PDE9 inhibitors with imidazotriazine skeleton and imidazopyrazine skeleton for treating peripheral diseases

Abstract

The present invention relates to PDE9 inhibitors and their use for treating benign prostatic hyperplasia and sickle cell disease. The inhibitory compound can be selected from: 3-(4-fluorophenyl)-6-((3-(pyridin-4-yloxy)azetidin-1-yl)methyl)imidazo[1,5-a]pyrazin-8(7H)-one, 6-[3-(pyridin-3-yloxy)azetidin-1-ylmethyl]-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one, 6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro- imidazo[1,5-a]pyrazin-8-one, (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, and (3R,4R)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one.

Description

[0001] This application is a divisional application of an application filed on July 6, 2016, with application number 201680039418.0, and invention name “PDE9 inhibitors with an imidazotriazine skeleton and an imidazopyrazine skeleton for treating peripheral diseases”.

[0002] Citation of Related Applications

[0003] This application claims priority to the following DK provisional patent applications: DK provisional patent application No. PA201500393, entitled “PDE9 inhibitors having an imidazotriazinone skeleton and an imidazopyrazinone skeleton for treating peripheral diseases,” filed on July 7, 2015, DK provisional patent application No. PA201500407, entitled “PDE9 inhibitors having an imidazotriazinone skeleton and an imidazopyrazinone skeleton for treating peripheral diseases,” filed on July 10, 2015, and DK provisional patent application No. PA201600209, entitled “PDE9 inhibitors having an imidazotriazinone skeleton and an imidazopyrazinone skeleton for treating peripheral diseases,” filed on April 7, 2016, the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention relates to cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 9 inhibitors (hereinafter referred to as PDE9 inhibitors) in the form of 3H-imidazo[5,1-f][1,2,4]triazin-4-one or 7H-imidazo[1,5-a]pyrazin-8-one and their use as medicaments for treating peripheral diseases. Furthermore, the present invention relates to pharmaceutical compositions comprising 3H-imidazo[5,1-f][1,2,4]triazin-4-one and 7H-imidazo[1,5-a]pyrazin-8-one. Background Art

[0005] Phosphodiesterase (PDE) is an enzyme family that degrades cyclic nucleotides and therefore regulates the cellular level of second messengers throughout the body. PDE represents an attractive drug target, which has been demonstrated by multiple compounds entering clinical trials and the market. PDE is encoded by 21 genes, which are functionally divided into 11 families that are different in terms of kinetic properties, substrate selectivity, expression, localization pattern, activation, regulatory factors, and inhibitor sensitivity. The function of PDE is to degrade cyclic monophosphate nucleosides, cyclic adenosine monophosphate (cAMP) and / or cyclic guanosine monophosphate (cGMP), which are important intracellular mediators involved in numerous vital processes (including controlling neurotransmission and smooth muscle contraction and relaxation).

[0006] PDE9 is cGMP-specific (Km for cAMP is over 1000-fold that of cGMP) and has been hypothesized to be a key regulator of cGMP levels because it has the lowest Km among PDEs of this nucleotide. PDE9 is expressed at low levels throughout the brain and has the potential to regulate basal cGMP.

[0007] In the periphery, PDE9 expression peaks in prostate, intestine, kidney, and hematopoietic cells, opening up therapeutic potential for various peripheral indications.

[0008] Benign prostate hyperplasia (BPH) is one of the most common conditions in the elderly male population and represents a major health problem (Ueckert S et al., Expert Rev Clin Pharmacol. 2013 May; 6(3): 323-32). BPH causes the formation of large nodules in the periurethral region of the prostate, which can lead to urinary tract obstruction. BPH is primarily the result of a stromal proliferation process, and an important cause of prostate enlargement is smooth muscle proliferation. Current drug treatments for BPH include α1 adrenergic blockers, 5-α-reductase inhibitors, and more recently, the PDE5 inhibitor tadalafil. PDE5 inhibitors are known to mediate smooth muscle relaxation via increased cGMP levels. cGMP-specific PDE9 is expressed at high levels in the prostate, and PDE9 inhibition may therefore provide potential anti-proliferative benefits for BPH.

[0009] PDE9 is widely distributed in the urothelium of the human lower urinary tract, and PDE9 inhibition may be beneficial in lower urinary tract dysfunctional epithelium (LUDE) diseases (Nagasaki et al., BJU Int. 2012 Mar; 109(6): 934-40). Dysfunction of the lower urinary tract epithelium can affect the bladder, urethra, labia or vaginal introitus in women and the prostate duct and urethra in men (Parsons LC et al., 2002).

[0010] PDE9 expression has been shown in the murine corpus cavernosum, and long-term PDE9 inhibition has been shown to result in an amplified NO-cGMP-mediated corpus cavernosum response and thus open up potential benefits in erectile dysfunction (DaSilva et al., Int J Impot Res. 2013 Mar-Apr; 25(2): 69-73). Currently approved treatments for erectile dysfunction are of the PDE5 inhibitor type, which increase cGMP in the smooth muscle cells lining the blood vessels supplying the corpus cavernosum of the penis.

[0011] cGMP PDE inhibition has been shown to enhance muscle microvascular blood flow and glucose uptake in response to insulin (Genders et al., Am J Physiol Endocrinol Metab. 2011 Aug;301(2):E342-50). Targeting cGMP-specific PDE9 expressed in muscle and blood vessels may provide a promising approach to enhance muscle insulin sensitivity and thus be beneficial in treating type 2 diabetes.

[0012] PDE9 inhibition could represent a novel first-line treatment for sickle cell disease (SCD), a genetic disorder that leads to a vaso-occlusive process that causes death in many SCD patients. SCD is caused by a point mutation in the hemoglobin (HBB) gene that produces abnormal sickle hemoglobin (HbS), which aggregates and forms stiff and sticky sickle-shaped red blood cells. Sickle-shaped red blood cells lead to chronic inflammation, increased cell adhesion, oxidative stress, and endothelial dysfunction, culminating in a vaso-occlusive process.

[0013] To date, there is no cure for SCD. Treatment options include blood transfusions and treatment with the anticancer agent hydroxyurea. Blood transfusions correct the anemia by increasing the number of normal, non-sickled red blood cells in the circulation. Regular blood transfusion therapy can help children at high risk prevent recurrent strokes. Hydroxyurea is approved for the treatment of SCD and has been shown to reduce the frequency of painful crises and hospitalizations. The hypothesized mechanism by which hydroxyurea improves SCD symptoms is twofold: a) increases the production of non-sickled fetal hemoglobin; and b) reduces cell adhesion. Specifically, hydroxyurea a) increases the production of non-sickled fetal hemoglobin via cGMP signaling, which has been shown to lead to increased blood cell survival; and b) increases nitric oxide and cGMP levels, thereby reducing adhesion and increasing survival. In summary, the evidence to date supports the view that both mechanisms by which hydroxyurea has a benefit in SCD are mediated via increased cGMP.

[0014] PDE9 is specifically expressed in the human hematopoietic system (including neutrophils, reticulocytes, erythroid cells, and erythroleukemic cells). Moreover, SCD patients show a clear and significantly increased expression of PDE9 in reticulocytes and neutrophils (Almeida et al., Br J Haematol. 2008 Sep; 142(5): 836-44). Evidence further supports a link between PDE9 and cell adhesion, as PDE9 inhibition leads to a reversal of the enhanced adhesion properties 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 via increased cGMP and reduced expression of endothelial adhesion molecules. Importantly, in animal models of SCD, the reduction in cell adhesion mediated by PDE9 inhibitors has the functional effect of increasing cell survival. In addition to demonstrating an effect on reducing cell adhesion comparable to that of hydroxyurea, PDE9 inhibition leads to increased production of non-sickled fetal hemoglobin. Finally, Almeida and colleagues demonstrated that treatment with hydroxyurea combined with PDE9 inhibition in a mouse model of SCD resulted in the PDE9 inhibitor having the added benefit of amplifying the cGMP-raising effect of hydroxyurea (Almeida et al., Blood. 2012 Oct 4;120(14):2879-88). In summary, PDE9 inhibition can both regulate the expression of fetal hemoglobin production and reduce cell adhesion, both mechanisms being critical for treating SCD.

[0015] WO 2013 / 053690 discloses PDE9 inhibitors having an imidazopyrazinone skeleton for use as medicaments, for example, in the treatment of patients suffering from cognitive impairment, in particular associated with neurodegenerative diseases such as cortical dementia (eg Alzheimer's disease) or subcortical dementia (eg AIDS-related dementia).

[0016] WO 2013 / 110768 discloses PDE9 inhibitors having an imidazotriazinone skeleton for use as medicaments, for example, in the treatment of patients suffering from cognitive impairment, in particular associated with neurodegenerative diseases such as cortical dementia (eg Alzheimer's disease) or subcortical dementia (eg AIDS-related dementia).

[0017] WO 2012 / 040230 discloses PDE9 inhibitors having an imidazotriazinone skeleton for use as drugs for treating PDE9-related diseases, including CNS and neurodegenerative disorders.

[0018] WO 2008 / 139293 and WO 2010 / 084438 both disclose amino-heterocyclic compounds that are PDE9 inhibitors and their use for treating neurodegenerative disorders and cognitive disorders. Summary of the Invention

[0019] There is a continuing need for improved treatments for the peripheral diseases benign prostatic hyperplasia (BPH), urothelial diseases, erectile dysfunction, type 2 diabetes, and sickle cell disease (SCD), for which the use of PDE9 inhibitors may be very useful. Because PDE9 is expressed at low levels throughout the brain, underlying basal cGMP, and thus signaling cascades have been shown to regulate synaptic transmission, it is clearly important that PDE9 inhibitors used to treat peripheral diseases have low blood-brain barrier penetration (BBB penetration) to avoid potential centrally mediated side effects.

[0020] The present invention provides novel PDE9 inhibitors that have been shown to have low blood-brain barrier penetration and are therefore particularly useful in treating peripheral diseases such as benign prostatic hyperplasia (BPH), urothelial diseases, erectile dysfunction, type 2 diabetes, and sickle cell disease (SCD). Moreover, the PDE9 inhibitors of the present invention are significantly more potent PDE9 inhibitors than PDE1 inhibitors, which is important because PDE1 is expressed in the heart and testes, and inhibition of these PDE1 isoforms is believed to be a potential cause of cardiovascular and reproductive side effects.

[0021] The present invention encompasses the following compounds:

[0022]

[0023] the racemate and enantiomerically pure modifications of compound P3,

[0024]

[0025] Another aspect of the present invention relates to the synthesis of P1, P2, P3 and P4. Yet another aspect of the present invention relates to the enantioselective synthesis of compound P3, including the conversion of intermediate compound rac-35 to (S,S)-35. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The absolute stereochemistry of Compound P3 Enantiomer 2 monohydrate is shown.

[0027] Figures 2A-2B is an optical micrograph of a crystallization batch ( Figure 2A ) and crystals used for data collection ( Figure 2B ).

[0028] Figure 3 is a ball-and-stick diagram of compound P3 enantiomer 2 monohydrate. DETAILED DESCRIPTION

[0029] Embodiments of the invention

[0030] The following notation is used: An embodiment of the present invention is described as Ei, where i is an integer representing the embodiment number. An embodiment Ei' that details a specific embodiment of a previously listed embodiment Ei is described as Ei'(Ei), for example E2(E1) means "in embodiment E2 of embodiment E1."

[0031] When an embodiment is a combination of two embodiments, the notation is similarly "Ei" (Ei and Ei'), for example, E3 (E2 and E1) means "in embodiment E3 of either embodiment E2 or E1".

[0032] When an embodiment is a combination of more than two embodiments, the notation is similarly Ei'' (Ei, Ei' and Ei"), for example E4 (E1, E2 and E3) means "in embodiment E4 of any one of embodiments E1, E2 and E3".

[0033] In a first embodiment E1, the present invention relates to compounds having the following structure:

[0034]

[0035] In racemic and enantiomerically enriched or pure form.

[0036] In embodiment E2 (E1), when the racemic mixture of P3 is separated by chiral HPLC (column: Chiralpak IA, 250 x 4.6 mm x 5 um; mobile phase: Hex / EtOH / DEA=70:30:0.2; flow rate of 1.0 mL / min), the enantiomerically pure variant of compound P3 is the first eluting compound (P3 enantiomer 1).

[0037] E3 (E1 and E2): A compound according to any one of embodiments E1 and E2 for use as a medicament.

[0038] E4: A compound according to any one of E1 and E2 or the following compound for use in treating benign prostatic hyperplasia or sickle cell disease:

[0039]

[0040] E5: A pharmaceutical composition comprising a therapeutically effective amount of any compound of E1 and E2 or compound P4, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0041] E6 (E5): The pharmaceutical composition is used to treat benign prostatic hyperplasia or sickle cell disease.

[0042] E7: Use of compound P4 or any of compounds E1 and E2 for the preparation of a medicament for treating benign prostatic hyperplasia or sickle cell disease.

[0043] E8: A method of treating a patient suffering from benign prostatic hyperplasia or sickle cell disease, comprising administering to a subject in need thereof a therapeutically effective amount of compound P4 or any of compounds E1 and E2.

[0044] E9: A compound selected from: 3-(4-fluorophenyl)-6-((3-(pyridin-4-yloxy)azetidin-1-yl)methyl)imidazo[1,5-a]pyrazin-8(7H)-one (P1), 6-[3-(pyridin-3-yloxy)azetidin-1-ylmethyl]-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P2), (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 (P3, enantiomer 1) and (3R,4R)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P3, enantiomer 2).

[0045] E10(E9): Compound (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 (P3, enantiomer 1).

[0046] E11 (E9): Compound (3R,4R)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P3, enantiomer 2).

[0047] E12 (E9, E10 and E11): A compound according to any one of embodiments E9 to E11 for use as a medicament.

[0048] E13: A compound selected from the group consisting of: 3-(4-fluorophenyl)-6-((3-(pyridin-4-yloxy)azetidin-1-yl)methyl)imidazo[1,5-a]pyrazin-8(7H)-one (P1), 6-[3-(pyridin-3-yloxy)azetidin-1-ylmethyl]-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P2), (3S,4S)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazole imidazo[1,5-a]pyrazin-8-one (P3, enantiomer 1), (3R,4R)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P3, enantiomer 2), and 2-[3-(4-fluoro-phenoxy)-azetidin-1-ylmethyl]-7-(tetrahydro-pyran-4-yl)-3H-imidazo[5,1-f][1,2,4]triazin-4-one (P4) for the treatment of benign prostatic hyperplasia or sickle cell disease.

[0049] E14: A pharmaceutical composition comprising a therapeutically effective amount of any of the following compounds and one or more pharmaceutically acceptable carriers, diluents or excipients: 3-(4-fluorophenyl)-6-((3-(pyridin-4-yloxy)azetidin-1-yl)methyl)imidazo[1,5-a]pyrazin-8(7H)-one (P1), 6-[3-(pyridin-3-yloxy)azetidin-1-ylmethyl]-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P2), (3S,4S)-6-(4-methyl-1-pyrimidin-2-ylmethyl [1,5-a]pyrazin-8-one (P3, enantiomer 1), (3R,4R)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P3, enantiomer 2), and 2-[3-(4-fluoro-phenoxy)-azetidin-1-ylmethyl]-7-(tetrahydro-pyran-4-yl)-3H-imidazo[5,1-f][1,2,4]triazin-4-one (P4).

[0050] E15 (E14): The pharmaceutical composition is used to treat benign prostatic hyperplasia or sickle cell disease.

[0051] E16: Use of any of the following compounds for the preparation of a medicament for the treatment of benign prostatic hyperplasia or sickle cell disease: 3-(4-fluorophenyl)-6-((3-(pyridin-4-yloxy)azetidin-1-yl)methyl)imidazo[1,5-a]pyrazin-8(7H)-one (P1), 6-[3-(pyridin-3-yloxy)azetidin-1-ylmethyl]-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P2), (3S,4S)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidone-3 -yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P3, enantiomer 1), (3R,4R)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P3, enantiomer 2), and 2-[3-(4-fluoro-phenoxy)-azetidin-1-ylmethyl]-7-(tetrahydro-pyran-4-yl)-3H-imidazo[5,1-f][1,2,4]triazin-4-one (P4).

[0052] E17: A method of treating a subject with benign prostatic hyperplasia or sickle cell disease, comprising administering to a subject in need thereof a therapeutically effective amount of any of the following compounds: 3-(4-fluorophenyl)-6-((3-(pyridin-4-yloxy)azetidin-1-yl)methyl)imidazo[1,5-a]pyrazin-8(7H)-one (P1), 6-[3-(pyridin-3-yloxy)azetidin-1-ylmethyl]-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P2), (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 (P3, enantiomer 1), (3R,4R)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (P3, enantiomer 2) and 2-[3-(4-fluoro-phenoxy)-azetidin-1-ylmethyl]-7-(tetrahydro-pyran-4-yl)-3H-imidazo[5,1-f][1,2,4]triazin-4-one (P4).

[0053] PDE9 inhibitors

[0054] In the context of the present invention, if the IC of any of the three PDE9 isoforms is reached 50The compound is considered a PDE9 inhibitor if the amount required to achieve the desired level is 10 micromolar or less, preferably less than 9 micromolar, such as 8 micromolar or less, such as 7 micromolar or less, such as 6 micromolar or less, such as 5 micromolar or less, such as 4 micromolar or less, such as 3 micromolar or less, more preferably 2 micromolar or less, such as 1 micromolar or less, especially 500 nM or less. In a preferred embodiment, the compound achieves an IC of PDE9. 50 The desired amount of the PDE9 inhibitor required for the desired level is 400 nM or less, such as 300 nM or less, 200 nM or less, 100 nM or less, or even 80 nM or less, such as 50 nM or less, for example 25 nM or less.

[0055] Throughout this application, the symbol IC 50 IC50 and IC50 are used interchangeably.

[0056] Isomers

[0057] When the compounds of the invention contain one or more chiral centers, unless otherwise specified, reference to any compound will encompass the enantiomerically or diastereomerically pure compound as well as mixtures of enantiomers or diastereomers in any ratio.

[0058] Pharmaceutically acceptable salts

[0059] The present invention also includes salts of the compounds, generally pharmaceutically acceptable salts. Such salts include pharmaceutically acceptable acid addition salts. Acid addition salts include salts of inorganic acids and organic acids.

[0060] Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, sulfamic acid, nitric acid, etc. Representative examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, glycolic acid, itaconic acid, lactic acid, methanesulfonic acid, maleic acid, malic acid, malonic acid, mandelic acid, oxalic acid, picric acid, pyruvic acid, salicylic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, tartaric acid, ascorbic acid, pamoic acid, bisimethylene salicylic acid, edisulphonic acid, gluconic acid, citraconic acid, aspartic acid, stearic acid, palmitic acid, EDTA, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, p-toluenesulfonic acid, theophylline acetic acid, and 8-halotheophylline (e.g., 8-bromotheoline), etc. More examples of pharmaceutically acceptable inorganic or organic acid addition salts are included in the pharmaceutically acceptable salts listed in Berge, SM et al., J. Pharm. Sci. 1977, 66, 2, the contents of which are incorporated herein by reference.

[0061] Furthermore, the compounds of the present invention can exist in unsolvated as well as solvated forms with pharmaceutically 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.

[0062] Pharmaceutical composition

[0063] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of any compound of the present invention and a pharmaceutically acceptable carrier or diluent. The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one of the specific compounds disclosed in the experimental section herein and a pharmaceutically acceptable carrier or diluent.

[0064] The compounds of the present invention can be administered individually or in combination with a pharmaceutically acceptable carrier, diluent or excipient in a single dose or multiple doses. The pharmaceutical composition according to the present invention can be formulated together with a pharmaceutically acceptable carrier or diluent and any other known adjuvant and excipient according to conventional techniques such as those described in the following document: Remington: The Science and Practice of Pharmacy (Remington: Pharmacy Science and Practice), 22nd edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2013.

[0065] The pharmaceutical composition may be specifically formulated for administration by any suitable route, such as 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 appreciated that the route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient.

[0066] Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, the composition can be prepared to have a coating such as an enteric coating, or it can be formulated to provide a controlled release of the active ingredient such as a sustained release or extended release according to methods known in the art. Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs.

[0067] Pharmaceutical compositions for parenteral administration include sterile injectable aqueous and non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. Other suitable administration forms include, but are not limited to, suppositories, sprays, ointments, creams, gels, inhalants, skin patches and implants.

[0068] A typical oral dosage is about 0.001 to about 100 mg / kg body weight per day. A typical oral dosage may also be about 0.01 to about 50 mg / kg body weight per day. A typical oral dosage may also be about 0.05 to about 10 mg / kg body weight per day. Oral dosages are generally administered in one or more doses (usually one to three doses per day). The exact dosage will depend on the frequency and mode of administration; the sex, age, weight and general condition of the subject being treated; the nature and severity of the condition being treated; and any concomitant diseases to be treated and other factors that will be apparent to one skilled in the art.

[0069] The formulations may also be presented in unit dosage form by methods known to those skilled in the art. For illustrative purposes, a typical unit dosage form for oral administration may contain about 0.01 to about 1000 mg, about 0.05 to about 500 mg, or about 0.5 mg to about 200 mg.

[0070] For parenteral routes of administration, such as intravenous, intrathecal, intramuscular and the like, typical doses are about half of those used for oral administration.

[0071] 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 and at least one pharmaceutically acceptable carrier or diluent. In one embodiment of the present invention, the compound used in the aforementioned method is one of the specific compounds disclosed in the experimental section herein.

[0072] The compounds of the present invention are generally used as free substances or as pharmaceutically acceptable salts thereof. Such salts are prepared in a conventional manner by treating a solution or suspension of the compounds of the present invention with a molar equivalent of a pharmaceutically acceptable acid. Representative examples of suitable organic and inorganic acids are described above.

[0073] For parenteral administration, solutions of the compounds of this invention in sterile aqueous solution, propylene glycol aqueous solution, vitamin E aqueous solution, or sesame oil or peanut oil can be used. Such aqueous solutions should be appropriately buffered when necessary, and the liquid diluent should first be made isotonic with enough saline or glucose. The aqueous solution is particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. The compounds of this invention can be easily incorporated into known sterile aqueous media using standard techniques known to those skilled in the art.

[0074] 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, gum arabic, 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, the carrier or diluent may include any sustained-release substance known in the art, such as glyceryl monostearate or glyceryl distearate, either alone or mixed with wax. The pharmaceutical composition formed by combining the compound of the present invention with a pharmaceutically acceptable carrier is easily administered in various dosage forms suitable for the disclosed routes of administration. The preparation can be conveniently presented in unit dosage form by methods known in the pharmaceutical field.

[0075] The formulations of the present invention suitable for oral administration can be presented as individual units, such as capsules or tablets, each containing a predetermined amount of active ingredient and optionally a suitable excipient. In addition, orally available formulations can be in the form of powders or granules, solutions or suspensions in water or non-aqueous liquids, or oil-in-water or water-in-oil liquid emulsions.

[0076] If a solid carrier is used for oral administration, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form, or it can be in the form of a troche or lozenge. The amount of solid carrier can vary widely, but will be in the range of about 25 mg to about 1 g per dosage unit. If a liquid carrier is used, the preparation can 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.

[0077] 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 a conventional adjuvant and / or diluent and then compressing the mixture in a conventional tablet press to prepare tablets. Examples of adjuvants or diluents include: corn starch, potato starch, talc, magnesium stearate, gelatin, lactose, glue, etc. Any other adjuvant or additive commonly used for such purposes, such as coloring agents, flavoring agents, preservatives, etc., can be used, provided that they are compatible with the active ingredient.

[0078] Compounds of the present invention

[0079] Table 1 lists the compounds of the present invention and the corresponding IC50 values ​​(nM), which were determined as described in the "PDE9 Inhibition Assay" section. Further, the concentrations of the compounds in plasma and brain are listed, which were determined as described in the "Blood-Brain Barrier Penetration" section. Each compound constitutes an independent embodiment of the present invention:

[0080]

[0081]

[0082] Table 1: Compounds of the present invention, IC50 values ​​and plasma / brain concentrations

[0083] Example

[0084] Example 1. Synthesis of compounds

[0085] The compounds of the present invention can be synthesized as follows.

[0086] Overall plan:

[0087]

[0088] Option 1:

[0089]

[0090] Scheme 2 (Compound (P1)):

[0091]

[0092] Scheme 3 (Compound (P2)):

[0093]

[0094] Scheme 4 (Compound (P3)):

[0095]

[0096] Scheme 5 (Compound (P4)):

[0097]

[0098] Synthesis steps:

[0099] List of abbreviations

[0100] aq water

[0101] NBS N-bromosuccinimide

[0102] Boc tert-butyloxycarbonyl

[0103] ℃ degrees Celsius

[0104] CDI N,N-Carbonyldiimidazole

[0105] δ H Chemical shift downfield from tetramethylsilane, in parts per million

[0106] DCM dichloromethane

[0107] DEAD Diethyl azodicarboxylate

[0108] Dppf Bis(diphenylphosphino)ferrocene

[0109] DIPEA N,N-Diisopropylethylamine

[0110] DMF N,N-dimethylformamide

[0111] eq equivalent

[0112] ESI electrospray ionization

[0113] Et ethyl

[0114] EtOAc

[0115] g grams

[0116] HPLC high-performance liquid chromatography

[0117] h hour

[0118] Hz Hertz

[0119] J coupling constant (in NMR spectroscopy)

[0120] LCMS liquid chromatography-mass spectrometry

[0121] LiHMDS Lithium bis(trimethylsilyl)amide

[0122] μ micro

[0123] m multiplet (spectral); meter; millimeter

[0124] M + Parent molecular ion

[0125] Me methyl

[0126] MeCN Acetonitrile

[0127] MeOH methanol

[0128] MHz Megahertz

[0129] min

[0130] mL milliliters

[0131] MS

[0132] MTBE methyl tert-butyl ether

[0133] N equivalent concentration (equivalent per liter)

[0134] NaOH sodium hydroxide

[0135] NBS N-bromosuccinimide

[0136] nm nanometer

[0137] NMR Nuclear Magnetic Resonance

[0138] PE petroleum ether, boiling point: 60~90℃

[0139] rt room temperature

[0140] s single peak (spectrum)

[0141] t triplet (spectrum)

[0142] T temperature

[0143] TEA triethylamine

[0144] TFA trifluoroacetic acid

[0145] THF Tetrahydrofuran

[0146] TLC thin layer chromatography

[0147] TMS Tetramethylsilane

[0148] TMS-Cl trimethylchlorosilane

[0149] Tol Toluene

[0150] General experimental methods

[0151] Recorded on a Bruker Avance III 400 MHz and a Bruker Fourier 300 MHz 1 H NMR spectrum using TMS as the internal standard.

[0152] LCMS was performed on an Agilent LC / MSD 1200 series (column: ODS2000 (50×4.6 mm, 5 μm) on a quadrupole mass spectrometer, operating in ES (+) or (−) ionization mode; T = 30° C.; flow rate = 1.5 mL / min; detection wavelength: 214 nm.

[0153] Synthesis of 6-chloro-pyrazin-2-ylamine (9)

[0154]

[0155] By compound 8 (450.0g, 3.02mol) in dense NH solution (3.0L) in 10L sealed pressure vessel at 135 ℃ and stir overnight.TLC and LC / MS show that starting raw material is fully converted.Reaction mixture is cooled to room temperature, and filtered, obtain white solid.This solid is washed with water (200mL x 3), then dry, obtain compound 9 (312g, yield is 80%) as solid.

[0156] 1 HNMR (400 MHz, DMSO-d6): δ7.82 (s, 1H), 7.12 (s, 1H), 6.93 (s, 2H). MS calculated value: 129 MS found value: 130 ([M+H] + ).

[0157] Synthesis of 6-chloro-5-iodo-pyrazin-2-ylamine (10)

[0158]

[0159] To a mixture of compound 9 (312.0 g, 2.4 mol) and KCO (664.0 g, 4.8 mol) in MeOH (1.0 L) was added ICl (704.0 g, 4.3 mol in 1.0 L DCM) at 0 ° C over 2 hours. The reaction mixture was then stirred at room temperature overnight. The reaction was quenched with NaSO aqueous solution (2 M, 1.5 L). The mixture was extracted with DCM (1.0 L x 3). The combined organic phases were dried over anhydrous NaSO, filtered and concentrated. The crude product was purified by silica gel column chromatography (PE / EA=10 / 1 to 4 / 1) to obtain compound 10 (460 g, 75% yield) as a solid.

[0160] 1 HNMR (400 MHz, DMSO-d6): δ 7.68 (s, 1H), 7.07 (s, 2H). MS calculated value: 255 MS found value: 256 ([M+H] + ).

[0161] Synthesis of 5-amino-3-chloro-pyrazine-2-carbonitrile (11)

[0162]

[0163] A mixture of compound 10 (460.0g, 1.8mol) and CuCN (177.0g, 1.98mol) in DMF (2.0L) was stirred at 150°C for 2 hours in an oil bath. LC / MS shows that the starting material is fully converted. The reaction mixture is cooled to room temperature and poured into EtOAc (1.5L). Concentrated NH is slowly added to the mixture obtained The aqueous solution (1.0L) is then extracted with EtOAc (1.0L x 2). The organic phases merged are washed with H o (1.5L x 5) and salt solution (1.5L), and through anhydrous Na sO dried. The organic phase is filtered and concentrated to obtain compound 11 (232g, 84% yield) as a solid.

[0164] 1 HNMR (400 MHz, DMSO-d6): δ 8.12 (s, 2H), 7.88 (s, 1H). MS calculated value: 154; MS found value: 155 ([M+H] + ).

[0165] Synthesis of 5-amino-3-methoxy-pyrazine-2-carbonitrile (12)

[0166]

[0167] In a round-bottomed flask, potassium tert-butoxide (168.0g, 1.5mol) was added portionwise to methanol (1.5L). The suspension was refluxed for 1 hour. Then under N2 atmosphere, compound 11 (232.0g, 1.5mol) was added. The suspension obtained was refluxed for 1.5 hours. After cooling to room temperature, the reaction mixture was concentrated under vacuum, and diluted with water (2.0L), then extracted with EtOAc (2.0L x5). The organic phase merged was Na2SO4 dried, filtered and concentrated to obtain 12 (170g, 75% yield) as a solid.

[0168] 1 HNMR (300 MHz, DMSO-d6): δ7.69 (s, 2H), 7.51 (s, 1H), 3.89 (s, 3H). MS calculated value: 150; MS found value: 151 ([M+H] + ).

[0169] Synthesis of (5-cyano-6-methoxy-pyrazin-2-yl)-carbamic acid tert-butyl ester (13)

[0170]

[0171] 4-Dimethylaminopyridine (1.0 g, 0.01 mol) was added to a mixture of compound 12 (120.0 g, 0.8 mol) in DCM (1.5 L) at room temperature. DCM (1.0 L) containing di-tert-butyl dicarbonate (327 g, 1.5 mol) was then added dropwise at 10-20 ° C for 2 hours. The reaction was then stirred at room temperature overnight. The suspension was dissolved and the reaction solution was diluted with 2 L of water. The DCM phase was separated and dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE / EtOAc=10:1) to give 13 (150 g, 75% yield).

[0172] 1 HNMR (300 MHz, DMSO-d6): δ10.78 (s, 1H), 8.70 (s, 1H), 3.97 (s, 3H), 1.49 (s, 9H). MS calculated value: 250; MS found value: 251 ([M+H] + ).

[0173] Synthesis of (5-aminomethyl-6-methoxy-pyrazin-2-yl)-carbamic acid tert-butyl ester (14)

[0174]

[0175] At room temperature, Raney Ni (10.0 g) was added to a mixture of compound 13 (30.0 g, 120 mmol) in a solution of concentrated NH in MeOH (500 mL). The suspension was stirred overnight at room temperature under 1 atm of H. The reaction mixture was diluted with a mixture of DCM / MeOH (1:1). The reaction mixture was filtered, and the filtrate was concentrated in vacuo. The residue was ground with PE / EtOAc=2 / 1 to give 14 (23 g, 75% yield) as a solid.

[0176] 1 HNMR (300 MHz, DMSO-d6): δ8.46 (s, 1H), 3.87 (s, 3H), 3.70 (s, 2H), 3.17 (s, 3H), 1.47 (s, 9H). MS calculated value: 254; MS found value: 255 ([M+H] + ).

[0177] 5-[(4-Fluoro-benzoylamino)-methyl]-6-methoxy-pyrazin-2-yl-carbamic acid tert-butyl ester (15) synthesis

[0178]

[0179] To a solution of compound 14 (4.52 g, 17.86 mmol) in DCM (200 mL) was added TEA (5.41 g, 58.53 mmol), followed by dropwise addition of 4-fluorobenzoyl chloride (3.4 g, 21.42 mmol). The resulting reaction mixture was stirred at room temperature for 2 hours. TLC detection revealed complete reaction. The reaction was quenched with water (100 mL). The organic phase was separated, and the aqueous phase was extracted with DCM (200 mL × 2). The combined organic phases were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography to give 15 (5.77 g, 85.9% yield) as a solid.

[0180] 1 HNMR (400 MHz, DMSO-d6): δ9.89 (s, 1H), 8.81 (t, J = 5.6 Hz, 1H), 8.46 (s, 1H), 7.94 (m, 2H), 7.29 (m, 2H), 4.49 (d, J = 5.6 Hz, 2H), 3.90 (s, 3H), 1.47 (s, 9H). MS calculated value: 376; MS found value: 377 ([M+H] + ).

[0181] Synthesis of N-(5-amino-3-methoxy-pyrazin-2-ylmethyl)-4-fluoro-benzamide (16)

[0182]

[0183] Compound 15 (5.77 g, 15.33 mmol) was dissolved in DCM (25 mL). TFA (25 mL) was added. The reaction was stirred at room temperature overnight. TLC detected that the reaction was complete. The solvent was removed. The residue was diluted with DCM (100 mL) and saturated NaHCO3 aqueous solution (100 mL). The organic phase was separated, and the aqueous phase was extracted with DCM (100 mL×2). The combined organic phases were dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with PE / EtOAc=6:1 to 1:1) to obtain 16 (3.9 g, 92.2% yield) as a solid.

[0184] 1 HNMR (300 MHz, CDCl3): δ 7.90-7.85 (m, 2H), 7.46 (s, 1H), 7.40 (t, J = 6.0 Hz, 1H), 7.11 (m, 2H), 4.60 (d, J = 6.0 Hz, 2H), 4.37 (s, 2H), 3.93 (s, 3H). MS calculated value: 276; MS found value: 277 ([M+H] + ).

[0185] Synthesis of 4-fluoro-N-(5-iodo-3-methoxy-pyrazin-2-ylmethyl)-benzamide (17)

[0186]

[0187] Compound 16 (3.9 g, 14.1 mmol) was dissolved in anhydrous THF (100 mL). Under N2 atmosphere, CuI (2.7 g, 14.1 mmol) was added, followed by isoamyl nitrite (4.9 g, 42.3 mmol) and CH2I2 (3.8 g, 14.1 mmol). The reaction mixture was heated at 75°C for 3 hours. The reaction was then cooled to room temperature and filtered. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with PE / EtOAc 5:1) to give 17 (2.0 g, 37% yield) as a solid.

[0188] 1 HNMR (400 MHz, CDCl3): δ8.34 (s, 1H), 7.88 (m, 2H), 7.36 (t, J = 4.4 Hz, 1H), 7.14 (m, 2H), 4.66 (d, J = 4.4 Hz, 2H), 4.04 (s, 3H). MS calculated value: 387; MS found value: 388 ([M+H] + ).

[0189] Synthesis of 3-(4-fluoro-phenyl)-6-iodo-8-methoxy-imidazo[1,5-a]pyrazine (18)

[0190]

[0191] Compound 17 (1.6 g, 4.13 mmol) was suspended in MeCNCH3CN (50 mL). POCl3 (6.3 g, 41.3 mmol) and TEA (1.25 g, 12.39 mmol) were added under N2 atmosphere, and the reaction mixture was heated at 85 ° C for 6 hours. The solvent was removed under reduced pressure. The residue was diluted with DCM (100 mL) and ice water (30 mL). Saturated Na2CO3 aqueous solution (100 mL) was then added. The organic phase was separated, and the aqueous phase was extracted with DCM (100 mL × 2). The combined organic phases were dried, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with PE / EtOAc=20:1 to 3:1) to obtain 18 (1.5 g, 97.8% yield) as a solid.

[0192] 1 HNMR (300 MHz, CDCl3): δ8.01 (s, 1H), 7.82 (s, 1H), 7.77-7.72 (m, 2H), 7.28-7.23 (m, 2H), 4.11 (s, 3H). MS calculated value: 369; MS found value: 370 ([M+H] + ).

[0193] Synthesis of 3-(4-fluoro-phenyl)-8-methoxy-imidazo[1,5-a]pyrazine-6-carboxylic acid methyl ester (19)

[0194]

[0195] To a mixture solution of 18 (4.11 g, 11.13 mmol), CuI (640 mg, 3.34 mmol) and Pd(dppf)2Cl2 (930 mg, 1.11 mmol) in MeOH (100 mL) was added TEA (14 mL). The reaction mixture was heated to 85 ° C under a CO atmosphere (3.0 MPa) for 16 hours. The reaction mixture was cooled to room temperature and concentrated in vacuo to give a crude product. The residue was purified by silica gel column chromatography (eluted with PE / EtOAc=1:1) to give 19 (2.3 g, 75% yield) as a solid.

[0196] 1 H NMR (400 MHz, CDCl3): δ8.59 (s, 1H), 7.87 (s, 1H), 7.78 (m, 2H), 7.28 (m, 2H), 4.21 (s, 3H), 3.96 (s, 3H). MS calculated value: 301; MS found value: 302 ([M+H] + ).

[0197] Synthesis of [3-(4-fluoro-phenyl)-8-methoxy-imidazo[1,5-a]pyrazin-6-yl]-methanol (20)

[0198]

[0199] A mixture of powdered anhydrous CaCl2 (4.23 g, 38.15 mmol) and NaBH4 (2.86 g, 76.3 mmol) in THF (100 mL) was stirred at room temperature for 1 hour. A solution of compound 19 (2.3 g, 7.63 mmol) in THF (25 mL) was added, followed by addition of MeOH (25 mL). The reaction mixture was stirred at room temperature for 1.5 hours. The mixture reaction was quenched with water (50 mL). After removing the organic solvent under reduced pressure, the resulting solution was dissolved in EtOAc (200 mL) and water (50 mL). The separated aqueous phase was extracted with EtOAc (3 x 100 mL). The combined organic phase was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluting with PE / EtOAc=2:1) ​​to obtain the desired product compound 20 (1.93 g, 93% yield), which was a solid.

[0200] 1H NMR (400 MHz, CDCl3): δ 7.81 (s, 1H), 7.79-7.74 (m, 3H), 7.25-7.22 (m, 2H), 4.56 (d, J = 4.4 Hz, 2H), 4.11 (s, 3H), 2.41 (t, J = 4.4 Hz, 1H). MS calculated value: 273; MS found value: 274 ([M+H] + ).

[0201] Synthesis of 6-chloromethyl-3-(4-fluoro-phenyl)-8-methoxy-imidazo[1,5-a]pyrazine (21)

[0202]

[0203] To a solution of 20 (1.88 g, 6.88 mmol) in dichloromethane (100 mL) was added dropwise thionyl chloride (4.5 mL) while cooling on an ice-water bath. After addition was complete, the mixture was stirred for another 2 hours. The reaction mixture was quenched with ice water, washed with salt water (20 mL), dried over Na SO and concentrated in vacuo to give 21 (2.01 g, 100% yield) as a solid.

[0204] 1 H NMR (400 MHz, CDCl3): δ7.87 (s, 1H), 7.83-7.79 (m, 3H), 7.30-7.27 (m, 2H), 4.50 (s, 2H), 4.12 (s, 3H). MS calculated value: 291; MS found value: 292 ([M+H] + ).

[0205] Synthesis of 6-chloromethyl-3-(4-fluoro-phenyl)-7H-imidazo[1,5-a]pyrazin-8-one (22)

[0206]

[0207] To a solution of 21 (1.87 g, 6.41 mmol) in MeOH (50 mL) was added 6N aqueous HCl, and the resulting solution was stirred for 1 h at 70° C. The mixture was concentrated to give the product 22 (1.60 g, 90% yield) as a white solid.

[0208] 1 H NMR (300 MHz, DMSO-d6): δ 11.29 (s, 1H), 8.07 (s, 1H), 7.83-7.87 (m, 2H), 7.74 (s, 1H), 7.46-7.50 (m, 2H), 4.59 (s, 2H). MS calculated value: 277; MS found value: 278 ([M+H] + ).

[0209] Synthesis of 4-(azetidin-3-yloxy)-pyridine hydrochloride (5)

[0210]

[0211] To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate 1 (4.55 g, 26.3 mmol) in THF (100 mL) was added pyridine-4-ol (2.0 g, 21.0 mmol), PPh (6.89 g, 26.3 mmol) and DEAD (4.57 g, 26.3 mmol). The resulting reaction mixture was stirred overnight at 70 ° C. TLC showed that the reaction was complete. The reaction mixture was concentrated under vacuum. The resulting oil was dissolved in a 1.0 M HCl aqueous solution (20 mL) and extracted with DCM (50 mL × 3). The combined organic phases were washed with HCl (aq) solution (0.5 M, 150 mL). The aqueous solution was combined and alkalized to pH ≈ 12 with NaOH (1.0 M) and extracted with DCM (100 mL × 3). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography to give 4 (2.81 g, yield 53%) as a solid.

[0212] 1 HNMR (400 MHz, DMSO-d6): δ 8.41 (d, J = 6.0 Hz, 2H), 6.88 (d, J = 6.0 Hz, 2H), 5.07-5.09 (m, 1H), 4.32-4.33 (m, 2H), 3.80-3.82 (m, 2H), 1.39 (s, 9H). MS calculated value: 250; MS found value: 251 ([M+H] + ).

[0213] To a solution of 4 (2.81 g, 11.2 mmol) in Et2O (100 mL) was added a solution of HCl in Et2O (20 mL). The resulting reaction mixture was stirred at room temperature overnight. TLC indicated that the reaction was complete. The reaction mixture was filtered and the solid was dried to afford 5 (1.82 g, 87% yield).

[0214] 1 HNMR (300 MHz, DMSO-d6): δ9.58 (s, 2H), 8.77-8.79 (m, 2H), 7.48-7.49 (m, 2H), 5.40-5.45 (m, 1H), 4.49-4.51 (m, 2H), 4.07-4.11 (m, 2H). MS calculated value: 150; MS found value: 151 ([M+H] + ).

[0215] 3-(4-Fluorophenyl)-6-((3-(pyridin-4-yloxy)azetidin-1-yl)methyl)imidazo[1,5-a] Synthesis of pyrazin-8(7H)-one (P1)

[0216]

[0217] To a mixture of compound 22 (1.5 g, 5.4 mmol) and 5 (1.31 g, 7.0 mmol) in MeCN (100 mL) was added DIPEA (6.96 g, 5.4 mmol). The reaction mixture was heated and refluxed overnight. The solvent was removed in vacuo. The residue was purified by reverse phase silica gel flash column chromatography (eluted with 5% to 95% aqueous MeCN) to give the desired product P1 (1.28 g, 62% yield) as a solid.

[0218] 1 H NMR (400 MHz, DMSO-d6): δ 10.7 (s, 1H), 8.37 (d, J = 6.0 Hz, 2H), 7.85 (s, 1H), 7.85-7.82 (m, 2H), 7.42 (m, 2H), 7.34 (s, 1H), 6.86 (d, J = 6.0 Hz, 2H), 4.93 (m, 1H), 3.88-3.77 (m, 2H), 3.42 (s, 2H), 3.18-3.14 (m, 2H). MS calculated value: 391; MS found value: 392 ([M+H] + ).

[0219] tert-Butyl (6-methoxy-5-{[(tetrahydro-pyran-4-carbonyl)-amino]-methyl}-pyrazin-2-yl)-carbamate Synthesis of ester (23)

[0220]

[0221] To a solution of compound 14 (28.4 g, 0.11 mol) in DCM (200 mL) was added TEA (49 mL, 0.34 mol), followed by the dropwise addition of tetrahydropyran-4-carbonyl chloride (17.5 g, 0.13 mol). The resulting reaction mixture was stirred at room temperature overnight. TLC showed that the reaction was complete. The reaction was quenched with water (100 mL). The organic phase was separated, and the aqueous phase was extracted with DCM (200 mL x 2). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE / EA=5 / 1 to 1 / 3) to give 23 (31 g, 75% yield) as a solid.

[0222] 1H NMR (DMSO-d6, 400 MHz): δ 9.89 (s, 1H), 8.47 (s, 1H), 8.10-8.07 (t, J = 5.2 Hz, 1H), 4.29-4.28 (d, J = 5.2 Hz, 2H), 3.87 (s, 3H), 3.85-3.82 (m, 2H), 3.32-3.25 (m, 2H), 2.45-2.43 (m, 1H), 1.60-1.55 (m, 4H), 1.48 (s, 9H). MS calculated value: 366; MS found value: 367 ([M+H] + ).

[0223] Synthesis of Tetrahydro-pyran-4-carboxylic acid (5-amino-3-methoxy-pyrazin-2-ylmethyl)-amide (24)

[0224]

[0225] Compound 23 (19.0 g, 0.08 mol) was dissolved in DCM (100 mL). TFA (100 mL) was added. The reaction was stirred at room temperature overnight. TLC showed that the reaction was complete. The solvent was removed. The residue was diluted with DCM (100 mL) and saturated NaHCO3 aqueous solution (100 mL). The aqueous phase was extracted with DCM (100 mL x 2). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE / EA=6 / 1 to 1 / 1) to obtain 24 (19 g, 85% yield) as a solid.

[0226] 1 H NMR (DMSO-d6, 400 MHz): δ 7.87 (t, J = 4.8 Hz, 1H), 7.36 (s, 1H), 6.26 (br.s, 2H), 4.16 (d, J = 4.8 Hz, 2H), 3.86-3.82 (m, 2H), 3.80 (s, 3H), 3.30-3.24 (m, 2H), 2.41 (m, 1H), 1.59-1.54 (m, 4H). MS calculated value: 266; MS found value: 267 ([M+H] + ).

[0227] Synthesis of Tetrahydropyran-4-carboxylic Acid (5-iodo-3-methoxy-pyrazin-2-ylmethyl)-amide (25)

[0228]

[0229] To a mixture of compound 24 (15.5 g, 58.4 mmol), CH2I2 (23.5, 87.6 mmol) and isoamyl nitrite (23.9 g, 204 mmol) in THF (600 mL) was added CuI (11.3 g, 39.6 mmol) under an N2 atmosphere. The reaction mixture was stirred at 80°C for 7 hours. The precipitate was filtered. The filtrate was concentrated and purified by column chromatography (MeOH / DCM=1 / 20) to obtain a crude product, which was then purified by reverse phase silica gel flash column chromatography (eluting with an aqueous solution of 5% to 95% MeCN) to obtain the desired product, compound 25 (4.5 g, 20% yield), as a solid.

[0230] 1 H NMR (DMSO-d6, 300 MHz): δ 8.41 (s, 1H), 8.16 (t, J = 5.4 Hz, 1H), 4.28 (d, J = 5.4 Hz, 2H), 3.92 (s, 3H), 3.87-3.81 (m, 2H), 3.30-3.24 (m, 2H), 2.49 (m, 1H), 1.60-1.56 (m, 4H). MS calculated value: 377 MS found: 378 ([M+H] + ).

[0231] Synthesis of 6-iodo-8-methoxy-3-(tetrahydro-pyran-4-yl)-imidazo[1,5-a]pyrazine (26)

[0232]

[0233] To a solution of compound 25 (4.5 g, 16.9 mmol) in MeCN (100 mL) was added POCl (18 g, 118 mmol). The reaction was stirred at reflux overnight under an N atmosphere. The solvent was removed under reduced pressure. The residue was treated with ice water (30 mL) and DCM (150 mL). The pH was adjusted to 7-8 with saturated NaCO solution. The separated aqueous phase was extracted with DCM (100 mL x 4). The combined organic phases were concentrated under reduced pressure to give the desired compound 26 (4.2 g, 99% yield) as a solid.

[0234] 1 H NMR (DMSO-d6, 400 MHz): δ8.46 (s, 1H), 7.64 (s, 1H), 3.98 (s, 3H), 3.94 (m, 2H), 3.53-3.47 (m, 3H), 1.81-1.77 (m, 4H). MS calculated value: 359; MS found value: 360 ([M+H] + ).

[0235] Synthesis of 8-methoxy-3-(tetrahydro-pyran-4-yl)-imidazo[1,5-a]pyrazine-6-carboxylic acid methyl ester (27)

[0236]

[0237] To a suspension of compound 26 (4.2 g, 11.7 mmol) in MeOH (100 mL) was added CuI (0.7 g, 3.0 mmol), Pd (dppf) Cl (1.0 g, 1.17 mmol) and TEA (16 mL). The reaction mixture was stirred for 16 hours in an oil bath set at 85 ° C under a CO atmosphere (3 MPa). The precipitate was filtered off and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography (eluted with EtOAc / PE = 2 / 1 to MeOH / DCM = 1 / 20) to give the desired compound 27 (2.7 g, 80% yield) as a solid.

[0238] 1 H NMR (CDCl 3, 400 MHz): δ8.32 (s, 1H), 7.70 (s, 1H), 4.17 (s, 3H), 4.14 (m, 2H), 3.98 (s, 3H), 3.66-3.60 (m, 2H), 3.31-3.26 (m, 1H), 2.17 -2.13 (m, 2H), 1.93 (m, 2H). MS calculated value: 291; MS found value: 292 ([M+H] + ).

[0239] Synthesis of [8-methoxy-3-(tetrahydro-pyran-4-yl)-imidazo[1,5-a]pyrazin-6-yl]-methanol (28)

[0240]

[0241] At room temperature in THF (100mL) by powdered anhydrous CaCl2 (2.4g, 21.5mmol) and NaBH4 (1.6g, 42.9mmol) mixture is stirred for 1 hour.Add a solution of compound 27 (2.4g, 4.29mmol) in THF (25mL), then add MeOH (25mL).The reaction mixture is stirred at room temperature for 1.5 hours.The mixture reaction is quenched with water (50mL).After removing the organic solvent under reduced pressure, the residue is distributed between EtOAc (200mL) and water (50mL).The separated aqueous phase is extracted with EtOAc (100x 3mL).Then the combined organic phase is concentrated under reduced pressure.The residue is purified by silica gel column chromatography (eluting with DCM / MeOH=100 / 1 to 30 / 1) to obtain the desired product compound 28, which is a white solid (1.87, in a yield of 80%).

[0242] 1 H NMR (CDCl 3,400 MHz): δ 7.65 (s, 1H), 7.43 (s, 1H), 4.58 (s, 2H), 4.13 (d, J = 12.0 Hz, 2H), 4.07 (s, 3H), 3.60 (dd, J = 10.4 Hz, 10.8 Hz, 2H), 3.24-3.17 (m, 1H), 2.60 (m, 1H), 2.18 -2.06 (m, 2H), 1.90 (d, J = 12.8 Hz, 2H). MS calculated value: 263; MS found value: 264 ([M+H] + ).

[0243] Synthesis of 6-chloromethyl-3-(tetrahydropyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one (30)

[0244]

[0245] At 0 ℃, SOCl2 (5mL) was added to the solution of compound 28 (1.9g, 7.11mmol) in DCM (100mL), and then the reaction mixture was stirred at room temperature for 5 hours. TLC and LC-MS showed that the starting raw material had been consumed. The concentrated mixture solution was then dissolved in HCl (aq.) solution (6N, 20mL). The mixture reaction was stirred at room temperature for 10 minutes. The reaction mixture was concentrated under reduced pressure to obtain the desired product compound 29 (1.90g, 95% yield), which was a solid.

[0246] 1 H NMR (DMSO-d6, 300 MHz): δ 11.49 (s, 1H), 8.28 (s, 1H), 8.00 (s, 1H), 4.55 (s, 2H), 3.97 (dd, J = 2.4 Hz, 2.8 Hz, 2H), 3.53-3.43 (m, 3H), 1.95-1.81 (m, 4H). MS calculated value: 267 MS found: 268 ([M+H] + ).

[0247] Synthesis of 3-(azetidin-3-yloxy)-pyridine hydrochloride (7)

[0248]

[0249] Compound 7 was prepared by a procedure analogous to that used to prepare amine 5.

[0250] Analytical data of 7: 1H NMR (DMSO-d6, 400 MHz): δ 9.73 (br d, 2H), 8.55 (d, J = 2.4 Hz, 2H), 8.47 (d, J = 4.4 Hz, 2H), 7.88-7.75 (m, 2H), 5.28 (t, J = 5.6 Hz, 1H), 4.50-4.43 (m, 2H), 4.08-4.00 (m, 2H). MS calculated value: 150, MS found: 151 ([M+H] + ).

[0251] 6-[3-(Pyridin-3-yloxy)-azetidin-1-ylmethyl]-3-(tetrahydropyran-4-yl)-7H-imidazo Synthesis of [1,5-a]pyrazin-8-one (P2)

[0252]

[0253] To a mixture of compound 30 (550 mg, 2.05 mmol) and 7 (500 mg, 2.67 mmol) in MeCN (200 mL) was added DIPEA (2.7 g, 20.5 mmol). The reaction mixture was refluxed overnight. The solvent was removed in vacuo. The crude product was purified by reverse phase silica gel flash column chromatography (eluted with 5% to 95% MeCN in water) to give the desired product P2 (360 mg, 46% yield) as a solid.

[0254] 1 H NMR (CDCl3, 300MHz): δ8.26 (d, J=4.0Hz 1H),8.22(s,1H),8.20(d,J=2.8Hz,1H),7.91(s,1H),7.24-7.21(m,1H),7.07(d,J=2.8Hz,1H),6.79(s,1H),4.86(m,1H),4.13(m,2H ),3.89(t,J=7.6Hz,2H),3.57(m,2H),3.50(s,2H),3.28(dd,J=2.4Hz,6.8Hz,2H),3.10-30.6(m,1H),2.14-2.08(m,2H),1.87(m,2H). MS calculated value: 381; MS found value: 382 ([M+H] + ).

[0255] Synthesis of 3H-imidazole-4-carboxylic acid methyl ester (32)

[0256]

[0257] To a solution of compound 31 (25 g, 0.22 mol) in MeOH (300 mL) was added H2SO4 (24 mL). The mixture was stirred under reflux for 18 hours. The pH of the reaction solution was then adjusted to 7. The reaction mixture was concentrated in vacuo. The residue was dissolved in 100 ml of MeOH and stirred at room temperature for 15 minutes. The mixture solution was filtered and the filtrate was concentrated to obtain a crude product 32 (28 g, 100% yield) as a solid, which was used in the next step without further purification.

[0258] 1 H NMR (400MHz, DMSO-d6): δ7.80 (s, 2H), 3.57 (s, 3H).

[0259] Synthesis of methyl 3H-imidazole-4-carboxylate (33)

[0260]

[0261] To a solution of compound 32 (22 g, 0.18 mol) in MeCN (500 mL) was added NBS (66 g, 0.37 mol). The mixture was stirred at 70 ° C for 4 hours. The reaction mixture was concentrated in vacuo. The crude product was purified by silica gel column chromatography (eluted with PE / EtOAc=5:1 to 1:1) to obtain compound 33 (20 g, 40% yield) as a solid.

[0262] 1 H NMR (400MHz, DMSO-d6): δ14.35(br,1H),3.81(s,3H).

[0263] Synthesis of racemic trans-1-benzyl-4-methyl-pyrrolidine-3-carboxylic acid ethyl ester (35)

[0264]

[0265] Toluene solution of 34 (69g, 0.29mol) is added butane-2-enoic acid ethyl ester (50g, 0.44mol) and TFA (25mL, 0.32mol). Under N2, the solution obtained is stirred at 50 ℃ and spend the night. Saturated NaHCO3 aqueous solution (300mL) is added to the reaction mixture, and aqueous phase is extracted with EtOAc (500mL x 3). The organic layer merged is washed with salt water (300mL), through Na2SO4 drying, filtered and concentrated in a vacuum. The crude product is purified by flash chromatography (PE / EA=20:1 to 6:1) to obtain the desired racemic trans product 35 (41g, yield 57%), which is an oil.

[0266] Synthesis of (S,S)-trans-1-benzyl-4-methyl-pyrrolidine-3-carboxylic acid ethyl ester (S,S)-(35)

[0267]

[0268] To a solution of Rac-35 (37 g, 0.15 mol) in 4-methyl-2-pentanone, (-)-dibenzoyl-L-tartaric acid (34.78 g, 0.65 eq.) was added, and the resulting reaction mixture was heated to 72 ° C for 1 hour, then cooled to RT and maintained at RT for 4 hours. The resulting solid was filtered out, and the filtrate was washed with concentrated sodium carbonate aqueous solution (55 mL). The aqueous phase was extracted with 4-methyl-2-pentanone (15 mL), and the combined organic phases were washed with brine (40 mL). The organic phase was then treated with (+)-dibenzoyl-D-tartaric acid (32.16 g) and heated to 72 ° C for 1 hour. The reaction mixture was cooled to RT and maintained at this temperature for 4 hours. The solid was filtered out and dried on the filter. The solid was then recrystallized by adding a mixture of MTBE-MeOH (2: 1, 270 mL), heated to 70 ° C for 1 hour, and the product was precipitated at room temperature for 4 hours. The resulting solid was filtered off, washed with MTBE and dried. Two more recrystallizations were performed following the same procedure to give the pure product as (+)-dibenzoyl-D-tartrate salt (>98% ee based on the isolated free base).

[0269] The free base was released by the following steps: the filtered solid was distributed between MTBE (250 mL) and concentrated aqueous sodium carbonate solution (250 mL), and the aqueous phase was extracted with MTBE (125 mL). The combined organic phases were washed with water (250 mL) and salt solution (50 mL), and evaporated to give the product as a clear oil (13.79 g, 0.056 mol).

[0270] Synthesis of racemic trans-4-methyl-pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl 3-methyl ester rac-(36)

[0271]

[0272] To a solution of 35 (41 g, 0.17 mol) and Boc O (43 g, 0.20 mol) in EtOH (500 mL) was added Pd / C (5%, 10.0 g). The reaction mixture was stirred at 50° C. under a H atmosphere (50 Psi) for 48 hours. The reaction mixture was filtered and concentrated in vacuo. The crude product was purified by flash chromatography (PE / EA=20 / 1) to give the desired racemic trans 36 (20 g, 46% yield) as an oil.

[0273] Synthesized via (S,S)-trans-4-methyl-pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl 3-methyl ester (S,S)-(36) (S,S)-trans-4-methyl-pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl ester (S,S)-(37)

[0274]

[0275] Under N2-protective atmosphere, a solution of (S,S)-35 (12.80 g, 51.8 mmol) and Boc2O (13.57 g, 1.2 eq) in EtOH (150 mL) was placed in an autoclave and Pd / C (5%, 2.56 g) was added. The reaction mixture was hydrogenated at 45-50°C and 15-20 Bar H2 pressure with stirring until no more hydrogen was absorbed (48 hours). The reaction mixture was cooled to RT and filtered, and the filtrate was washed with EtOH (50 mL). The filtrate was evaporated to approximately 25 mL at <45°C. Water (10 mL) and NaOH solution (2 mL) were added, and the resulting reaction mixture was stirred at RT for 2 hours (GC analysis showed that the starting material had completely disappeared at this time). Water (125 mL) was added, and the resulting mixture was extracted with MTBE (2 x 50 mL). The aqueous phase was treated with 2N HCl solution to achieve a pH value of 3-4 (about 25 mL), and the resulting solution was extracted with MTBE (2 x 150 mL). The combined organic extracts were washed with salt water (50 mL) and evaporated to about 20 mL. N-heptane (40 mL) was added, and the resulting reaction mixture was placed at 0 ° C for 2 hours, then filtered off and dried to obtain the product (S, S)-37 (9.48 g, 41.7 mmol) as a solid. In this step, ee was determined to be 97.5%. This material has the same NMR and LC / MS characteristics as rac-37 described below.

[0276] Synthesized via (S,S)-trans-4-methyl-pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl 3-methyl ester (S,S)-(36) (S,S)-trans-4-methyl-pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl ester (S,S)-(37)

[0277]

[0278] Under N2-protective atmosphere, a solution of (S,S)-35 (12.80 g, 51.8 mmol) and Boc2O (13.57 g, 1.2 eq) in EtOH (150 mL) was placed in an autoclave and Pd / C (5%, 2.56 g) was added. The reaction mixture was hydrogenated at 45-50°C and 15-20 Bar H2 pressure with stirring until no more hydrogen was absorbed (48 hours). The reaction mixture was cooled to RT and filtered, and the filtrate was washed with EtOH (50 mL). The filtrate was evaporated to approximately 25 mL at <45°C. Water (10 mL) and NaOH solution (2 mL) were added, and the resulting reaction mixture was stirred at RT for 2 hours (GC analysis showed that the starting material had completely disappeared at this time). Water (125 mL) was added, and the resulting mixture was extracted with MTBE (2 x 50 mL). The aqueous phase was treated with 2N HCl solution to achieve a pH value of 3-4 (about 25 mL), and the resulting solution was extracted with MTBE (2 x 150 mL). The combined organic extracts were washed with salt water (50 mL) and evaporated to about 20 mL. N-heptane (40 mL) was added, and the resulting reaction mixture was placed at 0 ° C for 2 hours, then filtered off and dried to obtain the product (S, S)-37 (9.48 g, 41.7 mmol) as a solid. In this step, ee was determined to be 97.5%. This material has the same NMR and LC / MS characteristics as rac-37 described below.

[0279] Synthesis of racemic trans-4-methyl-pyrrolidine-1,3-dicarboxylic acid 1-tert-butyl ester (37)

[0280]

[0281] A solution of compound 36 (10.0 g, 39.1 mmol) and NaOH (3.10 g, 78.2 mmol) in methanol / H o (50 / 5 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated and extracted with EA (150 mL). The aqueous phase was acidified to pH 5 with 2M HCl at 0°C and extracted with EtOAc (150 mL x 3). The combined organic layers were washed with salt water, dried and concentrated to give compound 37 (8.0 g, 90%) as an oil.

[0282] 1 H NMR (400MHz, DMSO-d6): δ12.43(s,1H),3.55-3.51(m,2H),3.47-3.27(m,1H),2.85-2 .78(m,1H),2.63-2.57(m,1H),2.34-2.28(m,1H),1.55(s,9H),1.03(d,J=4.8Hz,3H).

[0283] (S,S)-trans-3-(methoxy-methyl-carbamoyl)-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (S,S)-(38)Synthesis of (S,S)-trans-3-acetyl-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (S,S)-(39)

[0284]

[0285] To a solution of (S,S)-37 (5.0 g, 22.0 mmol) in DCM (50 mL) was added CDI (4.25 g, 1.2 eq) over 10 minutes, while maintaining the temperature below 5°C throughout the process. The reaction mixture was stirred for 1 hour, after which N,O-dimethylhydroxylamine hydrochloride (3.0 g, 1.4 eq) was added in small portions over approximately 10 minutes, maintaining the temperature below 5°C. The reaction was then allowed to warm to room temperature and stirred for 12 hours, at which point the starting material had been completely consumed. Water (50 mL) was added, the phases separated, and the aqueous phase extracted with DCM (35 mL). The combined organic phases were washed with water (50 mL) and concentrated to approximately 5 mL. THF (20 mL) was added, and the resulting solution was evaporated to dryness and dried under high vacuum. Dry THF (50 mL) was added, the solution cooled to 0°C, and MeMgCl (3 M, 11.35 mL, 1.5 eq) was added dropwise under a N2 atmosphere over 30 minutes, ensuring the temperature was below 5°C. The reaction mixture was then warmed to room temperature and stirred for 2 hours (at which point the Weinreb amide was fully converted). Saturated aqueous ammonium chloride (50 mL) was added dropwise below 25°C to quench the reaction, and the resulting reaction mixture was extracted with EtOAc (2 x 50 mL). The combined organic extracts were washed with brine (50 mL) and evaporated to approximately 5 mL. THF (25 mL) was added, and the resulting solution was evaporated to dryness in vacuo to give the product (S,S)-39 (4.91 g, 21.6 mmol) as an oil with approximately 98% ee. All spectral characteristics were identical to those of rac-39.

[0286] Racemic trans-3-(methoxy-methyl-carbamoyl)-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (38) Synthesis

[0287]

[0288] To a solution of 37 (8.0 g, 34.9 mmol) and O,N-dimethylhydroxylamine (4.0 g, 41.9 mmol) in DCM (50 mL) was added CDI (6.8 g, 41.9 mmol). The mixture was reacted and stirred at 20 ° C for 18 hours. Water (100 mL) was added to the mixture solution and extracted with DCM (100 mL x 3). The combined organic layers were washed with brine (30 mL), dried, and concentrated in vacuo. The crude product was purified by flash chromatography (PE / EtOAc=20 / 1) to give racemic trans 38 (8.0 g, 84% yield) as an oil.

[0289] 1 H NMR (400MHz, DMSO-d6): δ3.68(s,3H),3.60-3.48(m,2H),3.20-3.05(m,5H) ,2.84-2.73(m,1H),2.40-2.32(m,1H),1.39(s,9H),0.96(d,J=4.8Hz,3H).

[0290] Synthesis of racemic trans-3-acetyl-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (39)

[0291]

[0292] To a solution of 38 (8.0 g, 29.4 mmol) in THF (60 mL) was added MeMgBr (3.0 M, 13 mL, 38.2 mmol) at 0 ° C. The reaction mixture was stirred at room temperature for 2 hours. The mixture reaction was quenched with saturated NH4Cl aqueous solution (200 mL) and extracted with EtOAc (300 mL x 3). The combined organic layers were washed with brine, dried, and concentrated in vacuo. The crude product was purified by flash chromatography (PE / EtOAc=10 / 1) to obtain the desired racemic trans 39 (6.0 g, 94% yield), which was an oil.

[0293] 1 H NMR (400MHz, DMSO-d6): δ3.66-3.51(m,1H),3.49-3.39(m,1H),3.34-3.24(m,1H), 2.88-2.79(m,2H),2.34-2.30(m,1H),2.15(s,3H),1.36(s,9H),1.02-1.00(m,3H).

[0294] Synthesis of racemic trans-3-(2-bromoacetyl)-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (40)

[0295]

[0296] Under N2 atmosphere, at -78 ℃, LiHMDS solution (1M in THF, 40mL, 40mmol) is added to a solution of 39 (6.0g, 26.4mmol) in THF (100mL). The reaction mixture is stirred at this temperature for 1 hour. Then TMSCl (10mL, 26.4mmol) is added dropwise at -78 ℃, and the reaction temperature is raised to 0 ℃. After 1 hour, PhMe3NBr3 (11.0g, 29.1mmol) is added at 0 ℃. The mixture is stirred for another 1 hour, and then stirred at room temperature overnight. The reaction is quenched with water (200mL) and extracted with EtOAc (250mL x 3). The combined organic layer is washed with salt water, dried, and concentrated in vacuo. The crude product is purified by flash chromatography (PE / EtOAc=10 / 1) to obtain the desired racemic trans 40 (4.5g, 56% yield), which is an oil.

[0297] 1 H NMR (400MHz, CDCl3): δ4.05(s,2H),3.69-3.50(m,2H),3.36-3.30(m,1H),3.04-2.86(m,2H),2.51-2.43(m,1H),1.39(s,9H),1.10-1.05(m,3H).

[0298] Synthesis of (S,S)-trans-3-(2-bromoacetyl)-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (S,S)-(40)

[0299]

[0300] Under N2 atmosphere, at -78 ℃, LiHMDS solution (1M in THF, 21.12mL, 21.12mmol) was added dropwise to a solution of (S,S)-39 (3.96g, 17.4mmol) in THF (50mL). The reaction mixture was stirred at this temperature for 1 hour. TMSBr (6.43g, 42mmol) was then added dropwise at -78 ℃, and the reaction temperature was raised to 0 ℃. After 1 hour, NBS (2.76g, 15.5mmol) was added in small portions at 0 ℃. TLC showed that all starting materials had been consumed. Water (20mL) was added dropwise, the temperature was kept at RT, and the resulting reaction mixture was stirred for 30 minutes. Each phase was separated, and the aqueous phase was extracted with MTBE (2x 15mL). The combined organic phases were washed with salt water, dried, and concentrated in a vacuum. The residue was redissolved in MTBE (25 mL), washed with water (3 x 10 mL) and brine (10 mL), and concentrated in vacuo to give the product as an oil, which was purified by flash chromatography (PE / EtOAc = 10 / 1) to give the desired (S,S)-40 (6.4 g, 20.9 mmol) as an oil.

[0301] Racemic trans-2,5-dibromo-3-[2-(1-tert-butoxycarbonyl-4-methyl-pyrrolidin-3-yl)-2-oxo-ethyl Synthesis of methyl]-3H-imidazole-4-carboxylate (41)

[0302]

[0303] To a solution of 33 (4.1 g, 14.7 mmol) in DMF (30 mL) was added KCO (5.8 g, 42.5 mmol). After stirring for 15 minutes, compound 40 (4.5 g, 14.7 mmol) was added to the reaction mixture. The reaction was stirred at room temperature for 5 hours. The reaction mixture was diluted with EtOAc (200 mL) and washed with brine (200 mL x 2). The organic phase was then dried (NaSO), filtered, and concentrated in vacuo. The residue was purified by column chromatography (PE / EtOAc=10 / 0 to 3 / 1) to give racemic trans 41 (3.0 g, 40% yield) as a solid.

[0304] 1 H NMR (400MHz, DMSO-d6): δ5.41(s,2H),3.78(s,3H),3.68-3.66(m,1H),3.48-3.45(m,1H),3.34- 3.31(m,1H),3.20-3.25(m,1H),2.92-2.87(m,1H),2.50-2.46(m,1H),1.36(s,9H),1.07(m,3H).

[0305] (S,S)-trans-2,5-dibromo-3-[2-(1-tert-butoxycarbonyl-4-methyl-pyrrolidin-3-yl)-2-oxo-ethyl Synthesis of methyl]-3H-imidazole-4-carboxylate (S,S)-(41)

[0306]

[0307] To a solution of 33 (2.78 g, 9.79 mmol) in NMP (30 mL) was added Na2CO3 (3.11 g, 26.2 mmol). After stirring for 15 minutes, compound (S,S)-40 (4.5 g, 14.7 mmol) was added to the reaction mixture. The reaction was stirred at room temperature for 5 hours. The reaction mixture was diluted with EtOAc (200 mL) and washed with brine (200 mL x 2). The organic phase was then dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by column chromatography (PE / EtOAc = 10 / 0 to 3 / 1) to obtain the product as a crude solid, which was recrystallized from 2-propanol / n-heptane to obtain (S,S)-41 (3.03 g, 40% yield) as a solid. The ee of the material at this stage was determined to be greater than 99%. All spectral data were identical to those of rac-41.

[0308] Racemic trans-3-(1,3-dibromo-8-oxo-7,8-dihydro-imidazo[1,5-a]pyrazin-6-yl)-4-carboxylic Synthesis of tert-butyl-pyrrolidine-1-carboxylate (42)

[0309]

[0310] To a solution of 41 (3.0 g, 5.89 mmol) in MeOH (150 mL) was added NH4OAc (9.07 g, 117.8 mmol). The reaction mixture was heated to 130°C in a pressure vessel for 15 hours. The reaction mixture was filtered and concentrated to give the crude product. The residue was purified by column chromatography (DCM / MeOH = 100 / 1 to 10 / 1) to give racemic trans 42 (2.2 g, 80% yield) as a solid.

[0311] 1 H NMR (400MHz, DMSO-d6): δ10.98(br.s,1H),7.10(s,1H),3.63-3.54(m,2H),3 .39-3.34(m,1H),2.84-2.77(m,2H),2.50(m,1H),1.41(s,9H),0.96(m,3H).

[0312] (S,S)-trans-3-(1,3-dibromo-8-oxo-7,8-dihydro-imidazo[1,5-a]pyrazin-6-yl)-4-carboxylic acid Synthesis of tert-butyl pyrrolidine-1-carboxylate (S,S)-(42)

[0313]

[0314] To a solution of (S,S)-41 (3.03 g, 5.9 mmol) in 2-propanol (20 mL) was added NH4OAc (9.18 g, 118 mmol). The reaction mixture was heated at 105-110°C for 12 hours, after which it was poured into water (60 mL) with stirring and allowed to stand for 2 hours. The reaction mixture was filtered and concentrated to give the crude product. The residue was purified by column chromatography (DCM / MeOH = 100 / 1 to 10 / 1) and evaporated to give (S,S)-42 (2.1 g, 4.4 mmol) as a solid. This material was determined to have 99.3% ee and had spectral characteristics similar to those of rac-42.

[0315] Racemic trans-3-[1-bromo-3-(3,6-dihydro-2H-pyran-4-yl)-8-oxo-7,8-dihydro-imidazo] Synthesis of [1,5-a]pyrazin-6-yl]-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (43)

[0316]

[0317] To a mixture of compound 42 (2.2 g, 4.62 mmol) and 4- (4,4,5,5- tetramethyl - [1,3,2] dioxaborolane -2- bases) -3,6- dihydro -2H- pyrans (1.1 g, 5.08 mmol) in THF (200 mL) was added potassium phosphate (2.7 g, 13.86 mmol). The reaction mixture was degassed by purging with N for 5 min, followed by addition of Pd (dba) (0.8 g, 0.92 mmol) and Xanthphos (1.0 g, 1.84 mmol) to the mixture. The resulting suspension was degassed with N for 10 minutes. The mixture was then heated to 80 ° C for 15 hours under an N atmosphere. After cooling to room temperature, the reaction mixture was diluted with EtOAc (250 mL), and the precipitate was filtered out. The filtrate was concentrated. The crude residue was purified by silica gel column chromatography (eluted with EtOAc) to afford 43 (1.3 g, 60% yield) as a solid.

[0318] 1 H NMR (400MHz, DMSO-d6): δ10.80(m,1H),7.34(s,1H),6.42(s,1H),4.30-4.29(m,2H),3.92- 3.80(m,2H),3.63-3.33(m,4H),2.87-2.71(m,2H),2.50(m,1H),1.41(s,9H),0.95(m,3H).

[0319] (S,S)-trans-3-[1-bromo-3-(3,6-dihydro-2H-pyran-4-yl)-8-oxo-7,8-dihydro-imidazolinone] Synthesis of [1,5-a]pyrazin-6-yl]-4-methyl-pyrrolidine-1-carboxylic acid tert-butyl ester (S,S)-(43)

[0320]

[0321] To a mixture of compound (S, S) -42 (2.11 g, 4.42 mmol) and 4- (4,4,5,5- tetramethyl - [1,3,2] dioxaborolan -2- bases) -3,6- dihydro -2H- pyran (0.975 g, 4.64 mmol) in 1,4- dioxane (40 mL) and water (10 mL) was added potassium phosphate (2.57 g, 12.2 mmol). The reaction mixture was degassed by purging with N2 for 5 min, after which Pd2 (dba) 3 (0.8 g, 0.9 mmol) and Xanthphos (1.0 g, 1.8 mmol) were added to the mixture. The resulting suspension was degassed with N2 for 10 minutes. The mixture was then heated to 80 ° C for 15 hours under an N2 atmosphere. After cooling to room temperature, the reaction mixture was diluted with EtOAc (250 mL) and filtered through diatomaceous earth to remove the solid. The filtrate was concentrated. The crude residue was purified by silica gel column chromatography (eluting with EtOAc) to afford 43 (1.4 g, 2.92 mmol) as a solid. The material had greater than 99% ee at this stage.

[0322] Racemic trans-3-methyl-4-[8-oxo-3-(tetrahydro-pyran-4-yl)-7,8-dihydro-imidazo[1,5-a] Synthesis of tert-butyl pyrazin-6-yl-pyrrolidine-1-carboxylate (44)

[0323]

[0324] To a solution of 43 (1.3 g, 2.73 mmol) in DMF (100 mL) and methanol (30 mL) was added 10% Pd / C (0.8 g). The flask was filled with hydrogen (50 psi) and the mixture was stirred at 50° C. overnight. After cooling, the reaction mixture was filtered through celite. The filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluted with DCM / CH 3 OH=100 / 1-20 / 1) to give compound 44 (0.99 g, 90% yield) as a solid.

[0325] 1 H NMR (400MHz, CDCl3): δ10.80 (br d,1H),7.86(s,1H),6.79(s,1H),4.13-4.10(m,2H),3.83-3.79(m,3H),3.63-3.49(m,2H),3.13-3.03(m,2H),2 .77-2.75(m,2H),2.54-2.53(m,1H),2.11-2.06(m,2H),1.80-1.85(m,2H),1.48(m,9H),1.12(d,J=6.4Hz,3H).

[0326] (S,S)-trans-3-methyl-4-[8-oxo-3-(tetrahydro-pyran-4-yl)-7,8-dihydro-imidazo[1,5-a] Synthesis of tert-butyl pyrazin-6-yl-pyrrolidine-1-carboxylate (S,S)-(44)

[0327]

[0328] Under a N2-protective atmosphere, a solution of (S,S)-43 (1.15 g, 2.41 mmol) in methanol (50 mL) was placed in an autoclave, and 10% Pd / C (0.8 g) was added under a nitrogen atmosphere. The reaction mixture was hydrogenated at 45-50°C and 10-15 Bar H2 pressure with stirring until no more hydrogen was absorbed (24 hours). After cooling, the reaction mixture was filtered through celite. The filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluted with DCM / CH3OH=100 / 1-20 / 1) to give compound 44 (0.97 g, 2.41 mmol) as a solid. The ee was determined to be greater than 99%.

[0329] Racemic trans-6-(4-methyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a] Synthesis of pyrazin-8-one (45)

[0330]

[0331] To a solution of compound 44 (0.99 g, 2.49 mmol) in CH2Cl2 (20 mL) was added HCl / Et2O solution (20 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction was concentrated in vacuo to afford racemic trans 45 hydrochloride (0.75 g, 100% yield) as a solid.

[0332] 1 H NMR (400MHz, DMSO-d6): δ11.47(s,1H),9.93(s,2H),8.41(s,1H),7.92(s,1H),3.98-3.95(m,2H),3.85 -3.80(m,1H),3.58-3.44(m,3H),2.97-2.88(m,2H),2.60-2.50(m,3H),1.98-1.78(m,4H),1.08(m,3H).

[0333] (S,S)-trans-6-(4-methyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a] Synthesis of pyrazin-8-one (S,S)-(45)

[0334]

[0335] To a solution of compound (S,S)-44 (800 mg, 2.0 mmol) was added cold (0°C) HCl in MeOH (1.5 M, 10 mL), and the resulting reaction mixture was stirred while allowing to return to room temperature. After stirring for 2 hours, the reaction was concentrated in vacuo to afford (S,S)-45 hydrochloride (0.60 g, 2.0 mmol) as a solid.

[0336] 1 H NMR (400MHz, DMSO-d6): δ11.47(s,1H),9.93(s,2H),8.41(s,1H),7.92(s,1H),3.98-3.95(m,2H),3.85 -3.80(m,1H),3.58-3.44(m,3H),2.97-2.88(m,2H),2.60-2.50(m,3H),1.98-1.78(m,4H),1.08(m,3H).

[0337] Racemic trans-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)- Synthesis of 7H-imidazo[1,5-a]pyrazin-8-one (P3)

[0338]

[0339] To a solution of compound 45 (0.75 g, 2.49 mmol), 2-chloromethyl-pyrimidine (0.49 g, 2.99 mmol) in DMF (10 mL) and CH 3 CN (30 mL) was added K 2 CO 3 (1.7 g, 12.5 mmol). The mixture was stirred at 45° C. for 48 hours. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by flash column chromatography (gradient elution from DCM to 15% MeOH in DCM) to give racemic trans P3 (580 mg, 59% yield) as a solid.

[0340] 1 H NMR (400MHz, CD3OD): δ8.85(d,J=4.8Hz,2H),7.79(s,1H),7.42(t,J=4.8Hz,1H),7.36(s,1H),4.11-4.04(m,3H),3.93(d,J=15.2Hz,1H),3.684-3. 62(m,2H),3.41-3.32(m,2H),3.16-3.13(m,1H),2.85~2.80(m,2H),2.44- 2.40(m,1H),2.28-2.23(m,1H),2.04-1.86(m,4H),1.17(d,J=6.4Hz,3H). MS calculated value: 394.5; MS found value: 395.8 ([M+H] + ).

[0341] The racemic mixture of P3 (1.4 g) was separated by chiral HPLC (column: Chiralpak IA, 250 x 4.6 mm x 5 um; mobile phase: Hex / EtOH / DEA = 70:30:0.2) at a flow rate of 1.0 mL / min to give P3 enantiomer 1 ((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 or 6-[(3S, 4S)-4-methyl-1-(pyrimidin-2-ylmethyl]-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one] )pyrrolidin-3-yl]-3-tetrahydropyran-4-yl-7H-imidazo[1,5-a]pyrazin-8-one) (0.52 g, RT = 9.98 min) and P3 enantiomer 2 ((3R,4R)-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)-7H-imidazo[1,5-a]pyrazin-8-one, relative to P3 enantiomer 1) (0.49 g, RT = 12.6 min).

[0342] (S,S)-trans-6-(4-methyl-1-pyrimidin-2-ylmethyl-pyrrolidin-3-yl)-3-(tetrahydro-pyran-4-yl)- Synthesis of 7H-imidazo[1,5-a]pyrazin-8-one (S,S)-(P3)

[0343]

[0344] To a solution of compound (S,S)-45 (0.60 g, 2.0 mmol) and 2-chloromethyl-pyrimidine (0.40 g, 2.40 mmol) in DCM (15 mL) was added DIPEA (3.1 g, 24 mmol), and the mixture was stirred at RT for 24 hours (all starting materials were converted at this point). The reaction mixture was cooled to 5 ° C and deionized water (10 mL) was added. The pH of the aqueous phase was adjusted to pH 6.0 by adding hydrochloric acid (approximately 1 mL) while maintaining the temperature of the mixture at <25 ° C. The phases were separated and the organic phase was washed with salt water (3x5 mL) (these washings were discarded). The aqueous phase was extracted with dichloromethane (10 mL), and the organic phase from the extraction was washed with salt water (3x5 mL). The combined organic phases were dried over sodium sulfate (3 g) for 1 hour, filtered, and evaporated. The resulting residue was subjected to column chromatography (as described for rac-(P3)) to afford (S,S)-P3 (580 mg, 59% yield) as a solid after evaporation. This material had an ee greater than 99% and was identical in all respects to P3 enantiomer 1 (described above).

[0345] Synthesis of (aminooxy)(diphenyl)phosphine oxide (B)

[0346]

[0347] At -30 ℃, under nitrogen atmosphere, in 15 minutes, to the suspension of hydroxylamine hydrochloride (73.5g, 1.05mol) in dichloromethane (500mL), DIPEA (136g, 1.05mol) was added. A white precipitate was formed after the addition. After stirring at this temperature for 1 hour, a solution of diphenylphosphinyl chloride A (50g, 0.2mol) in dichloromethane (100mL) was added in 60 minutes. Under stirring, the mixture was heated to 0 ℃ in 1 hour. The reaction was quenched by adding water (200mL) in 10 minutes. After stirring the mixture for 0.5 hour, the precipitate was collected by filtration and washed with water (100mL x 2). The solid was then dried under reduced pressure to obtain a crude product. The crude product was ground with EtOH to obtain compound B (27g, 56% yield), which was a white solid.

[0348] 1 HNMR (400MHz, CD3OD): δ77.91-7.79(m,5H),7.62-7.50(m,7H).

[0349] MS calculated value: 233; MS found value: 234 ([M+H] + ).

[0350] Synthesis of methyl 3-amino-3H-imidazole-4-carboxylate (46)

[0351]

[0352] To compound 3H-imidazole-4-carboxylic acid methyl ester 32 (30.0g, 0.24mol) in THF (1.0L) was added dropwise LiHMDS (239mL, 10M in THF, 2.4mol) at -78 ℃ in 2 hours. Then the reaction mixture was stirred for another 2 hours at -78 ℃, and it was heated to -10 ℃. At this temperature, compound B (60.0g, 0.26mol) was added. Then the mixture reaction was stirred at ambient temperature overnight. After quenching with water (250mL), the reaction mixture was concentrated. The crude product was purified by silica gel column chromatography (DCM / MeOH=20 / 1) to obtain compound 46 (24g, 73% yield) as a solid.

[0353] 1 H NMR (400 MHz, DMSO-d6): δ7.82 (s, 1H), 7.51 (s, 1H), 6.20 (s, 2H), 3.79 (s, 3H). MS calculated value: 382; MS found value: 383 ([M+H] + MS calculated value: 141; MS found value: 142 ([M+H] + ).

[0354] Synthesis of 3-(2-Benzyloxy-acetylamino)-3H-imidazole-4-carboxylic acid methyl ester (47)

[0355]

[0356] To a solution of compound 46 (4.9 g, 30 mmol), benzyloxy-acetic acid (5.8 g, 30 mmol) and DIPEA (18.6 ml, 90 mmol) in DMF (100 mL) was added HATU (15.8 g, 36 mmol) while cooling on an ice-water bath. The mixture was then stirred at ambient temperature overnight. After removal of the solvent, the residue was purified by silica gel column chromatography (eluting with PE / EtOAc = 10:1 to 2:1) to give compound 47 (6.1 g, 61% yield) as an oil.

[0357] 1 H NMR (400 MHz, CDCl3): δ 9.93 (br.s, 1H), 7.74 (s, 1H), 7.67 (s, 1H), 7.39-7.33 (m, 5H), 4.70 (s, 2H), 4.23 (s, 2H), 3.83 (s, 3H). MS calculated value: 289; MS found value: 300 ([M+H] + ).

[0358] Synthesis of 3-(2-Benzyloxy-acetylamino)-3H-imidazole-4-carboxamide (48)

[0359]

[0360] Compound 47 (30.0 g, 100 mmol) and concentrated aqueous ammonia (300 mL) were combined in a sealed test tube and heated to 70° C. under microwave irradiation for 2 hours. The resulting mixture was concentrated in vacuo to afford compound 48 as a solid (26.3 g, 96% yield). MS calculated value: 274; MS found value: 275 ([M+H] + ).

[0361] Synthesis of 2-benzyloxymethyl-3H-imidazo[5,1-f][1,2,4]triazine-4-one (49)

[0362]

[0363] To a solution of compound 48 (28.0 g, 100 mmol) in EtOH (240 mL) was added a solution of KOH (19.8 g, 300 mmol) in water (200 mL). The resulting solution was heated to reflux for 3 hours. After removing the organic solvent in a vacuum, the mixture was poured into ice water and the pH was adjusted to 7.0 with a 1M HCl aqueous solution. The suspension was filtered and dried to give compound 49 (11.3 g, 44.1% yield) as a solid.

[0364] 1 H NMR (400 MHz, DMSO-d6): δ 12.05 (s, 1H), 8.45 (s, 1H), 7.74 (s, 1H), 7.39-7.29 (m, 5H), 4.59 (s, 2H), 4.36 (s, 2H). MS calculated value: 256; MS found value: 257 ([M+H] + ).

[0365] Synthesis of 2-benzyloxymethyl-7-iodo-3H-imidazo[5,1-f][1,2,4]triazine-4-one (50)

[0366]

[0367] To a solution of compound 49 (10.0 g, 38.2 mmol) in THF (240 mL) was added n-BuLi (46 mL) at -78 ° C, and the reaction was stirred for 1 hour at a temperature lower than -70 ° C. At this temperature, a solution of iodine (39.3 g, 153 mmol) in THF (120 mL) was added dropwise, and the reaction temperature was slowly warmed to room temperature. The reaction was quenched with saturated Na2SO3 aqueous solution (120 mL), then extracted with EtOAc (150 mL × 3). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo to give a crude product. The residue was purified by silica gel column chromatography (eluted with PE / EtOAc=10:1 to 2:1) to give compound 50 (4.75 g, 32.5% yield) as a solid.

[0368] 1 H NMR (400 MHz, DMSO-d6): δ 12.16 (br.s, 1H), 7.84 (s, 1H), 7.42-7.29 (m, 5H), 4.62 (s, 2H), 4.40 (s, 2H). MS calculated value: 382; MS found value: 383 ([M+H] + ).

[0369] 2-Benzyloxymethyl-7-(3,6-dihydro-2H-pyran-4-yl)-3H-imidazo[5,1-f][1,2,4]triazine-4- Synthesis of ketone (51)

[0370]

[0371] To a solution of compound 50 (4.75 g, 10.0 mmol) in dioxane (80 mL) was added dropwise a solution of CsCO (9.88 g, 30 mmol) in water (12 mL), followed by the addition of Pd(PPh) (2.36 g, 2.00 mmol) and 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyran (3.86 g, 18.0 mmol). The reaction mixture was degassed by purging with N for 15 min. The mixture was then heated to reflux for 16 hours. After removing the solvent in vacuo, the residue was purified by silica gel column chromatography (eluting with PE / EtOAc=10:1 to 1:5) to give compound 51 (2.1 mg, 76% yield) as a solid.

[0372] 1 H NMR (400 MHz, DMSO-d6): δ 12.10 (br.s, 1H), 7.78 (s, 1H), 7.39-7.30 (m, 5H), 7.25 (s, 1H), 4.62 (s, 2H), 4.41 (s, 2H), 4.27 (d, J = 2.8 Hz, 2H), 3.82 (t, J = 5.2 Hz, 2H), 2.63 (m, 2H). MS calculated value: 338; MS found value: 339 ([M+H] + ).

[0373] 2-Hydroxymethyl-7-(tetrahydro-pyran-4-yl)-3H-imidazo[5,1-f][1,2,4]triazin-4-one (52) synthesis

[0374]

[0375] To a solution of compound 51 (1.8 g, 5.0 mmol) in MeOH (70 mL) was added Pd(OH)2 (20% on carbon (wetted with approximately 50% water), 400 mg). The reaction flask was filled with hydrogen (50 psi) and the mixture was stirred in an oil bath heated to 70°C until LC / MS showed that the starting material had been consumed. The suspension was filtered through celite and the filtrate was washed with MeOH (100 mL x 2). The combined organic phases were concentrated in vacuo to give compound 52 (1.0 g, 79% yield) as a solid.

[0376] 1 H NMR (400 MHz, DMSO-d6): δ 11.65 (s, 1H), 7.68 (s, 1H), 4.30 (s, 2H), 3.96-3.92 (m, 2H), 3.51-3.17 (m, 3H), 1.88-1.81 (m, 4H). MS calculated value: 250; MS found value: 251 ([M+H] + ).

[0377] Synthesis of 2-chloromethyl-7-(tetrahydropyran-4-yl)-3H-imidazo[5,1-f][1,2,4]triazin-4-one (53)

[0378]

[0379] While cooling on ice-water bath, to compound 52 (1.0 g, 4 mmol) in CH Cl (50 mL) solution was added dropwise SOCl (15 mL). The resulting mixture was then stirred at ambient temperature overnight. The reaction mixture was concentrated in vacuo to give compound 53 (1.07 g, 100% yield) as a solid.

[0380] 1 H NMR (400 MHz, DMSO-d6): δ 12.50 (br.s, 1H), 8.02 (s, 1H), 4.57 (s, 2H), 3.95 (m, 2H), 3.57-3.48 (m, 3H), 1.91-1.81 (m, 4H). MS calculated value: 268; MS found value: 269 ([M+H] + ).

[0381] Synthesis of tert-butyl 3-(4-fluoro-benzyloxy)-azetidine-1-carboxylate (2)

[0382]

[0383] To a solution of compound 3-hydroxy-azetidine-1-carboxylic acid tert-butyl ester 1 (5.30g, 30mmol) in DMF (60mL) was added NaH (1.80g, 45mmol) while cooling on an ice-water bath. The suspension was then stirred at this temperature for 1 hour, followed by the addition of 1-chloromethyl-4-fluoro-benzene (8.94g, 60mmol). The resulting mixture was stirred overnight at ambient temperature. The reaction mixture was poured into water (200mL) and extracted with EtOAc (150mL×3). The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo to give a crude product. The residue was purified by silica gel column chromatography (eluted with PE / EtOAc=10:1 to 2:1) to give compound 2 (7.90g, 94% yield) as an oil.

[0384] 1 H NMR (300 MHz, DMSO-d6): δ7.41-7.37 (m, 2H), 7.21-7.14 (m, 2H), 4.40 (s, 2H), 4.33-4.29 (m, 1H), 4.02-3.97 (m, 2H), 3.68-3.66 (m, 2H), 1.37 (s, 9H). MS calculated value: 281; MS found value: 282 ([M+H] + ).

[0385] Synthesis of 3-(4-fluoro-benzyloxy)-azetidine (3)

[0386]

[0387] To a solution of compound 2 (2.68 g, 9.30 mmol) in dioxane (30 mL) was added HCl / dioxane (4 M, 9.25 mL) under an ice-water bath. The reaction mixture was then stirred at ambient temperature overnight. The reaction solution was concentrated in vacuo to afford the hydrochloride salt of compound 3 (1.2 g, 71% yield) as a solid.

[0388] 1 H NMR (300 MHz, DMSO-d6): δ 7.36 (m, 2H), 7.16 (m, 2H), 4.35 (s, 2H), 4.39 (m, 1H), 3.47 (t, J = 7.5 Hz, 2H), 3.38 (t, J = 7.2 Hz, 2H). MS calculated value: 181; MS found value: 182 ([M+H] + ).

[0389] 2-[3-(4-Fluoro-phenoxy)-azetidin-1-ylmethyl]-7-(tetrahydro-pyran-4-yl)-3H-imidazo Synthesis of [5,1-f][1,2,4]triazine-4-one (P4)

[0390]

[0391] To a solution of compound 53 (1.27 mg, 4.0 mmol) and compound 3 (1.8 g, 8.3 mmol) in CH 3 CN (20 mL) was added DIPEA (2.61 mL, 20 mmol). The resulting solution was heated to 70° C. for 2 hours. TLC indicated that the reaction was complete. The reaction was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluted with DCM / MeOH 100: 1 to 30: 1) to obtain the desired product P4 (1.23 g, 74% yield) as a solid.

[0392] 1 H NMR (400 MHz, DMSO-d6): δ 11.70 (br.s, 1H), 7.67 (s, 1H), 7.37 (m, 2H), 7.16 (m, 2H), 4.38 (s, 2H), 4.17 (m, 1H), 3.95-3.92 (m, 2H), 3.56 (t, J = 8.0 Hz, 2H), 3.54-3.46 (m, 4H), 3.37-3.35 (m, 1H), 3.06-3.03 (m, 2H), 1.86-1.80 (m, 4H). MS calculated value: 413; MS found value: 414 ([M+H] + ).

[0393] Example 2. X-ray crystal structure of P3 enantiomer 2

[0394] The single crystal X-ray structure of P3 enantiomer 2 was determined at 100K using crystals grown in the orthorhombic system with space group P212121. There is one compound molecule and one water molecule in the asymmetric unit. The final R1[I>2δ(I)]=3.09%. The absolute stereochemistry of the compound is Figure 1 Shown in.

[0395] P3 enantiomer 2 monohydrate

[0396] Detailed description of instruments and methods

[0397] Crystallization experiments were performed to obtain suitable crystals to determine the structure and absolute configuration of P3 enantiomer 2 by single crystal X-ray diffraction.

[0398] X-ray powder diffraction (XRPD)

[0399] X-ray powder diffraction patterns were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), a θ-2θ goniometer with V4 divergence and receiving slits, germanium monochromate (Gemonochromate), and a Lynxeye detector. The instrument was performance-checked using a certified Corundum standard (NIST 1976). Data were collected using DiffracPlus XRD Commander v2.6.1, and analyzed and displayed using Diffrac Plus EVA v15.0.0.0.

[0400] Samples were run under ambient conditions using the received powder as flat specimens. The sample was gently loaded into a cavity cut from a polished, zero-background (510) silicon wafer. The sample was rotated in its own plane during analysis. Data collection details were: angular range: 2 to 42° 2θ; step size: 0.05° 2θ; collection time: 0.5 s / step.

[0401] Single crystal X-ray diffraction (SCXRD)

[0402] Data were collected on an Oxford Diffraction Supernova dual-source (Cu at zero time) Atlas CCD diffractometer equipped with an Oxford Cryosystems Cobra cooling device. Data were collected using Cu Kα radiation. Structures were typically solved using the SHELXS or SHELXD programs and refined using the SHELXL program, part of the Bruker AXS SHELXTL suite (V6.10). Unless otherwise stated, hydrogen atoms attached to carbon were geometrically placed and allowed to be refined using the riding isotropic displacement parameter. Hydrogen atoms attached to heteroatoms were located in the difference Fourier synthesis and allowed to be freely refined using the isotropic displacement parameter.

[0403] Polarized Light Microscopy (PLM)

[0404] Samples were examined on a Nikon SMZ1500 polarized light microscope equipped with a digital video camera connected to a DS-L2 DS Camera control unit for image capture. A small amount of each sample was placed on a glass slide and mounted in immersion oil, isolating individual particles as much as possible. The samples were observed using partially polarized light coupled to a lambda false color filter at appropriate magnification.

[0405] Crystal screen

[0406] Attempt to dissolve P3 enantiomer 2 (5 mg) in the selected solvent at 50° C. The solution was placed in a refrigerator at 4° C. for 48 hours. The suspension was filtered and the resulting mother liquor was also placed at 4° C. Any crystals obtained were evaluated by optical microscopy.

[0407] The material was soluble in most solvent systems evaluated, except isopropyl acetate and cumene. Large prismatic crystals were obtained at 4°C from a range of solvents, including acetonitrile, tetrahydrofuran, and 1,4-dioxane. The crystal structure of P3 enantiomer 2 was solved using crystals obtained by cooling in acetonitrile.

[0408] Single crystal structure determination

[0409] A crystalline sample of P3 enantiomer 2 was obtained by dissolving 5 mg of the supplied material in 50 μl of acetonitrile and cooling at 4°C. The resulting crystals had a prismatic morphology. Crystals of sufficient size and quality for analysis were isolated by single crystal X-ray diffraction, with approximate dimensions of 0.25 x 0.15 x 0.11 mm. Optical micrographs of the received crystals and the single crystals used for data collection are available at Figure 1 Shown in.

[0410] The structure was determined at 100K in the orthorhombic system with space group P212121 with a final R1{I>2δ(I)] = 3.09%. The compound was identified as the monohydrate of P3 enantiomer 2, as Figure 1 and Figure 3 The asymmetric unit contains two molecules of the fully ordered P3 enantiomer and one molecule of water. Anisotropic atomic displacement ellipsoids are shown for non-hydrogen atoms at a 50% probability level. Hydrogen atoms are shown with arbitrarily small radii.

[0411] for Figure 1 The absolute stereochemistry of the P3 enantiomer 2 shown in FIG, with C12 and C13 (this numbering is not the numbering used in the IUPAC name) in the R configuration, has a Flack parameter = -0.03 (4). For the inversion structure (P3 enantiomer 1) with C12 and C13 in the S configuration, the Flack parameter = 1.03 (4).

[0412] The absolute structure was determined using a Bayesian statistical method for Bijvoet differences, which revealed a 1.000 probability that the absolute structure shown was correct, and a 0.000 probability that the absolute structure was a racemic pair or was incorrect. The Flack equivalent value and its uncertainty were calculated by this program to be -0.02 (4). This calculation was based on 1806 Bijvoet pairs, for a coverage of 100%.

[0413] Configuration analysis of P3 enantiomer 2 showed that the pyrimidine ring was planar, the pyrrolidine ring was enveloped on nitrogen, and the tetrahydropyran ring was chair-shaped.

[0414] As the opposite structure of P3 enantiomer 2, P3 enantiomer 1 has the following structure:

[0415] Example 3. In vitro testing

[0416] PDE9 inhibition assay

[0417] The PDE9 assay can be performed, for example, as follows: The assay is performed in 60 μL of sample containing a fixed amount of the relevant PDE enzyme (sufficient for conversion of 20-25% of the cyclic nucleotide substrate), buffer (50 mM HEPES 7.6; 10 mM MgCl2; 0.02% Tween 20), 0.1 mg / ml BSA, 225 pCi of 3H-labeled cyclic nucleotide substrate, tritiated cAMP at a final concentration of 5 nM, and varying amounts of inhibitor. The reaction was initiated by adding the cyclic nucleotide substrate and allowed to proceed for 1 hour at room temperature before being terminated by mixing with 15 μL 8 mg / mL yttrium silicate SPA beads (Amersham). The beads were allowed to stand in the dark for 1 hour, and the plate was then counted in a Wallac 1450 Microbeta counter. The measured signal can be converted to activity relative to an uninhibited control (100%), and the IC can be calculated using the Xlfit extension of Excel. 50 value.

[0418] In the present invention, the assay was performed in 60 μL of assay buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween 20) containing 10 nM dapoxetine sufficient for 20-25% conversion. 3 After 1 hour of incubation, the reaction was terminated by adding 15 μL of 8 mg / mL yttrium silicate SPA beads (Amersham). The beads were allowed to stand in the dark for 1 hour, and then the plate was counted in a Wallac 1450 Microbeta counter. IC values ​​were calculated by nonlinear regression using XLfit (IDBS). 50 value.

[0419] The experimental results showed that the tested compounds of the present invention inhibited PDE9 enzyme, IC 50 The value was lower than 100 nM.

[0420] PDE1 inhibition assay

[0421] The PDE1 assay can be performed as follows: The assay is performed in 60 μL of a sample containing a fixed amount of PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween 20), 0.1 mg / ml BSA, 15 nM tritiated cAMP, and varying amounts of inhibitor. The reaction is initiated by the addition of the cyclic nucleotide substrate and allowed to proceed for 1 hour at room temperature before being terminated by mixing with 20 μL (0.2 mg) of yttrium silicate SPA beads (PerkinElmer). The beads are allowed to stand in the dark for 1 hour, and the plate is then counted in a Wallac 1450 Microbeta counter.

[0422] The measured signal can be converted to activity relative to the uninhibited control (100%), and the IC can be calculated using XlFit (Model 205, IDBS). 50 value.

[0423] Example 4. In vivo testing

[0424] Blood-brain barrier penetration

[0425] Before starting the experiment, male CD mice (20-24g) were placed in pairs in cages with free access to food and water for an adaptation period of 3-7 days. Before administration, the animals were fasted overnight. During the test, the mice were kept in separate cages. Brain-plasma distribution was assessed 30 minutes and 2 hours (n=3 at each time point) after subcutaneous administration of the test compound at a dose of 10mg / kg. Appropriate solvents were used to dissolve each test compound so that the administration volume was 10ml / kg. During sampling, animals were anesthetized with isoflurane and systemic blood samples were collected into a blood sample collection container (vacutainer) containing sodium heparin as an anticoagulant by cardiac puncture. The blood was centrifuged at 3500rpm for 10 minutes at 4°C to obtain plasma. After decapitation, the brain was dissected out and transferred to a pre-weighed container, followed by tissue weight determination. Plasma and brain were stored at -80°C until quantitative bioanalysis was performed using LC-MS / MS. The results for plasma samples were expressed in ng / ml, and those for brain samples were expressed in ng / g.

Claims

1. An oral pharmaceutical composition comprising the compound (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 and one or more pharmaceutically acceptable carriers, wherein the content of the compound in the oral pharmaceutical composition is 0.01 mg to 1000 mg per unit dosage form.

2. An oral pharmaceutical composition comprising the compound (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 and one or more pharmaceutically acceptable diluents, wherein the content of the compound in the oral pharmaceutical composition is 0.01 mg to 1000 mg per unit dosage form.

3. An oral pharmaceutical composition comprising the compound (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 and one or more pharmaceutically acceptable excipients, wherein the content of the compound in the oral pharmaceutical composition is 0.01 mg to 1000 mg per unit dosage form.

4. The oral pharmaceutical composition according to any one of claims 1 to 3, wherein the content of the compound in the oral pharmaceutical composition is 0.05 mg to 500 mg per unit dosage form.

5. The oral pharmaceutical composition according to any one of claims 1 to 3, wherein the content of the compound in the oral pharmaceutical composition is 0.05 mg to 200 mg per unit dosage form.

6. Use of the oral pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a medicament for treating benign prostatic hyperplasia or sickle cell disease.

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