -R f O C (O) N (R g )-, -R f O C (O) N (R g )R h -, or -OC(O)NR g R h Refers to the format of Rf , R g and R h are each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, and hydrogen. Exemplary carbamates include, but are not limited to, aryl carbamates or heteroaryl carbamates (e.g., R f , R g , and R h wherein at least one of is independently selected from aryl or heteroaryl, such as pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl.

[0132] The term "carbonyl" as used herein refers to -C(O)-.

[0133] As used herein, the term "carboxy" or "carboxylate" refers to a j -COOH or its corresponding carboxylate (e.g., R j -COONa), R j may be independently selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, cycloalkyl, ether, haloalkyl, heteroaryl, and heterocyclyl. Exemplary carboxy includes, but is not limited to, R j Exemplary carboxy also includes aryl or heteroaryl carboxy, e.g., R j is an aryl group such as phenyl and tolyl, or a heteroaryl group such as pyridine, pyridazine, pyrimidine and pyrazine. The term "carboxy" includes "carboxycarbonyl", e.g., a carboxy group bound to a carbonyl group, e.g., salts such as -C(O)-COOH or -C(O)-COONa.

[0134] The term "dicarboxylic acid" as used herein refers to a group containing at least two carboxylic acid groups, such as saturated and unsaturated hydrocarbon dicarboxylic acids and their salts. Exemplary dicarboxylic acids include alkyl dicarboxylic acids. Dicarboxylic acids include, but are not limited to, succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, maleic acid, phthalic acid, aspartic acid, glutamic acid, malonic acid, fumaric acid (+) / (-)-malic acid, (+) / (-)-tartaric acid, isophthalic acid, and terephthalic acid. Dicarboxylic acids further include their carboxylic acid derivatives, such as anhydrides, imides, and hydrazides (e.g., succinic anhydride and succinimide).

[0135] The term "cyano" as used herein refers to --CN.

[0136] The term "ester" refers to an ester having the structure -C(O)O-, -C(O)OR i -, -R j C(O)OR i -or-R j C(O)O-, where O is not bonded to a hydrogen and R i and R j R may be independently selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, cycloalkyl, ether, haloalkyl, heteroaryl, and heterocyclyl. i can be hydrogen, but R j cannot be hydrogen. For example, the ester may be cyclic, e.g., having a carbon atom and R j , an oxygen atom and R i , or R i and R j may be linked to form a 3- to 12-membered ring. Exemplary esters include, but are not limited to, R i or R j wherein at least one of R is alkyl, e.g., -OC(O)-alkyl, -C(O)-O-alkyl-, and -alkyl-C(O)-O-alkyl-. Exemplary esters also include aryl or heteroaryl esters, e.g., Ri or R j at least one of is an aryl group, such as phenyl or tolyl, or a heteroaryl group, such as a pyridine, pyridazine, pyrimidine or pyrazine, such as a nicotinate ester. Exemplary esters are also represented by the structure -R j Reverse esters include those with C(O)O-, where the oxygen is attached to the parent molecule. Exemplary reverse esters include succinate, D-arginate, L-arginate, L-lysinate, and D-lysinate. Esters also include carboxylic acid anhydrides and acid halides.

[0137] The term "ether" refers to the structure -R k OR l - refers to R k and R l can be independently alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl, and ether. An ether is R k or R l Exemplary ethers include, but are not limited to, alkoxyalkyl and alkoxyaryl groups. Ethers can also be linked to the parent molecular group via, for example, R k and R l wherein one or both of said groups are ethers.

[0138] The terms “halo” or “halogen” or “hal” or “halide” as used herein refer to F, Cl, Br, or I.

[0139] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more halogen atoms.

[0140] As used herein, the terms "hydroxy" and "hydroxyl" refer to --OH.

[0141] The term "hydroxyalkyl," as used herein, refers to a hydroxy attached to an alkyl group.

[0142] The term "hydroxyaryl," as used herein, refers to a hydroxy attached to an aryl group.

[0143] As used herein, the term "ketone" refers to a compound having the structure -C(O)-R m (e.g., acetyl (-C(O)CH3 or -R m -C(O)-R n Ketones are represented by R m or R n R m or R n can be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl, or R m or R n can be linked to form, for example, a 3- to 12-membered ring.

[0144] The term "monoester" as used herein refers to the analog of a dicarboxylic acid where one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is the free carboxylic acid or a salt of a carboxylic acid. Examples of monoesters include, but are not limited to, the monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic acid, and maleic acid.

[0145] The term "nitro" as used herein refers to --NO.sub.2.

[0146] As used herein, the term "nitrate" refers to NO3 - Refers to...

[0147] As used herein, the term "perfluoroalkyl" refers to an alkyl group in which all of the hydrogen atoms have been replaced with fluorine atoms. Exemplary perfluoroalkyl groups include, but are not limited to, C1-C5 perfluoroalkyl, such as trifluoromethyl.

[0148] As used herein, the term "perfluorocycloalkyl" refers to a cycloalkyl group in which all of the hydrogen atoms have been replaced with fluorine atoms.

[0149] As used herein, the term "perfluoroalkoxy" refers to an alkoxy group in which all of the hydrogen atoms have been replaced with fluorine atoms.

[0150] As used herein, the term "phosphate" refers to a compound having the structure -OP(O)O2 2- , -R o OP(O)O2 2- , -OP(O)(OR q )O - , or -R o OP(O)(OR p )O - R o , R p and R q Each may independently be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl, or hydrogen.

[0151] The term "sulfide" as used herein refers to a compound having the structure -R q S- refers to R q can be alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl. The sulfide may be cyclic, for example, forming a 3- to 12-membered ring. As used herein, the term "alkyl sulfide" refers to an alkyl group bonded to a sulfur atom.

[0152] As used herein, the term “sulfinyl” refers to a group having the structure —S(O)O—, —R r S(O)O-, -R r S(O)OR s -OR-S(O)OR s - refers to R r and R s can be alkyl, alkenyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl, hydroxyl. Exemplary sulfinyl groups include, but are not limited to, R r or R s At least one of is alkyl, alkenyl, or alkynyl.

[0153] As used herein, the term “sulfonamide” refers to a compound having the structure —(R t )-NS(O)2-R v -or-R t (R u )NS(O)2-R v R t , R u and R v can be, for example, hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl and heterocyclyl. Exemplary sulfonamides are alkylsulfonamides (e.g., R v is alkyl), arylsulfonamides (e.g., when R v is aryl), cycloalkylsulfonamides (e.g., when R v is cycloalkyl), and heterocyclylsulfonamides (e.g., where R v is heterocyclyl).

[0154] As used herein, the term "sulfonate" refers to a salt or ester of a sulfonic acid. w SO3H, R w is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, or heterocyclyl (e.g., alkylsulfonyl). As used herein, the term "sulfonyl" refers to a group represented by the structure R x SO2-, R x can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, and heterocyclyl (e.g., alkylsulfonyl). As used herein, the term "alkylsulfonyl" refers to an alkyl group attached to a sulfonyl group. An "alkylsulfonyl" group can contain an alkenyl or alkynyl group, as appropriate.

[0155] As used herein, the term "sulfonate" refers to a compound represented by R w SO3 - Refers to. R wis alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, hydroxyl, alkoxy, aroxy, or aralkoxy, each of which is optionally substituted. A non-limiting example is triflate (trifluoromethanesulfonic acid, CF3SO3 - These include the benzenesulfonate salt, the tosylate (also known as the toluenesulfonate salt), and the like.

[0156] The term "thioketone" refers to a compound having the structure -R y -C(S)-R z - Ketones are R y or R z R y or R z can be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or aryl; R y or R z can be linked to form a ring, for example a 3- to 12-membered ring.

[0157] Each of the above groups may be substituted as appropriate. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds, with "permissible" being in the context of the chemical rules of valency known to those skilled in the art. It will be understood that "substituted" also includes those substitutions that result in stable compounds that do not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like. In some cases, "substituted" may generally refer to the replacement of a hydrogen with a substituent as described herein. However, "substituted," as used herein, does not encompass the substitution and / or modification of the functional group with which the molecule is identified, for example, such that the "substituted" functional group becomes a different functional group through substitution. For example, a "substituted phenyl group" must still include a phenyl moiety and, by this definition, cannot be modified by substitution, for example, to become a pyridine ring.

[0158] In a broad range of embodiments, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. Permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of the present teachings, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituent of organic compounds described herein that satisfies the valence of the heteroatom.

[0159] In various embodiments, the substituents are selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, (thioketone, each of which is optionally substituted with one or more suitable substituents). In some embodiments, the substituents are selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, each of which can be further substituted with one or more suitable substituents.

[0160] Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, thioketone, ester, heterocyclyl, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroar ... These include koxy, azido, alkylthio, oxo, acylalkyl, carboxyester, carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl, carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl, etc. In some embodiments, the substituent is selected from cyano, halogen, hydroxyl, and nitro.

[0161] As a non-limiting example, in various embodiments, NR a R b R b’ R a , R b , and R b’is selected from alkyl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl, each of the alkyl, alkenyl, alkynyl, cycloalkyl, and heterocyclyl may be optionally substituted with one or more substituents each independently selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone, and each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amido, amino, aryl, arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone may be further substituted with one or more suitable substituents. In some embodiments, when the amine is an alkylamine or cycloalkylamine, the alkyl or cycloalkyl can be substituted with one or more substituents independently selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonic acid, sulfonamide, and thioketone. In certain embodiments, when the amine is an alkylamine or cycloalkylamine, the alkyl or cycloalkyl can be substituted with one or more substituents independently selected from amino, carboxy, cyano, and hydroxyl. For example, the alkyl or cycloalkyl in the alkylamine or cycloalkylamine is substituted with an amino group to form a diamine.

[0162] As used herein, "suitable substituents" refers to groups that do not abrogate the synthetic or pharmaceutical usefulness of the compounds of the invention or intermediates useful in their preparation. Examples of suitable substituents include, but are not limited to, (C1-C 22 ), (C1-C8), (C1-C6) or (C1-C4) alkyl, alkenyl or alkynyl; (C6-C 22 ), (C6-C 18 ), (C6-C 14 ) or (C6-C 10 )Aryl;(C2-C 21 ), (C2-C 17 ), (C2-C 13 ) or (C2-C9)heteroaryl; (C3-C 22 ), (C3-C 12 ) or (C3-C8)cycloalkyl; (C1-C 22 ), (C1-C8), (C1-C6), (C1-C4)alkoxy; (C6-C 22 ), (C6-C 18 ), (C6-C 14 ) or (C6-C 10 )aryloxy;-CN;-OH;oxo;halo;carboxy;-NH((C1-C 22 ), (C1-C8), (C1-C6), or (C1-C4) alkyl), -N(C1-C 22 ), (C1-C8), (C1-C6), or (C1-C4) alkyl), -NH(C6) (aryl) or -N((C6-C 10 )aryl)2, such as amino; formyl; -CO((C1-C 22 ), (C1-C8), (C1-C6), or (C1-C4) alkyl), -CO2((C1-C 22 ), (C1-C8), (C1-C6)), or (C1-C4) alkyl) and -CO2(C6-C 10 )(aryl) and the like -CO(((C6-C 10 )aryl)esters. Those skilled in the art can easily select appropriate substituents based on the stability and pharmacological and synthetic activity of the compounds of the present invention.

[0163] Unless otherwise specified, chemical groups include their corresponding monovalent, divalent, trivalent and tetravalent radicals. For example, methyl refers to monovalent methyl (-CH3), divalent methyl (-CH2-, methylyl), trivalent methyl (-CH3-, methylyl),

[0164] [ka] and tetravalent methyl

[0165] [ka] Includes.

[0166] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, reaction conditions, and other properties or parameters used in the specification and claims should be understood to be modified in all instances by the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims should be understood to be approximate. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, the numerical parameters should be interpreted in light of the number of reported significant digits and the application of ordinary rounding techniques. For example, the term "about" can encompass a variation of ±10%, ±5%, ±2%, ±1%, ±0.5%, or ±0.1% of the numerical value of the number that it modifies. In various embodiments, the term "about" encompasses a variation of ±5%, ±2%, ±1%, or ±0.5% of the numerical value of the number. In some embodiments, the term "about" encompasses a variation of ±5%, ±2%, or ±1% of the numerical value of the number. In certain embodiments, the term "about" encompasses a ±5% variation of the numerical value of a number. In certain embodiments, the term "about" encompasses a ±2% variation of the numerical value of a number. In certain embodiments, the term "about" encompasses a ±1% variation of the numerical value of a number.

[0167] All numerical ranges herein include all numerical values ​​and ranges of numerical values ​​within the recited numerical ranges. As a non-limiting example, (C1-C6) alkyl also includes any one of C1, C2, C3, C4, C5, C6, (C1-C2) (C1-C3) (C1-C4) (C1-C5) (C2-C3) (C2-C4), (C2-C5), (C2-C6), (C3-C4), (C3-C5), (C3-C6), (C4-C5), (C4-C6)), and (C5-C6) alkyl.

[0168] Further, notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values ​​set forth in the Examples section are reported as precisely as possible. It should be understood, however, that such numerical values ​​inherently contain certain errors resulting from measuring devices and / or measuring techniques. List of abbreviations and terms 1H-NMR: Proton nuclear magnetic resonance spectroscopy ADME: Absorption, Distribution, Metabolism and Excretion AE: Adverse event AUC 0-24 : Area under the concentration-time curve from 0 to 24 hours after administration BBB: Blood-brain barrier Cmax: maximum plasma concentration cGMP: cyclic guanosine monophosphate DMSO: Dimethyl sulfoxide DSFC: Dorsal skin fold chamber F cell blood cell that uses fetal hemoglobin FIH: First in Human FTIR: Fourier transform infrared spectroscopy GC: Gas chromatography HBB: hemoglobin subunit β HbF: fetal hemoglobin HBG: gamma globin gene HbS: sickle hemoglobin hERG: human ether-a-go-go related gene HPLC: High Performance Liquid Chromatography HU: Hydroxyurea IC: inhibitory concentration IC50: Half minimum inhibitory concentration ICAM-1: Intercellular adhesion molecule-1 ICH: International Conference on Harmonisation ICP-MS: Inductively coupled plasma mass spectrometry IV: Intravenous MAD: Repeated administration MTD: Maximum tolerance NO: Nitric oxide NOAEL: No observed adverse effect level PD: Pharmacodynamics PDE9: phosphodiester-9 PEG: Polyethylene glycol PIC: Capsule powder PK: Pharmacokinetics(s) PKG: Protein kinase G RBC: Red blood cell RH: Relative humidity SCD: Sickle cell disease SD: standard deviation SEM: Standard error of the mean s GC: soluble guanylyl cyclase t1 / 2: half-life TK: Toxokinetics Tmax: time of maximum concentration VOCs: A vaso-occlusive crisis WBC: white blood cell w / w%: weight / weight percent EXAMPLES

[0169] The following examples are intended to illustrate, but not to limit, the present invention.Various other examples and modifications of the above description and examples will be apparent to those skilled in the art after reading the disclosure without departing from the spirit and scope of the present invention, and all such examples or modifications are intended to be included within the scope of the appended claims.All publications and patents referenced herein are incorporated herein by reference in their entirety.

[0170] Example 1. Synthesis of compound P3.1 List of abbreviations aq: water-based NBS: N-bromosuccinimide Boc: tert-butoxycarbonyl ℃: Celsius CDI: N,N-carbonyldiimidazole δH: Chemical shift in parts per million downfield from tetramethylsilane DCM: dichloromethane DEAD: Diethyl azodicarboxylate Dppf: Bis(diphenylphosphino)ferrocene DIPEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide eq: equivalent ESI: electrospray ionization Et: Ethyl EtOAc: ethyl acetate g: grams HPLC: High Performance Liquid Chromatography h: time Hz: Hertz J: Coupling constant (NMR spectroscopy) LCMS: Liquid Chromatography Mass Spectrometry LiHMDS: Lithium bis(trimethylsilyl)amide μ: Micro m: multiplet (spectrum); meter; millimeter M+: Parent molecular ion Me: Methyl MeCN: Acetonitrile MeOH: Methanol MHz: Megahertz min: minutes (s) mL: milliliter MS: Mass spectrometry MTBE: Methyl tert-butyl ether N: Normal (equivalents per liter) NaOH: Sodium hydroxide NBS: N-bromosuccinimide nm: nanometer (s) NMR: nuclear magnetic resonance PE: Petroleum Ether Boiling point: 60-90℃~207-208℃. RT: room temperature s: singlet (spectrum) t: triplet (spectrum) T: Temperature TEA: Triethylamine TFA: Trifluoroacetic acid THF: tetrahydrofuran TLC: Thin Layer Chromatography TMS: Tetramethylsilane TMS-Cl: Trimethylsilyl chloride Tol: Toluene

[0171] The compounds of the invention may be prepared using the methods disclosed in WO 2013 / 053690, WO 2013 / 110768 and / or WO 2017 / 005786. Alternatively, compound P3.1, which is the S,S enantiomer of P3, can be synthesized by the methods provided herein.

[0172] [ka] Compound P3.1 (chemical name: 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one or (3S,4S)-6-(4-methyl-1-pyrimidin-2-ylmethyl-)pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one)

[0173] The synthetic methods provided in this application do not involve chiral chromatographic separation of any intermediate or final product, nor do the methods involve separation of the racemate of the final product. Chiral chromatographic separation, as used herein, refers to a process or technique for the separation of a racemate into its enantiomers. Examples of chiral chromatographic separation include chiral HPLC, simulated moving bed (SMB) chromatography, or chiral supercritical fluid chromatography (SFC). When chiral chromatography such as chiral HPLC is used to determine chiral purity, chiral HP is not considered a chiral chromatographic separation in this respect.

[0174] In some embodiments, the synthetic methods provided in this application involve the resolution of racemic mixtures of intermediates based on diastereomeric salt formation. The diastereomeric salts of the intermediates can be separated based on their different solubilities. In some embodiments, washing and / or filtration techniques are used. Chiral chromatographic separation is not used to separate the diastereomeric salts.

[0175] In some embodiments, the synthetic method provided in the present application uses a chiral acid. The chiral acid reacts with the racemic mixture of intermediates to produce diastereomeric salts that can be separated without using chiral chromatographic separation, for example, based on their solubility. For example, the racemic mixture of intermediate 2 (Rac-2) can be reacted with a chiral acid to produce a solid intermediate that contains only the trans form of 2 (S,S-2).Non-limiting examples of chiral acids include (+)-2,3-dibenzoyl-D-tartaric acid 99.0% or more (T), dibenzoyl-L-tartaric acid 98%, (-)-O,O'-di-p-toluoyl-L-tartaric acid 97%, (+)-O,O'-di-p-toluoyl-D-tartaric acid, (+)-O,O'-di-pivaloyl-D-tartaric acid, (-)-O,O'-di-pivaloyl-D-tartaric acid, L-(+)-tartaric acid, (4R)- 2-Hydroxy-5,5-dimethyl-4-phenyl-1,3,2-dioxaphosphorinane 2-oxide 98%, L-(-)-Malic acid 97%, D-(+)-Malic acid, (R)-(-)-Mandelic acid, (S)-(+)-Mandelic acid, (R)-(-)-α-Methoxyphenylacetic acid, (S)-(+)-α-Methoxyphenylacetic acid, (R)-(+)-α-Methoxy-α-trifluoromethylphenylacetic acid, (S)-(-)-α-Methoxy-α-(trifluoromethyl)phenylacetic acid, (R)-(-)-2-Phenylpropionic acid, (S)-(+)-2-Phenylpropionic acid, (R)-1,4-Benzodioxane-2-carboxylic acid 97.0% or more (total of enantiomers, GC), (S)-1,4-Benzodioxane-2-carboxylic acid 97.0% or more (total of enantiomers) , GC), (R)-(-)-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate 98% or more, (S)-(+)-1,1'-binaphthyl-2,2'-98% or more, (S)-(+)-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate 97.0%, (1S)-(+)-3-bromocamphor-10-sulfonic acid hydrate 98%, (1R)-(+)-camphanic acid 98% , (1S)-(-)-camphanic acid 98%, (1R,3S)-(+)-camphanic acid 99%, (1R,3S)-(-)-camphanic acid 99%, (1R)-(-)-10-camphorsulfonic acid 98%, (1S)-(+)-10-camphorsulfonic acid 99%, (R)-(-)-5-oxo-2-tetrahydrofuran carboxylic acid, (S)-(+)-5-oxo-2-tetrahydrofuran carboxylic acid, D-(-)-quinic acid. A person skilled in the art may carry out any suitable method known in the art to screen and select chiral acids that provide good chemical yields and high stereochemical purity.In one embodiment, the synthetic methods provided in the present application use (+)-O,O-dibenzoyl-D-tartaric acid.

[0176] The yield of compound P3.1 is at least about 50%, about 60%, about 70%, about 80%, or about 90%. Yield, as used herein, refers to the ratio of the actual product weight and the theoretical product weight calculated from the amount of starting materials of the reaction. For example, the yield of compound P3.1 synthesized from intermediate 12 is the actual weight of compound P3.1 divided by the theoretical weight of compound P3.1 calculated from the amount of intermediate 12.

[0177] The chiral purity of compound P3.1 is greater than about 95.0%, about 96.0%, about 97.0%, about 98.0%, about 99.0%, about 99.5%, about 99.8%, or about 99.9%. The chiral purity of compound P3.1 is calculated by dividing the amount of pure (S,S) enantiomer by the total amount of all enantiomers. Chiral purity can be measured using any suitable technique, for example, chiral HPLC, polarimeter, or chiral NMR shift reagents.

[0178] The purity of compound P3.1 is greater than about 95.0%, about 96.0%, about 97.0%, about 98.0%, about 99.0%, about 99.5%, about 99.8% or about 99.9%. The purity of compound P3.1 is calculated by dividing the amount of compound P3.1 by the total amount of the product including all impurities. The purity of compound P3.1 can be determined by any suitable method, such as HPLC, GC (gas chromatography), mass spectrometry, or NMR.

[0179] Compound P3.1 prepared from the synthetic methods disclosed in this application is substantially free of impurities. The impurities may include chemical impurities and / or physical impurities. The chemical impurities may include starting materials, intermediates, solvents, reagents, and / or any by-products. The level of impurities may be less than about 5%, about 2%, about 1%, about 0.5%, about 0.2%, or about 0.1%.

[0180] The synthesis of compound P3.1 (also referred to as (S,S)-P3) follows the following steps, which do not involve chiral chromatographic separation of intermediates Rac-2, (S,S)-2, (S,S)-3, (S,S)-4, (S,S)-10, (S,S)-11, (S,S)-12, or the final product P3.1.

[0181] Synthesis of intermediate (S,S)-4:

[0182] [ka]

[0183] Synthesis of intermediate 9:

[0184] [ka]

[0185] Synthesis of final compound P3.1:

[0186] [ka]

[0187] Racemic trans-1-benzyl-4-methyl-pyrrolidine-3-carboxylic acid ethyl ester (1)

[0188] [ka]

[0189] A solution of but-2-enoic acid ethyl ester (125 g, 1.10 mol) in toluene (1700 g) was prepared at 20 °C under nitrogen and TFA (7.68 g, 0.068 mol) was added while keeping the temperature below 25 °C. Compound 1 (200 g, 0.84 mol) was added while keeping the reaction mixture below 35 °C (an exothermic reaction was observed), followed by stirring the reaction for 2-5 h while keeping the temperature at 25-35 °C. Near complete consumption of 1 was confirmed by HPLC (criterion: ratio of 1:2 should be less than 3%). AcOH (5.60 g, 0.093 mol) was then added while keeping the reaction temperature at 10-35 °C, followed by stirring the reaction at room temperature for 30-60 min. 10% sodium carbonate solution (200 g) was added while keeping the reaction temperature at 10-35 °C, and the resulting mixture was stirred at room temperature for 1-3 h. The phases were separated and the organic layer was isolated and evaporated to approximately 400-600 g while keeping the temperature below 70 °C to give a toluene solution of 2 that was analyzed by HPLC. Yields were generally in the range of 55-85%.

[0190] (3S,4S)-trans-1-benzyl-4-methyl-pyrrolidine-3-carboxylic acid ethyl ester (S,S)-(2)

[0191] [ka]

[0192] 4-Methylpentan-2-one (640 g) was charged to a reactor at room temperature and, with stirring, the toluene solution of 2 from the previous step (100 g, 0.404 mol) was added under nitrogen. The reaction was evaporated to a total mass of 200-300 g while keeping the temperature below 65° C., followed by 4-methylpentan-2-one (160 g) followed by (−)-dibenzoyl-L-tartaric acid (94.1 g, 0.263 mol) and the resulting reaction mixture was heated to 65-75° C. and stirred at this temperature for 1-2 hours. The reaction mixture was then cooled to 25-30° C. over a period of 5 hours and then stirred at 25-30° C. for 3-5 hours. The solids were filtered and the filter cake was washed with 4-methylpentan-2-one (80 g). The filtrate was charged to a reactor and 10% aqueous sodium carbonate solution (150 g) was added. The resulting reaction mixture was stirred for 1-2 hours, the phases were separated, and the aqueous phase was extracted with 4-methylpentan-2-one (40.5 g). The combined organic phase was washed with water (100 g). At this point, a sample was analyzed by HPLC to confirm that the level of (-)-dibenzoyl-L-tartaric acid did not exceed 0.5%. The organic phase was then treated with (+)-dibenzoyl-D-tartaric acid (86.7 g, 0.242 moles) under nitrogen, and the resulting reaction mixture was heated to 65-75°C and maintained at this temperature with stirring for 1-2 hours. The reaction mixture was then cooled to 25-30°C over 3 hours and stirred at 25-30°C for 3-5 hours. The solid was filtered and washed with 4-methylpentan-2-one (80 g). At this point, the separated solid was analyzed by chiral HPLC to confirm that the chiral purity of (S,S)-2 was greater than 99.5% before proceeding.

[0193] If the chiral purity was less than 99.5%, the following steps were carried out. Ethanol (400 g) was charged to a reactor, the isolated solid was added, and the resulting reaction mixture was heated to 70-78° C. and stirred at this temperature for 1-4 hours. The resulting solution was cooled to 20-25° C. over a period of 5-10 hours and stirred at 20-25° C. for 1-5 hours. The solid was filtered, and the cake was filtered, washed with EtOH, and dried. At this point, the isolated solid was analyzed by chiral HPLC to determine if the chiral purity of (S,S)-2 was greater than 99.5% before proceeding. If the chiral purity was less than 99.5%, the recrystallization from EtOH was repeated.

[0194] A 10% sodium carbonate solution (400 g) was charged to the reactor and the solid cake was added followed by MTBE (488 g). The mixture was stirred for 30 minutes and then the phases were separated. The aqueous phase was charged to the reactor and MTBE (244 g) was added. The resulting mixture was stirred for 30 minutes and then the phases were separated. The combined organic phase was evaporated to a total mass of about 30-50 g at a temperature below 40 °C. (S,S)-2 was isolated as an oil. HPLC analysis indicated that the yield was in the range of 20-50% and the chiral purity was greater than 99.5%.

[0195] Alternatively, (S,S)-2 can be prepared by asymmetric enzymatic hydrolysis. No chiral acid is used in this alternative method. An enzyme selectively hydrolyzes the ester of only the unwanted (S,S)-2 enantiomer. This step would replace the separation using chiral acid. The synthetic scheme is shown below. HLE stands for human leukocyte elastase, a serine protease enzyme. (R,R)-2, (S,S)-acid and (R,R)-acid are by-products.

[0196] [ka]

[0197] Reaction conditions: For each vial, 10 mg of Rac-2 was dissolved in 0.1 mL of MTBE, and approximately 5 mg of HLE enzyme and 1 mL of buffer (pH = 7) were added. Such mixtures were incubated for 24 h at 23 (20-25) °C in an orbital shaker and then extracted with 1 mL of dichloromethane (DCM). The upper layer (aqueous phase) was discarded and the lower layer (DCM phase) was evaporated by nitrogen flow and subjected to HPLC analysis. The extraction step removed the acid by-products. The esters, (S,S)-2 and (R,R)-2, were extracted from the buffer with DCM, whereas the acid by-products remained in the aqueous phase.

[0198] Various enzymes were tested. The ratio between (S,S)-2 and (R,R)-2 was measured and the enantiomeric excess (ee) was calculated. ee is a measure of purity used for chiral substances. It reflects the degree to which a sample contains more of one enantiomer than the other.

[0199] [Table 2]

[0200] The enzyme with the best ee was further tested to confirm the stereospecificity. Reaction conditions: For each vial, 500 mg of Rac-2 was mixed with 10 mL of buffer (pH=7) and 50 mg of enzyme. Such mixture was incubated for 24 h at 23 (20-25) °C in an orbital shaker and then extracted by 2 mL of DCM. The upper layer (aqueous phase) was discarded and the lower layer (DCM phase) was evaporated by nitrogen flow and subjected to HPLC analysis. The best enzyme showed a stereospecificity (ee) of more than 99%.

[0201] [Table 3]

[0202] Further tests were performed by scaling down the reaction volume, for example by reducing the buffer load, to obtain higher yields. Reaction conditions: For each vial, 0.1, 0.2, 0.5, 1, 2 g of Rac-2 were mixed with 2 mL of buffer (pH=7) and 0.1X of enzyme. Such mixtures were incubated for 24 hours at 23 (20-25) °C in an orbital shaker, and then dissolved to 20 mL using EtOH. The mixture was filtered and the filtrate was subjected to HPLC analysis and assay for ee. The stereospecificity (ee) was over 99%, but the yield (in situ) was not high enough.

[0203] [Table 4]

[0204] Co-solvents were then screened for better in situ yields. MTBE was replaced by other solvents. Reaction conditions: For each vial, 0.1 g of Rac-2 was dissolved in 0.1 mL of co-solvent and mixed with 0.9 mL of buffer (pH = 7) and 10 mg of HLE enzyme. Such mixture was incubated at 23 (20-25) °C for 24 h in an orbital shaker, and then dissolved to 5 mL using EtOH. The mixture was filtered and the filtrate was used for analysis.

[0205] [Table 5]

[0206] Although the stereospecificity was good, the yields using enzymatic hydrolysis in situ were not high enough (<40%) and cosolvents did not improve the yields. Therefore, resolution using a chiral acid to prepare (S,S)-2 is preferred.

[0207] (3S,4S)-trans-4-methyl-pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl ester (S,S)-(3)

[0208] [ka]

[0209] THF (700 g) was placed in the primary reactor R1, which had previously been purged with nitrogen. (S,S)-2 (100 g, 0.40 mol) was added at 20-25 °C, the reactor was evaporated and purged with nitrogen three times. Wet pregnanediol / C (10%, 10 g) was added to the reactor at 20-25 °C, the reactor was evaporated and purged with hydrogen three times. The reaction mixture was hydrogenated at 10-18 °C with stirring at a pressure of 0.3-0.4 MPa and a temperature of 45-50 °C. At this point, HPLC analysis was performed to confirm that less than 0.2% of the starting material remained. The reaction mixture was filtered through a plug of Celite (50-100 g) and the filter was washed with THF (230 g). The combined filtrates were transferred to the secondary reactor R2 and cooled to 0-10 °C. Boc2O (90 g, 0.41 mol) was charged to the reactor at 0-10 °C and the reaction mixture was warmed to 20-30 °C and stirred at this temperature for 1-4 h. At this point, HPLC analysis confirmed that less than 1% of the debenzylated intermediate remained. The reaction mixture was concentrated in vacuo below 45 °C and THF (500 g) was added to the reactor. The resulting reaction mixture was evaporated in vacuo below 45 °C to give the product (S,S)-3. HPLC analysis confirmed that the water content was less than 1%. Yields were generally in the range of 80-95%.

[0210] (3S,4S)-trans-3-(2-chloroacetyl)-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester

[0211] [ka]

[0212] Sodium chloroacetate (68 g, 0.58 mol) was placed in a 5000 mL reactor R1 under nitrogen and dry THF (890 g) was added followed by (S,S)-3 (100 g, 0.39 mol). After triethylamine (58 g, 0.58 mol) was added, the temperature was adjusted to -5°C to 5°C. tert-Butylmagnesium chloride (914 mL, 868.3 g, 4.0 equiv.) in THF was added dropwise over 1 h while maintaining the reaction at a temperature interval of -5°C to 5°C. The reaction mixture was warmed to 5°C to 10°C and stirred at this temperature for 2 h. At this point, HPLC analysis was performed to ensure that the starting material was nearly completely consumed. The temperature was readjusted to -5°C to 5°C and the reaction was quenched by the dropwise addition of 15% (w / w) aqueous ammonium chloride solution (2200 g to 2775 g) at -5°C to 5°C. The temperature was adjusted to 0°C-5°C, and the resulting reaction mixture was stirred for 1-2 hours, then left to stand for 2-3 hours to separate the phases. The aqueous layer was transferred to a second reactor, R2, and n-heptane (1000 mL) was added, and the reactor was stirred at 0°C-5°C for 0.5-1 hour, and then left to stand for 0.5-1 hour. The aqueous layer was separated. Aqueous sodium sulfate solution (10% (w / w), 100 g) was placed in the first reactor R1 containing the THF solution at 0°C-5°C, and the reactor was stirred at 0°C-5°C for 0.5-1 hour. Then, it was left to stand for 0.5-1 hour at 0°C-5°C. The aqueous layer was transferred to a second reactor R2, and the organic phase in R1 was concentrated under vacuum to about 100-200 g at a temperature below 30°C. Concurrently, the second reactor R2 was stirred at 0°C-5°C for 0.5-1 h and then allowed to stand at 0°C-5°C for 0.5-1 h. The aqueous phase was separated and the organic phase was transferred to R1. Saturated aqueous sodium sulfate (10% (w / w), 50-100 g) was added and the reactor was stirred at 0°C-5°C for 0.5-1 h and then allowed to stand at 0°C-5°C for 0.5-1 h. The aqueous phase was separated and the organic phase was filtered through a plug of sodium sulfate (50-100 g). The organic filtrate was evaporated under vacuum at a temperature below 30°C to give the product in yields ranging from 60-100%.

[0213] Tetrahydropyran-4-carbaldehyde (6)

[0214] [ka]

[0215] Dry toluene (500 g) was placed in a dried reactor under nitrogen and tetrahydropyran-4-carbonitrile 5 (100 g, 0.90 mol) was added. The resulting solution was cooled to -5°C to 5°C and a solution of DIBAL-H in toluene (1.0 M, 800 g, 1.0 mol) was added dropwise at -5°C to 5°C and the resulting reaction mixture was stirred at this temperature for 1-2 h. The reaction mixture was then warmed to 20°C to 25°C and stirred at this temperature for 1-2 h. The reaction mixture was then cooled to -5°C to 5°C and quenched by the dropwise addition of a solution of AcOH (195 g) in toluene (180 g) at -5°C to 5°C (Caution: Exothermic, gas evolution). A 25% solution of sodium tartrate tetrahydrate (1000 g) was added slowly at -5°C to 5°C. The reaction mixture was warmed to 20°C to 25°C and stirred at this temperature for 8-16 h. The layers were separated and the aqueous layer was extracted twice with EtOAc (900 mL) at 20-25° C. The two EtOAc extracts were combined with the original organic phase, and the combined organic phase was evaporated in vacuo to approximately 100-200 g to give the product 6. The product was generally isolated in 50-80% yield.

[0216] 2-Tetrahydropyran-4-yl-5-(trifluoromethyl)-1H-imidazole (7)

[0217] [ka]

[0218] Water (730 g) was charged to the main reactor R1 under nitrogen, followed by sodium acetate (159 g, 1.94 mol) and 1,1-dibromo-3,3,3-trifluoroacetone (260 g, 0.96 mol), added dropwise while maintaining the reaction temperature at 20°C-30°C. The reaction mixture was then warmed to 80°C-85°C and stirred at this temperature for 1-2 h before being cooled to 20°C-25°C. Compound 6 (100 g, 0.88 mol) was charged to the secondary reactor R2, followed by MeOH (1150 g) and a 25% aqueous solution of ammonia (614 g, 4.38 mol) was slowly added while maintaining the temperature in the range of 15°C-30°C. The mixture in R1 was then added dropwise to R2 over 2 h at 15°C-30°C. The reaction was then stirred at 25°C-30°C for 18-24 h. The reaction was then evaporated under vacuum at a temperature below 45°C to approximately 500-800 g, then cooled to 25°C-30°C and MTBE (100 g) was charged with stirring, followed by heptane (180 g). The reaction mixture was stirred at 20°C-30°C for 2-3 hours and then filtered. The filter was washed with water (320 g) and heptane (120 g). At this point, HPLC analysis was performed to confirm the purity of 7 was greater than 96%. The filter cake was dried under vacuum at 40°C-45°C for 8-24 hours. At this point, Karl Fischer titration indicated the water content was less than 0.5%. Compound 7 was generally isolated at 50-80% purity. Purity was greater than 96%.

[0219] Methyl 2-tetrahydropyran-4-yl-1H-imidazole-5-carboxylate (8)

[0220] [ka]

[0221] 30% sodium methoxide in methanol (245 g, 1.36 mol) was charged under nitrogen to the main reactor R1, compound 7 (100 g, 0.45 mol) was added followed by MeOH (590 g) and the resulting reaction mixture was warmed to 60°C-65°C and stirred for 5-10 h before being cooled to 40°C-45°C. At this point, HPLC analysis confirmed that the level of 7 was less than 1%. The reaction mixture was filtered and the filter was washed with MeOH (170 g). The filtrate was charged under nitrogen to the secondary reactor R2 and the pH was adjusted to 5-6 by dropwise addition of AcOH (30 g) at a temperature of 0°C-10°C. 150 g of water was added while maintaining the temperature in the range of 0°C-10°C, after which the reaction was warmed to 30°C-35°C and stirred at this temperature for 6-18 h. The reaction mixture was concentrated under vacuum at a temperature below 40 °C to approximately 200-300 g. Water (200 g) was added and the reaction was stirred at 15 °C-25 °C for 1-2 h. Solid sodium bicarbonate (45 g) was added with stirring until the pH reached 7-8 and the reaction was stirred at 15-25 °C for 1-2 h. The resulting solid was filtered, washed with water (100 g) and dried under vacuum at 40 °C-45 °C for 18-24 h. Karl Fischer titration confirmed that the water content was less than 1%. Compound 8 was generally isolated in 70-90% yield.

[0222] 4-Bromo-2-tetrahydropyran-4-yl-1H-imidazole-5-carboxylate methyl (9)

[0223] [ka]

[0224] Dichloromethane (1330 g) was charged to the primary reactor R1, compound 8 (100 g, 0.475 mol) was charged to the reactor, followed by N-bromosuccinimide (84.7 g, 0.475 mol) while maintaining the temperature in the range of 20 °C to 25 °C. The reaction mixture was then stirred at 20 °C to 25 °C for 1 to 5 hours. At this point, HPLC analysis indicated that less than 1% of the compound remained. An aqueous solution of sodium carbonate (10%) and sodium sulfite (0.3%) (1100 g) was added at a temperature of 20 °C to 25 °C, and the reaction was stirred at this temperature for 1 to 3 hours. The organic (bottom) phase was transferred to the secondary reactor R2, and R1 was charged with dichloromethane (266 g) and stirred at 20 °C to 25 °C for 0.5 to 2 hours. The bottom (organic) layer was charged into R2, followed by the addition of 10% sodium carbonate solution (220 g) at 20 °C-25 °C, and the resulting mixture was stirred at 20 °C-25 °C for 1-3 h. The bottom (organic) layer was transferred to R1, dichloromethane (266 g) was charged into R2, and the mixture was stirred at 20 °C-25 °C for 1-3 h. The bottom (organic) layer was transferred to R1, followed by the addition of 10% sodium carbonate aqueous solution (220 g). The resulting mixture was stirred at 20 °C-25 °C for 1-3 h, and the bottom (organic) layer was then transferred to the empty R2. Dichloromethane (266 g) was charged into R1, the reaction mixture was stirred at 20 °C-25 °C for 1-3 h, and the bottom (organic) layer was then transferred to R2. R2 was concentrated under vacuum to 100-200 g at a temperature below 40 °C. The residue was cooled to 20 °C-25 °C, and heptane (200 g) was added. The resulting mixture was stirred at 20°C-25°C for 3-12 h, and the solid was filtered and dried under vacuum at 40°C-45°C for 20-24 h. Karl Fischer titration confirmed that the water content was less than 0.5%. Yields of 9 were generally in the range of 70-100%.

[0225] (3S,4S)-3-(1-bromo-8-oxo-3-tetrahydropyran-4-yl)-7H-imidazo[1,5-a]pyrazin-6-yl)-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (S,S)-(10)

[0226] [ka]

[0227] NMP (516 g) was charged to the primary reactor R1 under nitrogen, followed by compound 9 (100 g, 0.346 mol). Potassium carbonate (72 g, 0.519 mol) and potassium iodide (5.74 g, 0.035 mol) were added and the resulting reaction mixture was cooled to -5 to 5 °C with stirring. Compound (S,S)-4 (109 g, 0.415 mol) was dissolved in MTBE (152 g) and added dropwise at -5 to 5 °C and stirred at this temperature for 12 to 16 h. HPLC analysis at this point confirmed that compound 9 levels were less than 1%. Diatomaceous earth (50 to 100 g) was charged to the reactor and the resulting mixture was stirred at -5 to 5 °C for 1 to 2 h. The filter was washed with MTBE (152 g). The combined filtrate was transferred to the secondary reactor R2 and evaporated under vacuum at below 40 °C to 500-600 g, and the residue was cooled to 20-30 °C. Ammonium acetate (107 g, 0.345 mol) was added at 10-30 °C under nitrogen, and the resulting reaction mixture was warmed to 100-110 °C and stirred at this temperature for 12-16 h. At this point, the reaction mixture was cooled to 20-30 °C, and HPLC analysis confirmed that conversion of the non-cyclized products to cyclized products had occurred (i.e., less than 1% of the uncyclized intermediate remained). DCM (800 g) was charged into the reaction mixture at 20-30 °C, followed by water (1000 g) at 20-30 °C. The resulting mixture was stirred for 0.5-1 h and allowed to settle for 0.5-1 h. The bottom layer was transferred to the primary reactor R1. DCM (400 g) was charged to R2 at 20-30 °C, the resulting mixture was stirred for 0.5-1 h and allowed to stand for 0.5-1 h. The bottom layer was transferred to R1. The combined organic phases in R1 were treated with 10% aqueous sodium sulfate solution (300 g) at 20-30 °C, the resulting mixture was stirred for 0.5-1 h and allowed to stand for 0.5-1 h. The top layer (aqueous) was discarded. 10% aqueous sodium sulfate solution (300 g) was charged to R1 at 20-30 °C, the resulting mixture was stirred for 0.5-1 h and allowed to stand for 0.5-1 h. The bottom (organic) layer was transferred to the emptied secondary reactor R2 and concentrated under vacuum to about 200-300 g at a temperature below 40 °C. Water (1500-2000 g) was added to R1, and the reaction mixture in R2 was added dropwise with stirring at 20-30° C. over 1 hour, and the resulting mixture was stirred for 1-2 hours. The solid was filtered and washed with water (100 g).Isopropyl acetate (440 g) was charged into the primary reactor R1 at 20-30 °C, and the wet cake was similarly charged into R1. The resulting mixture was warmed to 50-60 °C and stirred at this temperature for 2-24 h. The resulting reaction mixture was cooled to 20-30 °C, the mixture was filtered, and the filter was washed with isopropyl acetate (100 g). The solid was dried at 40-45 °C for 18-24 h. The yield of compound (S,S)-10 was generally in the range of 45-75%. The purity was greater than 96%.

[0228] (3S,4S)-trans-6-(4-methyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one hydrochloride (3S,4S)-(12)

[0229] [ka]

[0230] (3S,4S)-10 (100 g, 0.21 mol) was charged to the primary reactor R1 under nitrogen and MeOH (2400 g) was added. Pd / C catalyst (10%, 10 g) was charged to the reactor under nitrogen and the resulting reaction mixture was hydrogenated (0.3-0.4 MPa) at 20-30 °C for 12-24 h. At this point, HPLC analysis was performed to confirm that less than 1% of the starting material remained. The reaction mixture was filtered through a pad of Celite (50-100 g) and the Celite was washed with MeOH (200 g). The combined filtrate was placed in the secondary reactor R2 and evaporated to approximately 400 milliliters in vacuum below 40 °C. A solution of HCl (30-35 g) in MeOH (200 g) was added dropwise into the reaction mixture at a temperature of 0-10 °C. The reaction mixture was then warmed to 20-25 °C and stirred at this temperature for 8-12 h. HPLC analysis at this stage helped to confirm that less than 1% of intermediate (3S,4S)-11 remained in solution. The reaction mixture was concentrated in vacuum to a volume of about 200-250 milliliters at a temperature below 40 °C. The reaction mixture was cooled to 20-25 °C, and the resulting reaction mixture was added dropwise to EtOAc (1800 g) with stirring at 20-25 °C, and the resulting mixture was stirred at 20-25 °C for 12-18 h. The solid was filtered under a nitrogen atmosphere and washed with EtOAc (100 g). The solid was dried under a stream of nitrogen at 20-30 °C for 18-24 h. The yields of (3S,4S)-12 were generally in the range of 70-100%.

[0231] (3S,4S)-trans-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P3.1)

[0232] [ka]

[0233] Dichloromethane (1330 g) was charged to the primary reactor R1 at room temperature under nitrogen, compound (S,S)-12 (100 g, 0.238 mol) was added followed by 2-chloromethylpyrimidine hydrochloride (47.2 g, 0.286 mol) and the reaction mixture was cooled to 0-10° C. with stirring. N,N-Diisopropylethylamine (153 g, 1.19 mol) was added dropwise at a temperature of 0-10° C. and the reaction mixture was warmed to 20-30° C. and stirred at this temperature for 44-48 h. At this point, HPLC analysis was performed to ensure that no more than 5% (S,S)-12 remained. (If this test failed, a 3.9 g portion of 2-chloromethylpyrimidine hydrochloride was added and the reaction was stirred for an additional 44-48 h at 20-30 °C. Water (500 g) was added to the reaction mixture at 20-30 °C and the reaction was stirred at this temperature for 1-2 h. The bottom layer was transferred to a second reactor, R2. Dichloromethane (266 g) was charged to R1 and the mixture was stirred for 1-2 h at 20-30 °C. The bottom layer was transferred to R2, followed by the addition of water (200 g). The reaction mixture was cooled to 0-10 °C. Concentrated HCl (approximately 80 g) was added dropwise with stirring at a temperature of 0-10 °C until the pH of the reaction mixture reached 2-4, and the reaction mixture was stirred at 0-10 °C for 1-2 h. Stirring was stopped and the phases were allowed to separate. The lower layer was poured into the emptied R1, followed by water (100 g). After stirring R1 at 0-10 °C for 1-2 h, the upper layer was transferred to R2. The reaction mixture in R2 was warmed to 10-20 °C and slowly charged with sodium carbonate (50 g) at 10-20 °C until a pH of 8-9 was reached (Caution: Gas evolution). DCM (266 g) was charged into R2 and the reactor was stirred at 10-30° C. for 1-2 h. The bottom (organic) layer was transferred to R1. DCM (266 g) was charged to R2 and the reactor was stirred at 10-30 °C for 1-2 h. The bottom (organic) layer was transferred to R1. DCM (266 g) was charged to R2 and the reactor was stirred at 10-30 °C for 1-2 h. The bottom (organic) layer was transferred to R1. The mixture in R1 was filtered through a plug of sodium sulfate (50 g) into R2 and the filter was washed with DCM (133 g). The combined organic filtrate was concentrated under vacuum to about 266-400 g at a temperature below 45 °C. Isopropanol (600 g) was charged to R2 and the resulting mixture was concentrated under vacuum at a temperature below 45 °C. The resulting mixture was cooled to 20-30 °C and seed crystals (100 milligrams) were added. The resulting reaction mixture was stirred at 20-30 °C for 1-4 h. Heptane (100 g) was charged to the reaction mixture at 20-30° C., followed by (100 mg). The resulting reaction mixture was stirred at 20-30° C. for 1-4 hours. Heptane (300 g) was charged to the reaction mixture at 20-30 °C, and the resulting reaction mixture was stirred at 20-30 °C for 6-12 h. The solid was filtered and washed with a mixture of isopropanol:heptane (1:3, 100 g). The solid was dried in vacuum at 40-45 °C for 18-24 h. HPLC analysis of the resulting solid confirmed a purity greater than 97.5%. The yield of (S,S)-P3.1 was in the range of 50-80%.

[0234] Example 2. Formulation of Compound P3.1

[0235] Stability testing of Compound P3.1 has been completed. Samples of Compound P3.1 were dispensed into double walled polyethylene pouches, which were then tied and heat sealed in aluminum pouches. Samples were stored at ambient temperature and at 40-45°C (without humidity control) and tested over a 3 month period. There was no change in the appearance or purity of the material at either room temperature or accelerated conditions over the course of the study, indicating that the drug substance is not readily affected by accelerated temperature conditions.

[0236] In another stability study, compound P3.1 was dissolved in purified water at approximately 40 mg / mL and evaluated for purity over a period of 8 days. Samples were stored under both refrigerated and ambient conditions and tested at T=0, day 2, and day 8. No significant changes to the compound purity or solution appearance were observed over the course of the study.

[0237] In yet another stability study, the study design includes sample storage at 25°C ± 2°C / 60% relative humidity (RH) ± 5%RH, and 40°C ± 2°C / 75%RH ± 5%RH. Samples are stored in bags equivalent to those used for packaging compound P3.1. The study is designed to evaluate the stability of compound P3.1 at accelerated temperatures for up to 6 months and at a prescribed storage temperature of 25°C for 36 months.

[0238] Compound P3.1 packaging is prepared by filling the compound directly into opaque white gelatin capsules (powder in capsules, PIC). No binders, fillers, or other excipients are added. Capsules contain 10-100 mg of Compound P3.1.

[0239] Packaging will be monitored with stability studies from 6 to 36 months. Conditions include 25°C / 60% RH and 40°C / 75% RH (6 months only). Studies will include appearance, assay and related substances, dissolution and moisture analysis. A 5°C arm will also be included, but will not be tested unless there are indications of product instability in the 25°C arm of the study.

[0240] Alternatively, the dosage form is prepared by mixing compound P3.1 with selected excipients. The excipients that may be used are summarized in Table 2 below.

[0241] Table 2: Suggested excipients for future drug manufacturing

[0242] [Table 6]

Claims

1. A process for the synthesis of 6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one (compound P3.1), which process does not involve chiral chromatographic separation of enantiomers and further comprises: A. Synthesis of intermediate 4 using Scheme 1 below: B. Synthesis of intermediate 9 using Scheme 2 below, and C. Synthesis of compound P3.1 using Scheme 3 below: A method comprising the steps of: 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】

2. 2. The method of claim 1, wherein the chiral purity of compound P3.1 is greater than 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.5%, 99.8%, or 99.9%.

3. 2. The method of claim 1, wherein the purity of compound P3.1 is greater than 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, 99.5%, 99.8%, or 99.9%.

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