Compounds as PARP1 inhibitors

Abstract

The present invention relates to compounds that inhibit enzymes of the poly (ADP-ribose) polymerase (PARP) family and pharmaceutically acceptable salts thereof. The invention also relates to the use of these compounds or pharmaceutically acceptable salts thereof in the treatment of disease.

Description

Technical Field

[0001] The present invention relates to compounds that inhibit poly (ADP-ribose) polymerase (PARP) family enzymes and pharmaceutically acceptable salts thereof. The present invention also relates to the use of these compounds or pharmaceutically acceptable salts thereof in treating diseases. Background Art

[0002] The poly (ADP-ribose) polymerase (PARP) family consists of about 17 proteins, including PARP-1, PARP-2, PARP-3, PARP-4 (vPARP), PARP-5 (tankyrase-1, tankyrase-2), PARP-7, PARP-10, etc. These proteins all show a certain degree of homology in their catalytic domains, but differ in their cellular functions.

[0003] Among the various functions attributed to PARP-1 and PARP-2, their main role is to promote DNA repair through ADP-ribosylation and thus coordinate multiple DNA repair proteins. After exposure to radiation, oxygen free radicals or nitric oxide (NO), etc., the activation of PARP is induced through DNA single-strand breaks. DNA damage leads to PARP activation, thereby repairing DNA single-strand breaks, so PARP can lead to drug resistance that may appear in various types of cancer treatment. Specifically, it is reported that PARP inhibitors can be used to specifically kill tumors that lack DNA double-strand repair factors (such as BRCA-1 and BRCA-2), and therefore have been developed as patient-specific anticancer agents for various types of cancer (including breast cancer, ovarian cancer, prostate cancer, etc.), which have abnormalities in DNA double-strand damage repair factors.

[0004] Inhibition of PARP family enzymes has been used as a strategy to selectively kill cancer cells by inactivating complementary DNA repair pathways. Many preclinical and clinical studies have demonstrated that tumor cells carrying deleterious alterations in BRCA1 or BRCA2, key tumor suppressor proteins involved in repairing double-stranded DNA breaks (DSBs) by homologous recombination (HR), are selectively sensitive to small molecule inhibitors of the PARP family of DNA repair enzymes. Such tumors have a defective homologous recombination repair (HRR) pathway and rely on PARP enzyme function for survival. Although PARP inhibitor therapy is primarily targeted at BRCA-mutated cancers, PARP inhibitors have been clinically tested in non-BRCA-mutated tumors, i.e., tumors that exhibit homologous recombination deficiency (HRD).

[0005] It is believed that PARP inhibitors with improved selectivity for PARP1 may have improved efficacy and reduced toxicity compared to other clinical PARP1 / 2 inhibitors. It is also believed that selective and strong inhibition of PARP1 will lead to the capture of PARP1 on DNA, causing DNA double-strand breaks (DSBs) through the collapse of the S-phase replication fork. It is also believed that PARP1-DNA capture is an effective mechanism for selective killing of tumor cells with HRD. Therefore, there is an unmet medical need for effective and safe PARP inhibitors. In particular, PARP inhibitors that are selective for PARP1. Summary of the invention

[0006] The present invention relates to compounds or pharmaceutically acceptable salts having PARP inhibitory activity and are therefore useful for treating diseases and conditions in which PARP function is of pharmacological significance. In addition, the compounds described herein have a higher selectivity for PARP1 over PARP2.

[0007] In one aspect, the present invention provides a compound or a pharmaceutically acceptable salt having PARP inhibitory activity. Preferably, the compounds described herein have a higher selectivity for PARP1 than other PARP family members (eg, PARP2).

[0008] In another aspect, the present invention provides a composition comprising a compound or a pharmaceutically acceptable salt of formula I. Preferably, the composition further comprises at least one pharmaceutically acceptable diluent, excipient or inert carrier.

[0009] In another aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, or a composition thereof, for use as a medicament.

[0010] In another aspect, the present invention provides a method of treatment comprising administering a therapeutically effective amount of a compound thereof to a patient in need thereof. Preferably, the patient in need thereof suffers from cancer. Preferably, the cancer lacks the HR-dependent DNA DSB repair pathway. More preferably, the cancer cell has a BRCA1 or BRCA2 defective phenotype, or the cancer cell lacks BRCA1 or BRCA2. More preferably, the cancer is selected from any one of breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, and lung cancer.

[0011] In another aspect, the present invention provides a method for treating or preventing diseases and conditions in which inhibition of PARP1 is beneficial, comprising administering a therapeutically effective amount of a compound thereof to a patient in need thereof. Preferably, the patient in need thereof suffers from cancer. More preferably, the patient is heterozygous for a mutation in a gene encoding a component of the HR-dependent DNA DSB repair pathway, or the patient is heterozygous for a mutation in a gene encoding a component of the HR-dependent DNA DSB repair pathway.

[0012] In another aspect, the present invention provides a use of the compound in the preparation of a medicament for treating cancer. More preferably, the cancer lacks the HR-dependent DNA DSB repair pathway. DETAILED DESCRIPTION

[0013] (I) Compound

[0014] In one aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof having PARP inhibitory activity. Preferably, the compounds described herein have a higher selectivity for PARP1 than other PARP family members (eg, PARP2).

[0015] In some embodiments, the compound is a compound of Formula I:

[0016]

[0017] or a pharmaceutically acceptable salt thereof, wherein

[0018] X1, X2 or X3 are independently selected from N or CH;

[0019] X5 is selected from N, CH or CF;

[0020] A is selected from -CH2-, -O- or -NR7-, wherein R7 is selected from H or C1-C6 alkyl, or A is absent;

[0021] B is selected from a substituted or unsubstituted The substituent at any position of the B ring is R5;

[0022] R1 is selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C3-C5 cycloalkyl;

[0023] R2 is One of X4 is N and one of X4 is CH;

[0024] Each R3 is independently selected from H or unsubstituted or substituted -C1-C6 alkyl, the substituent of -C1-C6 alkyl is selected from H, -O-CH3, -CN, -OH, or two R3 are connected to form a C3-C5 cycloalkyl;

[0025] Each R 4a is independently selected from H, CN, halogen, C1-C6 alkyl, -O-alkyl, C1-C6 haloalkyl, or -C1-C6 alkoxy; preferably, R 4a is independently selected from H, CN, halogen, C1-C3 alkyl, -O-C1-C3 alkyl, C1-C3 haloalkyl, or -C1-C3 alkoxy; more preferably, each R 4aIndependently selected from H, -CH3, -CN, F.

[0026] R5 is selected from H, C1-C6 alkyl, =O, -(CH2) 1-3 OH, or halogen.

[0027] In some embodiments, the compound of formula I is selected from

[0028]

[0029]

[0030] or a pharmaceutically acceptable salt thereof.

[0031] In some embodiments, the compound of formula I, wherein

[0032] A is -O-;

[0033] B is selected from a substituted or unsubstituted and the substituent at any position of the B ring is R5;

[0034] R1 is selected from C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, C3-C5 cycloalkyl;

[0035] R2 is One of X4 is N and one of X4 is CH;

[0036] Each R3 is independently selected from H or unsubstituted or substituted -C1-C6 alkyl, the substituent of -C1-C6 alkyl is selected from H, -O-CH3, -CN, -OH, or two R3 are connected to form a C3-C6 cycloalkyl;

[0037] Each R 4a is independently selected from H, CN, halogen, C1-C3 alkyl, -O-C1-C3 alkyl, C1-C3 haloalkyl, or -C1-C3 alkoxy; preferably, each R 4a Independently selected from H, -CH3, -CN, F;

[0038] R5 is selected from -CH3, -CH2OH, or -F.

[0039] In some embodiments, the compound of formula I, wherein the formula I is

[0040]

[0041] A is selected from -O-;

[0042] R1 is selected from C1-C3 alkyl, C3-C5 cycloalkyl;

[0043] R2 is One of X4 is N and one of X4 is CH;

[0044] Each R3 is independently selected from H or unsubstituted or substituted -C1-C6 alkyl, the substituent of -C1-C6 alkyl is selected from H, -O-CH3, -CN, -OH, or two R3 are connected to form a C3-C5 cycloalkyl;

[0045] Each R 4a Independently selected from H, -CH3, -CN, F.

[0046] In some embodiments, in the compound of formula I, two R3 are not H at the same time.

[0047] In some embodiments, the compound of formula I, wherein

[0048] R2 is Each R 4a Independently selected from H, -CH3, -CN, F;

[0049] Each R3 is independently selected from H or -C1-C3 alkyl, wherein two R3 are not H at the same time.

[0050] In some embodiments, the compound of formula I, wherein the formula I is

[0051]

[0052] A is selected from -O-;

[0053] R1 is selected from C1-C3 alkyl, C3-C5 cycloalkyl;

[0054] R2 is

[0055] Each R3 is independently selected from H or unsubstituted or substituted -C1-C6 alkyl, the substituent of -C1-C6 alkyl is selected from H, -O-CH3, -CN, -OH, or two R3 are connected to form a C3-C5 cycloalkyl;

[0056] Each R 4a Independently selected from H, -CH3, -CN, F.

[0057] In some embodiments, the compound of formula I wherein, preferably,

[0058] R1 is selected from C1-C3 alkyl;

[0059] Each R3 is independently selected from H or unsubstituted -C1-C3 alkyl;

[0060] Each R 4a Independently selected from H, -CH3, F.

[0061] In some embodiments, the compound of Formula I is selected from Table 1, or a pharmaceutically acceptable salt thereof.

[0062] Table 1

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] In order to minimize the risk of off-target effects, it is desirable that the drug molecule has selectivity for a specific target. Advantageously, the compound of formula I is selective for PARP1 over PARP2. In one embodiment, a compound of formula I is provided that has a 10-fold selectivity for PARP1 over PARP2. In one embodiment, a compound of formula I is provided that has a 100-fold selectivity for PARP1 over PARP2.

[0074] (II) Pharmaceutical Composition

[0075] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof. The composition further comprises at least one pharmaceutically acceptable diluent, excipient or inert carrier. Preferably, the composition is used to treat cancer.

[0076] In one specific variation, the composition is a solid formulation suitable for oral administration. In another specific variation, the composition is a liquid formulation suitable for oral administration. In yet another specific variation, the composition is a tablet. In yet another specific variation, the composition is a liquid formulation suitable for parenteral administration.

[0077] The present invention also provides a pharmaceutical composition comprising a compound according to any of the above embodiments and variations, wherein the composition is suitable for administration by a route selected from the group consisting of oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, liposomes, by inhalation, intravaginal, intraocular, by topical delivery (e.g., by catheter or stent), subcutaneous, intrafatty, intraarticular, and intrathecal.

[0078] (III) Treatment methods

[0079] In another aspect, the present invention provides a method of treatment comprising administering a therapeutically effective amount of a compound thereof to a patient in need thereof. Preferably, the patient in need thereof suffers from cancer. Preferably, the cancer lacks the HR-dependent DNA DSB repair pathway. More preferably, the cancer cell has a BRCA1 or BRCA2 defective phenotype, or the cancer cell lacks BRCA1 or BRCA2. More preferably, the cancer is selected from any one of breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer, and lung cancer.

[0080] In another aspect, the present invention provides a method for treating or preventing diseases and conditions in which inhibition of PARP1 is beneficial, comprising administering a therapeutically effective amount of a compound thereof to a patient in need thereof. Preferably, the patient in need thereof suffers from cancer. More preferably, the patient is heterozygous for a mutation in a gene encoding a component of the HR-dependent DNA DSB repair pathway, or the patient is heterozygous for a mutation in a gene encoding a component of the HR-dependent DNA DSB repair pathway.

[0081] (IV) Preparation

[0082] In another aspect, the invention provides the use of the compound in the preparation of a medicament for treating a disease or condition in which inhibition of PARP1 is beneficial. In some embodiments, the cancer is breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer (e.g., gastric cancer and colorectal cancer), or lung cancer. In various embodiments, the cancer is breast cancer, ovarian cancer, pancreatic cancer, or prostate cancer.

[0083] (V) General Definitions

[0084] Throughout this disclosure and claims, the word "comprise" and variations such as "comprises" and "comprising" are to be interpreted in an open, inclusive sense, i.e., as "including but not limited to," unless the context requires otherwise.

[0085] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment disclosed herein. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" at various locations throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0086] For example, "C u-v " or (C u -C v ) indicates that the following group has from u to v carbon atoms. For example, "C 1-6 "Alkyl" means that the alkyl group has from 1 to 6 carbon atoms.

[0087] "Alkyl" refers to a straight or branched chain hydrocarbon group consisting of carbon and hydrogen atoms, which is saturated and has from 1 to 12 carbon atoms (C 1-12 alkyl), in certain embodiments 1 to 8 carbon atoms (C 1-8 Alkyl) or 1 to 6 carbon atoms (C 1-6 alkyl), or 1 to 4 carbon atoms (C 1-4 alkyl), or 1 to 3 carbon atoms (C 1-3 alkyl), and it is connected to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), 1,1-dimethylethyl (tert-butyl), n-pentyl, hexyl, 3-methylhexyl, 2-methylhexyl, etc.

[0088] "Fused" refers to a carbocyclic, heterocyclic, aromatic or heteroaromatic ring structure described herein that is attached to an existing ring structure in a compound disclosed herein via two adjacent atoms that are shared by the fused ring structure and the existing ring structure.

[0089] "Halo" or "halogen" refers to bromo, chloro, fluoro or iodo.

[0090] "Haloalkyl" refers to an alkyl group as defined above substituted with one or more halo groups as defined above, for example, trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.

[0091] "Alkoxy" refers to an oxygen moiety having a further alkyl substituent. The alkoxy groups of the present invention may be optionally substituted.

[0092] "Cycloalkyl" refers to a saturated monocyclic, bicyclic, spirocyclic or bridged carbocyclic ring with a defined number of carbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. In one embodiment of the invention, the cycloalkyl is selected from: cyclopropane, cyclobutane and cyclohexane. In another embodiment, the cycloalkyl is cyclopropane, cyclobutane or cyclopentane. In another embodiment, the cycloalkyl is cyclopropane or cyclobutane. In another embodiment, the cycloalkyl is cyclopropane. In another embodiment, the cycloalkyl is cyclobutane. In another embodiment, the cycloalkyl is cyclopentane. In another embodiment, the cycloalkyl is cyclohexane. In another embodiment, the cycloalkyl is cycloheptane.

[0093] "Oxo" refers to a =0 substituent.

[0094] In this specification, unless otherwise specified, the term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with human and animal tissues within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0095] In this specification, unless otherwise indicated, the phrase "effective amount" refers to an amount of a compound or composition sufficient to significantly and positively alter the symptoms and / or condition to be treated (e.g., to provide a positive clinical response). The effective amount of an active ingredient for a pharmaceutical composition will vary with the specific condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the specific active ingredient employed, the specific pharmaceutically acceptable excipient / carrier used, and similar factors within the knowledge and expertise of the attending physician.

[0096] Unless otherwise stated, as used herein, the term "treatment (treating)" refers to the reversal, alleviation, inhibition of progression, delay of progression, delay of onset, or prevention of the disorder or condition to which such term is applied, or one or more symptoms of such disorder or condition. Unless otherwise stated, as used herein, since "treatment (treating)" is defined above, the term "treatment (treatment)" refers to the act of treating (treating). The term "treatment (treating)" also includes adjuvant therapy and neoadjuvant therapy to the subject. For the avoidance of doubt, "treatment (treatment)" mentioned herein includes reference to curative, palliative and preventive treatments, as well as reference to the administration of drugs for such treatments.

[0097] The compounds of formula I may form stable pharmaceutically acceptable acid or base salts, and in such cases, administration of the compound in salt form may be appropriate.

[0098] Salts may be formed by conventional methods, for example, by reacting the free base form of the product with one or more equivalents of a suitable acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is removed in vacuo or by freeze drying or by exchanging the anion of the existing salt for another anion on a suitable ion exchange resin.

[0099] Compounds of formula I may have more than one chiral center, and it is understood that the present application encompasses all individual stereoisomers, enantiomers and diastereomers and mixtures thereof. Thus, it is understood that as long as compounds of formula I can exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the present application includes in its definition any such optically active or racemic forms having the above-mentioned activity. The present application encompasses all such stereoisomers having activity as defined herein.

[0100] It should also be understood that certain compounds of Formula I and their pharmaceutical salts can exist in solvated and non-solvated forms, such as hydrated and anhydrous forms. It should be understood that the compounds herein encompass all such solvated forms. For clarity, this includes both solvated (e.g., hydrated) forms of the free form of the compound and solvated (e.g., hydrated) forms of the salts of the compound.

[0101] Formula I as described herein is intended to encompass all isotopes of its constituent atoms. For example, FI (or hydrogen) includes any isotopic form of hydrogen, including 1 H. 2 H(D) and 3 H(T); C includes any isotopic form of carbon, including 12 C. 13 C and 14 C; O includes any isotopic form of oxygen, including 16 O. 17 O and 18 O; N includes any isotopic form of nitrogen, including 13 N. 14 N and 15 N. It is to be understood that the present application encompasses all such isotopic forms.

[0102] The compound of formula I or its pharmaceutically acceptable salt will usually be administered via oral route in the form of a pharmaceutical preparation comprising an active ingredient or its pharmaceutically acceptable salt or solvate, or a solvate of such a salt, in a pharmaceutically acceptable dosage form. Depending on the condition to be treated and the patient, the composition can be administered in varying doses.

[0103] Pharmaceutical formulations of the compounds of formula I described above may be prepared for oral administration, particularly in the form of tablets or capsules, and particularly involving techniques aimed at providing colon-targeted drug release.

[0104] The pharmaceutical formulations of the compounds of formula I described above may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mark Publishing Company, Easton, Pennsylvania, (1985).

[0105] Pharmaceutical preparations suitable for oral administration may include one or more physiologically compatible carriers and / or excipients, and may be in solid or liquid form. Tablets and capsules may be prepared with adhesives, fillers, lubricants and / or surfactants (e.g., sodium lauryl sulfate). Liquid compositions may contain conventional additives, such as suspending agents, emulsifiers and / or preservatives. Liquid compositions may be encapsulated in, for example, gelatin to provide unit dosage forms. Solid oral dosage forms include tablets, two-piece hard shell capsules, and soft elastic gelatin (SEG) capsules. Such two-piece hard shell capsules may be manufactured, for example, by filling the compound of formula (I) into a gelatin or hydroxypropyl methylcellulose (HPMC) shell.

[0106] The dry shell formulation typically comprises gelatin at a concentration of about 40% to 60% w / w, a plasticizer (e.g., glycerol, sorbitol, or propylene glycol) at a concentration of about 20% to 30%, and water at a concentration of about 30% to 40%. Other materials such as preservatives, dyes, opacifiers, and flavors may also be present. The liquid fill material comprises a solid drug that has been dissolved, solubilized, or dispersed (using a suspending agent such as beeswax, hydrogenated castor oil, or polyethylene glycol 4000) or a liquid drug in a carrier or carrier combination (e.g., mineral oil, vegetable oil, triglyceride, ethylene glycol, polyol, and surfactant).

[0107] In therapeutic treatment of humans, a suitable daily dose of a compound of formula I or a pharmaceutically acceptable salt thereof is about 0.0001 to 100 mg / kg body weight.

[0108] Oral formulations are preferred, particularly tablets or capsules, which can be formulated by methods known to those skilled in the art to provide a dose of active compound in the range of 0.1 mg to 1000 mg.

[0109] Example

[0110] The present invention is further described and explained by citing the following examples. It should be noted that the following examples are only illustrative and are not intended to limit the scope of the present invention in any way.

[0111] Example 1: Preparation of 5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)azetidin-3-yl)oxy)-3-fluoro-N-methylpicolinamide (Compound 2)

[0112] Step 1: 3-Fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carboxylic acid methyl ester

[0113]

[0114] A solution of 5-bromo-3-fluoropyridine-2-carboxylic acid methyl ester (4.00 g, 17.1 mmol, 1.0 equiv), bis(pinacol)diboron (6.51 g, 25.6 mmol, 1.5 equiv), potassium acetate (2.52 g, 25.6 mmol, 1.50 equiv), Pd2(dba)3 (626 mg, 0.68 mmol, 0.04 equiv) and tricyclohexylphosphine (383 mg, 1.37 mmol, 0.08 equiv) in dioxane (40 mL) was stirred overnight at 90 ° C under N2 atmosphere. The resulting mixture was extracted with EtOAc (3×200 mL). The combined organic layers were washed with saturated brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated. The crude residue was purified by reverse phase chromatography (column: XBridge preparative grade C18 OBD column, 30*100 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: MeCN; flow rate: 60 mL / min; gradient: 35% B to 65% B, 65% B in 7 minutes; wavelength: 254 / 220 nm; RT (min): 6.9) to obtain 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carboxylic acid methyl ester (2.50 g, 52% yield).

[0115] Step 2: 3-Fluoro-5-hydroxypyridine-2-carboxylic acid methyl ester

[0116]

[0117] A solution of 3-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carboxylic acid methyl ester (2.50 g, 8.89 mmol, 1.0 equiv) in THF (25 mL) was treated with hydrogen peroxide (30% aqueous solution, 15 mL) at room temperature overnight. The reaction solution was then diluted with water (50 mL) and extracted with ethyl acetate (2×50 mL), washed with 5% aqueous sodium thiosulfate solution (50 mL) and saturated brine (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography, eluting with CH2Cl2 / MeOH (10:1) to obtain 3-fluoro-5-hydroxypyridine-2-carboxylic acid methyl ester (1 g, 66% yield).

[0118] Step 3: 5-{[1-(tert-Butyloxycarbonyl)azetidin-3-yl]oxy}-3-fluoropyridine-2-carboxylic acid methyl ester

[0119]

[0120] A solution of 3-fluoro-5-hydroxypyridine-2-carboxylic acid methyl ester (400 mg, 2.34 mmol, 1.0 equiv), 3-(methylsulfonyloxy)azetidine-1-carboxylic acid tert-butyl ester (587 mg, 2.34 mmol, 1.0 equiv) and K2CO3 (969 mg, 7.01 mmol, 3.0 equiv) in DMF (4 mL) was heated at 80 ° C overnight. The mixture was cooled to room temperature and diluted with water (100 mL). The resulting mixture was extracted with EtOAc (3 × 100 mL). The combined organic layer was washed with water (2 × 10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain 5-{[1-(tert-butyloxycarbonyl)azetidine-3-yl]oxy}-3-fluoropyridine-2-carboxylic acid methyl ester (300 mg, 39% yield) as a yellow oil. The crude product was used directly in the next step without further purification.

[0121] Step 4: tert-Butyl 3-{[5-fluoro-6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylate

[0122]

[0123] A solution of 5-{[1-(tert-butyloxycarbonyl)azetidine-3-yl]oxy}-3-fluoropyridine-2-carboxylic acid methyl ester (250 mg, 0.77 mmol, 1.0 equivalent) in MeOH (2.5 mL) was treated with a MeOH solution of methylamine (0.5 mL, 31 wt.%) and the resulting mixture was stirred at room temperature for 1.5 hours. The reaction solution was acidified to pH 5 with HCl (0.2 M) and concentrated under reduced pressure. The residue was purified by reverse flash chromatography (column, C18 silica gel; mobile phase, aqueous solution of MeCN (0.1% FA), gradient of 10% to 50% in 10 minutes) to obtain 3-{[5-fluoro-6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylic acid tert-butyl ester (150 mg, 60% yield) as a light yellow oil.

[0124] Step 5: 5-(azetidin-3-yloxy)-3-fluoro-N-methylpyridine-2-carboxamide

[0125]

[0126] A solution of tert-butyl 3-{[5-fluoro-6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylate (150 mg, 0.46 mmol, 1.0 equiv) in HCl in 1,4-dioxane (3 mL, 4.0 M) was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure to afford 5-(azetidin-3-yloxy)-3-fluoro-N-methylpyridine-2-carboxamide hydrochloride (100 mg, 96% yield).

[0127] Step 6: 5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)azetidin-3-yl)oxy)-3-fluoro-N-methylpicolinamide

[0128]

[0129] A solution of 7-(chloromethyl)-3-ethyl-1H-1,5-naphthyridin-2-one (100 mg, 0.45 mmol, 1.0 equiv), 5-(azetidin-3-yloxy)-3-fluoro-N-methylpyridine-2-carboxamide hydrochloride (121 mg, 0.54 mmol, 1.2 equiv), potassium iodide (14.9 mg, 0.09 mmol, 0.2 equiv) and DIPEA (391 μL, 2.25 mmol, 5.0 equiv) in MeCN (1 mL) was stirred at 80 °C for 2 h. The mixture was concentrated under reduced pressure and then purified by preparative HPLC (MeCN in water (0.1% FA), gradient 10% to 50% over 10 min) to afford 5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)azetidin-3-yl)oxy)-3-fluoro-N-methylpicolinamide (36.8 mg, 20% yield).

[0130] LC-MS(ES + )m / z:412.35(M+H) +

[0131] 1 H NMR (400MHz, DMSO-d6) δ11.86(s,1H),8.45(m,1H),8.36(m,1H),8.13(m,1H),7.74(s,1H),7.56(m,1H),7. 40(m,1H),5.04(m,1H),3.82–3.78(m,4H),3.29–3.11(m,2H),2.75(m,3H),2.59–2.53(m,2H),1.18(t,3H).

[0132] Example 2: Preparation of (R)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-N-methylpicolinamide (Compound 4) and (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-N-methylpicolinamide (Compound 5)

[0133] Step 1: tert-Butyl 3-[(6-cyanopyridin-3-yl)oxy]azetidine-1-carboxylate

[0134]

[0135] A solution (10 mL) of tert-butyl 3-hydroxyazetidine-1-carboxylate (1.70 g, 9.83 mmol, 1.0 equiv) in DMF was treated with NaH (60%) (655 mg, 16.4 mmol, 2.0 equiv) at 0 ° C for 30 minutes, and then a solution of 5-fluoropyridine-2-carbonitrile (1.00 g, 8.19 mmol, 1.0 equiv) in DMF (10 mL) was added dropwise at 0 ° C. The reaction was stirred at room temperature for 2 hours. The reaction was quenched with water (50 mL) at 0 ° C. The resulting mixture was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain tert-butyl 3-[(6-cyanopyridin-3-yl)oxy]azetidine-1-carboxylate (1.00 g, 44% yield). The crude product was used directly in the next step without further purification.

[0136] Step 2: 5-{[1-(tert-Butyloxycarbonyl)azetidin-3-yl]oxy}pyridine-2-carboxylic acid

[0137]

[0138] A solution of tert-butyl 3-[(6-cyanopyridin-3-yl)oxy]azetidine-1-carboxylate (1.00 g, 3.63 mmol, 1.0 equiv) in EtOH (10 mL) was treated dropwise with aqueous NaOH (55 mL, 2.0 M) at room temperature. The mixture was heated to reflux overnight. The mixture was acidified to pH 5 with concentrated HCl. The resulting mixture was extracted with CH2Cl2 (2×100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain 5-{[1-(tert-butoxycarbonyl)azetidine-3-yl]oxy}pyridine-2-carboxylic acid (700 mg, 66% yield).

[0139] Step 3: tert-Butyl 3-{[6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylate

[0140]

[0141] A solution of 5-{[1-(tert-butyloxycarbonyl)azetidine-3-yl]oxy}pyridine-2-carboxylic acid (700 mg, 2.38 mmol, 1.0 equiv) and methylamine hydrochloride (208 mg, 3.09 mmol, 1.3 equiv) in DMF (7.00 mL) was treated with DIPEA (1.36 mL, 7.85 mmol, 3.3 equiv) at -15 ° C, and then a solution of T3P (1.97 g, 6.18 mmol, 2.6 equiv) in DMF (7.00 mL) was added dropwise at -15 ° C. The resulting mixture was stirred at room temperature for 2 hours. A saturated sodium carbonate solution was added to the resulting mixture and stirred at room temperature for 15 minutes. The precipitated solid was collected by filtration and washed with water (2 × 5 mL). The crude product was purified by reverse phase chromatography (column: XBridge preparative grade C18 OBD column, 30*100 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: MeCN; flow rate: 60 mL / min; gradient: 35% B to 65% B in 7 minutes) to obtain tert-butyl 3-{[6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylate (500 mg, 68% yield).

[0142] Step 4: 5-(azetidin-3-yloxy)-N-methylpyridine-2-carboxamide

[0143]

[0144] A solution of tert-butyl 3-{[6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylate (500 mg, 1.63 mmol, 1.0 equiv) in HCl in 1,4-dioxane (5 mL, 4.0 M) was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure to obtain 5-(azetidine-3-yloxy)-N-methylpyridine-2-carboxamide, hydrochloride (250 mg, 74% yield). The crude product was used directly in the next step without further purification.

[0145] Step 5: 5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-N-methylpicolinamide (Compound 43)

[0146]

[0147] A solution of 7-(1-chloroethyl)-3-ethyl-1H-1,5-naphthyridin-2-one (100 mg, 0.42 mmol, 1.0 equiv), 5-(azetidin-3-yloxy)-N-methylpyridine-2-carboxamide hydrochloride (87.5 mg, 0.42 mmol, 1.0 equiv), potassium iodide (14.0 mg, 0.084 mmol, 0.2 equiv) and DIPEA (368 μL, 2.11 mmol, 5.0 equiv) in MeCN (1.0 mL) was stirred at 80° C. for 2 hours. The resulting mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC (MeCN in water (10 mmol / L NH4HCO3), gradient 10% to 50% over 10 min; detector, UV 254 nm) to afford 5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-N-methylpicolinamide (77.3 mg, 45% yield).

[0148] LC-MS(ES + )m / z:408.35(M+H) +

[0149] 1 H NMR(400MHz,DMSO-d6)δ11.86(s,1H),8.45(m,1H),8.36(m,1H),8.18(s,1H),8.13(m,1H),7.74(s,1H),7.56(m,1H),7.40(m,1 H),5.04(m,1H),3.87–3.78(m,1H),3.58-3.54(m,2H),3.19(m,1H),3.16(m,1H),2.75(m,3H),2.57–2.52(m,2H),1.18(m,6H).

[0150] Step 6:

[0151]

[0152] The enantiomeric mixture (40 mg) was separated by chiral SFC (column: CHIRAL ARTAmylose-SA, 2*25 cm, 5 μm; mobile phase A: MtBE (10 mM NH3-MeOH), mobile phase B: MeOH; flow rate: 20 mL / min; gradient: 50% B for 40 min; wavelength: 220 / 242 nm; RT1 (min): 13.02; RT2 (min): 29.36; sample solvent: MeOH:DCM=1:1; injection volume: 1 mL; number of runs: 2) to obtain:

[0153] (R)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-N-methylpicolinamide (Compound 4) (16.9 mg, 42.3% yield).

[0154] LC-MS(ES + )m / z:408.35(M+H) +

[0155] 1 H NMR(400MHz,DMSO-d6)δ11.86(s,1H),8.45(m,1H),8.36(m,1H),8.18(s,1H),8.13(m,1H),7.74(s,1H),7.56(m,1H),7.40(m,1 H),5.04(m,1H),3.87–3.78(m,1H),3.58-3.54(m,2H),3.19(m,1H),3.16(m,1H),2.75(m,3H),2.57–2.52(m,2H),1.18(m,6H).

[0156] Chiral analysis SFC conditions for compound 4:

[0157] Column: CHIRALPAK IA-3, 50×3 mm, 1.6 μm

[0158] Mobile phase: (MtBE:DCM=1:1)(0.1% DEA):MeOH=80:20

[0159] Flow rate: 1.67ml / min

[0160] RT = 0.92 min; 100%

[0161] (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-N-methylpicolinamide (Compound 5) (14.8 mg, 37.0% yield).

[0162] LC-MS(ES + )m / z:408.35(M+H) +

[0163] 1H NMR(400MHz,DMSO-d6)δ11.86(s,1H),8.45(m,1H),8.36(m,1H),8.18(s,1H),8.13(m,1H),7.74(s,1H),7.56(m,1H),7.40(m,1 H),5.04(m,1H),3.87–3.78(m,1H),3.58-3.54(m,2H),3.19(m,1H),3.16(m,1H),2.75(m,3H),2.57–2.52(m,2H),1.18(m,6H).

[0164] Chiral analysis SFC conditions for compound 5:

[0165] Column: CHIRALPAK IA-3, 50×3 mm, 1.6 μm

[0166] Mobile phase: (MtBE:DCM=1:1)(0.1% DEA):MeOH=80:20

[0167] Flow rate: 1.67ml / min

[0168] RT = 1.24 minutes; 99.8%

[0169] Alternative preparation of compound 5:

[0170] Step 8: (R)-N-((S)-1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)-2-methylpropane-2-sulfenamide

[0171]

[0172] A solution of 7-acetyl-3-ethyl-1,5-naphthyridin-2(1H)-one (150 mg, 0.69 mmol, 1.0 equiv) and (R)-2-methylpropane-2-sulfenamide (92.4 mg, 0.76 mmol, 1.1 equiv) in THF (5 mL) was treated with Ti(OEt)4 (316 mg, 1.38 mmol, 2.0 equiv) at 0°C. The resulting mixture was stirred at 65°C overnight. To the above mixture was added lithium tri-sec-butylborohydride (395 mg, 2.08 mmol, 3.0 equiv) dropwise at -78°C. The resulting mixture was stirred at room temperature for another 15 minutes. The reaction was quenched with MeOH at -50°C. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in DMSO (2 mL). The crude product was purified by preparative HPLC (column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient from 10% to 50% in 10 minutes; detector, UV 254 nm) to afford (R)-N-((S)-1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)-2-methylpropane-2-sulfenamide (80 mg, 36% yield).

[0173] Step 9: (S)-7-(1-aminoethyl)-3-ethyl-1,5-naphthyridin-2(1H)-one

[0174]

[0175] A solution of (R)-N-((S)-1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)-2-methylpropane-2-sulfenamide (80 mg, 0.24 mmol, 1.0 equiv) in MeOH (1 mL) was treated with a solution of hydrogen chloride in 1,4-dioxane (4.0 M, 0.5 mL) at room temperature. After stirring the reaction mixture at room temperature for 2 h, the reaction solution was concentrated under reduced pressure to give the crude (S)-7-(1-aminoethyl)-3-ethyl-1,5-naphthyridin-2(1H)-one hydrochloride (50 mg, 93% yield).

[0176] Step 10: (S)-3-ethyl-7-(1-(3-hydroxyazetidin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one

[0177]

[0178] A solution of (S)-7-(1-aminoethyl)-3-ethyl-1,5-naphthyridine-2(1H)-one hydrochloride (50 mg, 0.23 mmol, 1.0 equiv) and epichlorohydrin (21.2 mg, 0.23 mmol, 1.0 equiv) in IPA (1 mL) was treated with NaHCO3 (29.0 mg, 0.34 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred at 80 ° C overnight. The resulting mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC (column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient from 10% to 50% in 10 minutes; detector, UV 254 nm) to obtain (S)-3-ethyl-7-(1-(3-hydroxyazetidin-1-yl)ethyl)-1,5-naphthyridine-2(1H)-one (20 mg, 32% yield).

[0179] Step 11: (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)pyridinecarbonitrile

[0180]

[0181] A solution of (S)-3-ethyl-7-(1-(3-hydroxyazetidin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one (20 mg, 0.073 mmol, 1.0 equiv) in DMF (1 mL) was treated with NaH (60%) (3.51 mg, 0.146 mmol, 2.0 equiv) at 0° C. for 30 min, and then 5-fluoropyridine-2-carbonitrile (13.4 mg, 0.109 mmol, 1.5 equiv) was added at 0° C. The reaction was quenched with water at 0° C. The crude product was purified by preparative HPLC (column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient 10% to 50% in 10 min; detector, UV 254 nm) to afford (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)pyridinecarbonitrile (10 mg, 36% yield).

[0182] LC-MS(ES + )m / z:376.2(M+H) +

[0183] Step 12: (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)picolinic acid

[0184]

[0185] A solution of (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)pyridinecarbonitrile (10 mg, 0.027 mmol, 1 eq) in EtOH (1 mL) was treated with NaOH (0.01 mL, 10 M) at room temperature. The resulting mixture was stirred at 80 °C overnight. The mixture was acidified to pH = 5 with HCl (aq). The crude mixture was concentrated and purified by preparative HPLC (column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient 10% to 50% in 10 min; detector, UV 254 nm) to afford (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)picolinic acid (5 mg, 48% yield).

[0186] LC-MS(ES + )m / z:394.0(M+H) +

[0187] Step 13: (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-N-methylpicolinamide (Compound 5)

[0188]

[0189] To a solution of (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)picolinic acid (5 mg, 0.013 mmol, 1.0 eq) and CH3NH2.HCl (0.94 mg, 0.014 mmol, 1.1 eq) in DMF (1 mL) was added DIEA (8.82 μL, 0.052 mmol, 4.0 eq). The reaction mixture was stirred at room temperature for 10 min, then T3P (1.27 mg, 0.033 mmol, 2.5 eq) was added at -15 °C. The resulting mixture was stirred at room temperature for 2 h. The crude mixture was concentrated and the product was purified by preparative HPLC (column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient 10% to 50% in 10 min; detector, UV 254 nm) to afford (S)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-N-methylpicolinamide (2.0 mg, 39% yield).

[0190] LC-MS(ES + )m / z:408.15(M+H) +

[0191] 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),8.57(q,J=4.8Hz,1H),8.40(d,J=1.8Hz,1H), 8.21(d,J=2.9Hz,1H),7.93(d,J=8.7Hz,1H),7.73(s,1H),7.60(d,J=1.9Hz,1H),7. 38(dd,J=8.7,2.9Hz,1H),4.95(p,J=5.5Hz,1H),3.85(p,J=5.5Hz,1H),3.03-3.65( dd,J=8.0,5.0Hz,4H),2.78(d,J=4.8Hz,3H),2.58–2.51(m,2H),1.23–1.12(m,6H).

[0192] Chiral Analysis SFC:

[0193] Column: CHIRALPAK IA-U, 50×3 mm, 1.6 μm

[0194] Mobile phase: (MtBE:DCM=2:1)(0.1% DEA):MeOH=70:30

[0195] Flow rate: 1.2ml / min

[0196] RT = 1.07 min; 97.6%

[0197] Example 3: Preparation of 6-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)amino)-N-methylnicotinamide (Compound 10)

[0198] Step 1: 5-{[1-(tert-Butyloxycarbonyl)pyrrolidin-3-yl]amino}pyridine-2-carboxylic acid methyl ester

[0199]

[0200] A mixture of 5-bromopyridine-2-carboxylic acid (1.00 g, 4.95 mmol, 1.0 equiv), RuPhos Pd G3 (207 mg, 0.248 mmol, 0.05 equiv), RuPhos (231 mg, 0.495 mmol, 0.1 equiv), Cs2CO3 (4.84 g, 14.9 mmol, 3.0 equiv) and tert-butyl 3-aminopyrrolidine-1-carboxylate (921 mg, 4.95 mmol, 1.0 equiv) in 1,4-dioxane (20 mL) was stirred overnight at 110 ° C under a nitrogen atmosphere. The reaction was cooled to room temperature and then quenched by the addition of water (20 mL). The resulting mixture was extracted with EtOAc (3×50 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by reverse phase chromatography (10-50% MeCN in water (10 mM NH4HCO3)) to afford methyl 5-{[1-(tert-butoxycarbonyl)pyrrolidin-3-yl]amino}pyridine-2-carboxylate (1.00 g, 63% yield) as a yellow oil.

[0201] Step 2: tert-Butyl 3-{[6-(methylcarbamoyl)pyridin-3-yl]amino}pyrrolidine-1-carboxylate

[0202]

[0203] 5-{[1-(tert-butoxycarbonyl)pyrrolidin-3-yl]amino}pyridine-2-carboxylic acid methyl ester (1.00g, 4.36mmol, 1.0 equivalent) and a solution of methylamine in MeOH (10M, 5.0mL) were stirred overnight at room temperature under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. This gave 3-{[6-(methylcarbamoyl)pyridin-3-yl]amino}pyrrolidine-1-carboxylic acid tert-butyl ester (800mg) as a yellow oil, which was used in the next step without further purification.

[0204] Step 3: N-methyl-5-(pyrrolidin-3-ylamino)pyridine-2-carboxamide hydrochloride

[0205]

[0206] A mixture of tert-butyl 3-{[6-(methylcarbamoyl)pyridin-3-yl]amino}pyrrolidine-1-carboxylate (800 mg, 4.06 mmol, 1.0 equiv) and HCl in 1,4-dioxane (4.0 M, 10.2 mL) was stirred at room temperature for 2 h under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure to afford N-methyl-5-(pyrrolidin-3-ylamino)pyridine-2-carboxamide hydrochloride (600 mg) as a white solid, which was used without further purification.

[0207] Step 4: 6-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)amino)-N-methylnicotinamide

[0208]

[0209] A solution of 3-ethyl-7-(hydroxymethyl)-1H-1,5-naphthyridin-2-one (100 mg, 0.49 mmol, 1.0 equiv) and SOCl2 (400 μL, 5.51 mmol, 11.2 equiv) in DCM (2 mL) was stirred at room temperature for 2 hours under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure. A mixture of N-methyl-5-(pyrrolidin-3-ylamino)pyridine-2-carboxamide hydrochloride (107 mg, 0.49 mmol, 1.0 equiv), potassium iodide (16.3 mg, 245 μmol, 0.20 equiv) and DIPEA (425 μL, 2.45 mmol, 5.0 equiv) in MeCN (2 mL) was stirred at 80 ° C for 2 hours under a nitrogen atmosphere. The precipitated solid was collected by filtration and washed with water (10 mL). The residue was purified by preparative HPLC (10% to 31% MeCN in water (0.05% formic acid)) to afford 6-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)amino)-N-methylnicotinamide (51.1 mg, 25% yield).

[0210] LC-MS(ES + )m / z:407.3[M+H] +

[0211] 1H NMR (300MHz, DMSO-d6): δ11.87(s,1H),8.40(m,1H),8.30–8.22(m,1H),7.9 2(m,1H),7.77–7.68(m,2H),7.57(s,1H),6.94(m,1H),6.65(m,1H),3.98(br s,1H),3.76(s,2H),2.89–2.85(m,1H),2.76(m,3H),2.67–2.63(m,1H),2.57–2. 53(m,2H),2.45–2.40(m,1H),2.30–2.24(m,1H),1.69–1.58(m,1H),1.18(m,3H).

[0212] Example 4: Preparation of (Enantiomer 1)-5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (Compound 16) and (Enantiomer 2)-5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (Compound 17)

[0213] Step 1: 5-{[1-(tert-Butyloxycarbonyl)pyrrolidin-3-yl](methyl)amino}pyridine-2-carboxylic acid methyl ester

[0214]

[0215] To a stirred solution of 5-bromopyridine-2-carboxylic acid methyl ester (1.50 g, 6.94 mmol, 1.0 equiv) and 3-(methylamino)pyrrolidine-1-carboxylic acid tert-butyl ester (1.39 g, 6.94 mmol, 1.0 equiv) in 1,4-dioxane (15 mL) at 25 °C was added RuPhos (323 mg, 0.69 mmol, 0.1 equiv), RuPhos Pd G3 (290 mg, 0.35 mmol, 0.05 equiv) and K3PO4 (4.40 g, 20.8 mmol, 3.0 equiv) under N2 atmosphere. The resulting mixture was stirred at 100 °C for 12 hours and then cooled to room temperature. The resulting mixture was filtered and the filter cake was washed with 1,4-dioxane (3×5 mL). The filtrate was concentrated under reduced pressure. The residue was purified by reverse phase chromatography (10-50% MeCN in water (10 mM NH4HCO3)) to afford methyl 5-{[1-(tert-butoxycarbonyl)pyrrolidin-3-yl](methyl)amino}pyridine-2-carboxylate (700 mg, 30% yield) as an off-white solid.

[0216] Step 2: tert-Butyl 3-{methyl[6-(methylcarbamoyl)pyridin-3-yl]amino}pyrrolidine-1-carboxylate

[0217]

[0218] A stirred solution of methyl 5-{[1-(tert-butoxycarbonyl)pyrrolidin-3-yl](methyl)amino}pyridine-2-carboxylate (700 mg, 2.09 mmol, 1.0 equiv) and CH3NH2 in MeOH (10 M, 6 mL) was stirred overnight at room temperature under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure to afford tert-butyl 3-{methyl[6-(methylcarbamoyl)pyridin-3-yl]amino}pyrrolidine-1-carboxylate (600 mg, 67% yield) as an off-white solid which was used without further purification.

[0219] Step 3: N-methyl-5-[methyl(pyrrolidin-3-yl)amino]pyridine-2-carboxamide hydrochloride

[0220]

[0221] The stirred solution of 3-{methyl[6-(methylcarbamoyl)pyridin-3-yl]amino}pyrrolidine-1-carboxylic acid tert-butyl ester (600 mg, 1.79 mmol, 1.0 equivalent) in a solution of HCl in 1,4-dioxane (4.0 M, 5 mL) was stirred at 80 ° C for 1 hour and then cooled to room temperature. The resulting mixture was concentrated in vacuo. This gave N-methyl-5-[methyl(pyrrolidin-3-yl)amino]pyridine-2-carboxamide HCl salt (300 mg, 86% yield) as a white solid, which was used without further purification.

[0222] Step 4: 5-({1-[(7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]pyrrolidin-3-yl}(methyl)amino)-N-methylpyridine-2-carboxamide

[0223]

[0224] SOCl2 (135 μL, 1.85 mmol, 5.0 equiv) was added dropwise to a stirred solution of 3-ethyl-7-(hydroxymethyl)-1H-1,5-naphthyridin-2-one (75.4 mg, 0.37 mmol, 1.0 equiv) and DMF (2.80 μL, 36.9 μmol, 0.1 equiv) in DCM (2 mL) under N2 atmosphere at room temperature. The resulting mixture was stirred for 6 hours and then concentrated under reduced pressure. Potassium iodide (12.3 mg, 73.8 μmol, 0.2 equiv), N-methyl-5-[methyl(pyrrolidin-3-yl)amino]pyridine-2-carboxamide hydrochloride (100 mg, 0.37 mmol, 1.0 equiv) and MeCN (2 mL) were added under N2 atmosphere at room temperature, followed by DIPEA (386 μL, 2.22 mmol, 6.0 equiv). The resulting mixture was stirred at 80° C. for 2 h and then concentrated under reduced pressure. The residue was purified by preparative HPLC (10% to 40% MeCN in water (0.1% NH4HCO3)) to afford 5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (53.1 mg, 34% yield).

[0225] Step 5: (Enantiomer 1)-5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (Compound 16) and (Enantiomer 2)-5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (Compound 17)

[0226]

[0227] The enantiomers of 5-({1-[(7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]pyrrolidin-3-yl}(methyl)amino)-N-methylpyridine-2-carboxamide (50 mg) were separated by chiral SFC (column: CHIRALPAK IA-3, 4.6×50 mm, 3 μm; mobile phase: 1:1 MTBE (0.1% diethylamine) / MeOH) to give (enantiomer 1)-5-((1-((7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]pyrrolidin-3-yl}(methyl)amino)-N-methylpyridine-2-carboxamide. ((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (13.1 mg, 26% yield) and (Enantiomer 2)-5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (14.9 mg, 30% yield). The absolute configuration of the chiral center of each isolated enantiomer is unknown.

[0228] Data for (Enantiomer 1)-5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (Compound 16):

[0229] LC-MS(ES + )m / z:421.3[M+H] +

[0230] 1 H NMR (400MHz, DMSO-d6) δ11.89(s,1H),8.41(m,1H),8.30(m,1H),8.11(m,1H),7.84–7.71(m,2H),7.63(m,1H),7.23(m,1H),4.62(m,1H),3.79( m,1H),3.64(m,1H),2.97(s,3H),2.90(m,1H),2.77(m,3H),2.72(m,1H) ,2.60–2.52(m,3H),2.38–2.20(m,2H),1.80–1.68(m,1H),1.18(m,3H).

[0231] Chiral analysis SFC conditions of compound 16:

[0232] Column: CHIRALPAK IA-3, 50×4.6 mm, 3.0 μm

[0233] Mobile phase: MtBE (0.1% DEA): MeOH = 50:50

[0234] Flow rate: 1.67ml / min

[0235] RT = 4.51 minutes; 100%

[0236] Data for (Enantiomer 2)-5-((1-((7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)pyrrolidin-3-yl)(methyl)amino)-N-methylpicolinamide (Compound 17):

[0237] LC-MS(ES + )m / z:421.3[M+H] +

[0238] 1 H NMR (400MHz, DMSO-d6) δ11.89(s,1H),8.41(m,1H),8.30(m,1H),8.11(m,1H),7.84–7.71(m,2H),7.63(m,1H),7.23(m,1H),4.62(m,1H),3.79( m,1H),3.64(m,1H),2.97(s,3H),2.90(m,1H),2.77(m,3H),2.72(m,1H) ,2.60–2.52(m,3H),2.38–2.20(m,2H),1.80–1.68(m,1H),1.18(m,3H).

[0239] Chiral analysis SFC conditions for compound 17:

[0240] Column: CHIRALPAK IA-3, 50×4.6 mm, 3.0 μm

[0241] Mobile phase: MtBE (0.1% DEA): MeOH = 50:50

[0242] Flow rate: 1.67ml / min

[0243] RT = 1.95 min; 100%

[0244] Example 5: Preparation of (Enantiomer 1)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 45) and (Enantiomer 2)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 46)

[0245] Step 1: tert-Butyl 3-[(6-chloro-2-fluoropyridin-3-yl)oxy]azetidine-1-carboxylate

[0246]

[0247] To a stirred solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (2.00 g, 11.5 mmol, 1.0 equiv), 6-chloro-2-fluoropyridine-3-ol (1.70 g, 11.5 mmol, 1.0 equiv) and PPh3 (3.63 g, 13.9 mmol, 1.2 equiv) in THF (20 mL) was added DIAD (2.72 mL, 13.9 mmol, 1.2 equiv) under a nitrogen atmosphere at 0 ° C. The resulting mixture was stirred at room temperature overnight. The resulting mixture was filtered and the filter cake was washed with DCM (3×3 mL). The filtrate was concentrated under reduced pressure and then purified by reverse phase flash chromatography (column, C 18 ; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient from 10% to 70% in 15 minutes; detector, UV 254 nm) to obtain tert-butyl 3-[(6-chloro-2-fluoropyridin-3-yl)oxy]azetidine-1-carboxylate (1.00 g, 29% yield) as a light yellow solid.

[0248] Step 2: tert-Butyl 3-[(6-cyano-2-fluoropyridin-3-yl)oxy]azetidine-1-carboxylate

[0249]

[0250] To a stirred solution of tert-butyl 3-[(6-chloro-2-fluoropyridin-3-yl)oxy]azetidine-1-carboxylate (1.00 g, 3.30 mmol, 1.0 equiv) and Zn(CN)2 (0.39 g, 3.30 mmol, 1.0 equiv) in DMAc (10 mL) was added dppf (0.18 g, 0.33 mmol, 0.1 equiv) and Pd2(dba)3 (0.15 g, 0.16 mmol, 0.05 equiv) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 125 °C overnight. The resulting mixture was filtered and the filter cake was washed with DMAc (3×5 mL). The filtrate was concentrated under reduced pressure and then purified by preparative HPLC (column: XSelect CSH Prep C 18 OBD column, 19*150 mm, 5 μm; mobile phase A: water (0.05% FA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 10% B to 31% B, 31% B in 7 minutes; wavelength: 254 / 220 nm) to obtain tert-butyl 3-[(6-cyano-2-fluoropyridin-3-yl)oxy]azetidine-1-carboxylate (700 mg, 72% yield) as an off-white solid.

[0251] Step 3: tert-Butyl 3-((6-carbamoyl-2-fluoropyridin-3-yl)oxy)azetidine-1-carboxylate

[0252]

[0253] Under nitrogen atmosphere at room temperature, 30% of H2O2 aqueous solution (7mL) was added dropwise to a stirred solution of 3-[(6-cyano-2-fluoropyridin-3-yl)oxy]azetidine-1-carboxylic acid tert-butyl ester (700mg, 2.38mmol, 1.0 equivalent) and K2CO3 (0.13g, 0.95mmol, 0.4 equivalent) in DMSO (14mL). The resulting mixture was stirred at room temperature for 3 hours and then diluted with water (30mL). The precipitated solid was collected by filtration and washed with H2O (3×5mL). This gave tert-butyl 3-((6-carbamoyl-2-fluoropyridin-3-yl)oxy)azetidine-1-carboxylate (320mg, 43% yield) as an off-white solid. The crude product was used in the next step without further purification.

[0254] Step 4: tert-Butyl 3-{[2-fluoro-6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylate

[0255]

[0256] To a stirred solution of tert-butyl 3-[(6-carbamoyl-2-fluoropyridin-3-yl)oxy]azetidine-1-carboxylate (320 mg, 1.03 mmol, 1.0 eq) and benzyltriethylammonium chloride (234 mg, 1.03 mmol, 1.0 eq) and dimethyl sulfate (117 μL, 1.23 mmol, 1.2 eq) in toluene (4.5 mL) and MeCN (4.5 mL) was added NaOH (41.1 mg, 1.03 mmol, 1 eq) at room temperature under nitrogen atmosphere. The resulting mixture was stirred overnight and then concentrated in vacuo. The residue was purified by preparative HPLC (column: XSelect CSH Prep C 18 OBD column, 19*150 mm, 5 μm; mobile phase A: water (0.05% FA), mobile phase B: ACN; flow rate: 25 mL / min; gradient: 10% B to 31% B, 31% B in 7 minutes; wavelength: 254 / 220 nm) to obtain tert-butyl 3-{[2-fluoro-6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylate (200 mg, 60% yield) as an off-white solid.

[0257] Step 5: 5-(azetidin-3-yloxy)-6-fluoro-N-methylpicolinamide

[0258]

[0259] 3-{[2-fluoro-6-(methylcarbamoyl)pyridin-3-yl]oxy}azetidine-1-carboxylic acid tert-butyl ester (200mg, 0.61mmol, 1.0 equivalent) and dioxane solution of hydrogen chloride (4.0M, 7mL) were added to a 40mL vial at room temperature. The resulting mixture was stirred for another 2 hours at room temperature. The resulting mixture was concentrated in vacuo. This gave 5-(azetidine-3-yloxy)-6-fluoro-N-methylpicolinamide hydrochloride (170mg) as an off-white solid. The crude product was used directly in the next step without further purification.

[0260] Step 6: 5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 21)

[0261]

[0262] To a 20 mL vial was added 5-(azetidin-3-yloxy)-6-fluoro-N-methylpicolinamide hydrochloride (170 mg, 0.75 mmol, 1.7 eq), 7-(1-chloroethyl)-3-ethyl-1H-1,5-naphthyridin-2-one (105 mg, 0.44 mmol, 1.0 eq), KI (22.1 mg, 0.13 mmol, 0.3 eq) and MeCN (8 mL) at room temperature. To the above mixture was added DIEA (425 μL, 2.44 mmol, 5.5 eq) dropwise at room temperature and the resulting mixture was stirred at 80 °C overnight. The resulting mixture was concentrated under reduced pressure and the residue was purified by preparative TLC (CH3CN / H2O 3:1) to afford 5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 21) (19.1 mg, 10% yield).

[0263] LC-MS(ES + )m / z:426.2(M+H) +

[0264] 1H NMR (400MHz, DMSO-d6) δ11.85(s,1H),8.48(q,J=4.7Hz,1H),8.42(d,J=1.8Hz,1H) ,7.85(d,J=8.2Hz,1H),7.75(s,1H),7.65–7.51(m,2H),5.00(t,J=5.5Hz,1H),3.8 3(d,J=7.0Hz,1H),3.59(td,J=6.6,4.1Hz,2H),3.18(dd,J=8.1,5.0Hz,1H),3.08( dd,J=8.1,5.0Hz,1H),2.78(d,J=4.8Hz,3H),2.58–2.53(m,2H),1.24–1.16(m,6H).

[0265] Step 7: (Enantiomer 1)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 45) and (Enantiomer 2)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 46)

[0266]

[0267] The enantiomers of 5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 21) (18 mg) were separated by SFC (column: CHIRALPAK IA, 2*25 cm, 5 μm; mobile phase A: MtBE:DCM=2:1, mobile phase B: MeOH; flow rate: 20 mL / min; 30% B for 10 min; wavelength: 330 / 224 nm; RT1 (min): 4.0; RT2 (min): 7.0) to obtain:

[0268] (Enantiomer 1)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 45) (5.6 mg, 31% yield).

[0269] LC-MS(ES + )m / z:426.3(M+H) +

[0270] 1H NMR (400MHz, DMSO-d6) δ11.85(s,1H),8.48(d,J=5.0Hz,1H),8.41(s,1H),7.84(d,J=8.1Hz,1H),7.73(s,1H),7.64–7.51(m,2H), 4.99(s,1H),3.83(s,1H),3.58(s,2H),3.17(d,J=7.5Hz,1H),3.06(s,1H),2.76(d,J=4.8Hz,3H),2.61–2.52(m,2H),1.19(m,6H).

[0271] Chiral analysis SFC conditions for compound 45:

[0272] Column: CHIRALPAK IA-U, 50×3.0mm, 1.6μm

[0273] Mobile phase: MtBE / DCM (2:1) (0.1% DEA): MeOH = 70:30

[0274] Flow rate: 1.2ml / min

[0275] RT = 0.459 min; 100%

[0276] (Enantiomer 2)-5-((1-(1-(7-ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 46) (5.7 mg, 32% yield).

[0277] LC-MS(ES + )m / z:426.2(M+H) +

[0278] 1 H NMR (400MHz, DMSO-d6) δ11.88(s,1H),8.48(d,J=31.0Hz,2H),8.41(s,1H),7.87(d,J=8.2Hz,1H),7.77(s,1H),7.61(d,J=11.1Hz ,2H),5.02(s,1H),3.86(s,1H),3.61(s,2H),3.24–3.06(m,2H),2.79(d,J=4.8Hz,3H),2.58(t,J=7.2Hz,2H),1.57–0.96(m,6H).

[0279] Chiral analysis SFC conditions of compound 46:

[0280] Column: CHIRALPAK IA-U, 50×3.0mm, 1.6μm

[0281] Mobile phase: MtBE / DCM (2:1) (0.1% DEA): MeOH = 70:30

[0282] Flow rate: 1.2ml / min

[0283] RT = 0.703 min; 99.7%

[0284] The absolute configuration of the chiral center of each isolated enantiomer is unknown.

[0285] Example 6: Preparation of (Enantiomer 1)-5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 49) and (Enantiomer 2)-5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 50)

[0286] Step 6: 7-Chloro-3-ethyl-1,6-naphthyridin-2(1H)-one

[0287]

[0288] A solution of 4-amino-6-chloropyridine-3-carboxaldehyde (10.0 g, 64.0 mmol, 1.0 equiv.), DIEA (55.8 mL, 320 mmol, 5.0 equiv.), DMAP (1.56 g, 12.8 mmol, 0.20 equiv.) in DCM (100 mL) was treated with butyryl chloride (21.9 mL, 211 mmol, 3.30 equiv.) at 0 ° C under a nitrogen atmosphere for 4 hours, and then butyryl chloride (19.7 mL, 190 mmol, 3.0 equiv.) was added dropwise at 0 ° C. The resulting mixture was stirred at room temperature overnight. The reaction was quenched by adding water (100 mL) at room temperature. The precipitated solid was collected by filtration and washed with water (3 × 50 mL). This gave 7-chloro-3-ethyl-1,6-naphthyridine-2 (1H) -one (2.40 g, 36% yield) as a white solid. The crude product was used without further purification.

[0289] Step 7: 7-(1-ethoxyvinyl)-3-ethyl-1,6-naphthyridin-2(1H)-one

[0290]

[0291] A solution of 7-chloro-3-ethyl-1,6-naphthyridin-2(1H)-one (2.40 g, 11.5 mmol, 1.0 eq.), Pd(PPh3)2Cl2 (0.81 g, 1.15 mmol, 0.1 eq.) and tributyl(1-ethoxyvinyl)stannane (10.4 g, 28.8 mmol, 2.5 eq.) in dioxane (50 mL) was stirred overnight under nitrogen atmosphere at 110° C. The resulting mixture was used directly in the next step without further purification.

[0292] Step 8: 7-Acetyl-3-ethyl-1,6-naphthyridin-2(1H)-one

[0293]

[0294] Under nitrogen atmosphere at room temperature, a mixture of 7-(1-ethoxyvinyl)-3-ethyl-1,6-naphthyridine-2(1H)-one (60 mL, mixture) and HCl (2 mL) in dioxane (10 mL) was stirred for 1 hour. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography (column, C18 silica gel; mobile phase, aqueous solution of MeCN (10 mmol / LNH4HCO3), 10% to 50% gradient in 10 minutes; detector, UV 254 nm) to obtain 7-acetyl-3-ethyl-1,6-naphthyridine-2(1H)-one (700 mg, 16% yield) as a brown solid.

[0295] Step 9: 3-ethyl-7-(1-hydroxyethyl)-1,6-naphthyridin-2(1H)-one

[0296]

[0297] A solution of 7-acetyl-3-ethyl-1,6-naphthyridin-2(1H)-one (700 mg, 3.24 mmol, 1.0 equivalent) and NaBH4 (490 mg, 13.0 mmol, 4.0 equivalent) in MeOH (10 mL) was stirred for 4 hours at room temperature under a nitrogen atmosphere. The resulting mixture was concentrated under reduced pressure and purified by reverse phase flash chromatography (column, C18 silica gel; mobile phase, aqueous solution of MeCN (10 mmol / L NH4HCO3), gradient from 10% to 50% in 10 minutes; detector, UV 254 nm) to obtain 3-ethyl-7-(1-hydroxyethyl)-1,6-naphthyridin-2(1H)-one (500 mg, 71% yield) as a yellow solid.

[0298] Step 10: 7-(1-bromoethyl)-3-ethyl-1,6-naphthyridin-2(1H)-one

[0299]

[0300] A solution of 3-ethyl-7-(1-hydroxyethyl)-1,6-naphthyridine-2(1H)-one (500 mg, 2.30 mmol, 1.0 equiv) in DCM (5 mL) was treated with 1,2-dibromo-1,1,2,2-tetrachloroethane (1.64 g, 5.04 mmol, 2.2 equiv) at 0 ° C under a nitrogen atmosphere for 20 minutes, and then a solution of PPh3 (1.20 g, 4.58 mmol, 2.0 equiv) in DCM (5 mL) was added dropwise at 0 ° C. The resulting mixture was stirred at room temperature for another 4 hours. The resulting mixture was concentrated under reduced pressure to obtain 7-(1-bromoethyl)-3-ethyl-1,6-naphthyridine-2(1H)-one (400 mg, crude) as a brown solid, which was used without further purification.

[0301] Step 11: 5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide

[0302]

[0303] A solution of 7-(1-bromoethyl)-3-ethyl-1,6-naphthyridin-2(1H)-one (400 mg, 1.42 mmol, 1.0 equiv), DIEA (1.24 mL, 7.12 mmol, 5.0 equiv), KI (47.3 mg, 0.285 mmol, 0.20 equiv) and 5-(azetidin-3-yloxy)-6-fluoro-N-methylpicolinamide hydrochloride (320 mg, 1.43 mmol, 1.0 equiv) in MeCN (10 mL) was stirred at 80 °C under nitrogen atmosphere for 2 h. The resulting mixture was concentrated under reduced pressure and the residue was purified by reverse phase flash chromatography (column, C18 silica gel; mobile phase, MeCN in water (10 mmol / L NH4HCO3), gradient from 10% to 40% in 10 minutes; detector, UV 254 nm) to afford 5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (150 mg, 25% yield).

[0304] LC-MS(ES + )m / z:426.3(M+H) +

[0305] Step 12: (Enantiomer 1)-5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 49) and (Enantiomer 2)-5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 50)

[0306]

[0307] The enantiomers of 5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (100 mg) were separated by SFC (column: CHIRAL ART cellulose-SB, 4.6*100 mm, 3.0 μm; mobile phase: MtBE (0.1% DEA):EtOH=80:20; flow rate: 1.67 mL / min) to obtain:

[0308] (Enantiomer 1)-5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 49) (30 mg, 30% yield).

[0309] LC-MS(ES + )m / z:426.0(M+H) +

[0310] 1 H NMR (400MHz, DMSO-d6): δ11.92(s,1H),8.71(s,1H),8.48(d,J=5.0Hz,1H),7.85(d,J=8.2Hz, 1H),7.78(s,1H),7.56(dd,J=10.2,8.2Hz,1H),7.23(s,1H),5.02(t,J=5.4Hz,1H),3.79(t,J= 6.9Hz,1H),3.68(t,J=7.1Hz,1H),3.57(q,J=6.4Hz,1H),3.23(dd,J=8.1,4.7Hz,1H),3.11(dd ,J=8.3,4.8Hz,1H),2.77(d,J=4.7Hz,3H),2.52(s,1H),2.50–2.44(m,1H),1.26–1.12(m,6H).

[0311] SFC RT = 1.993 min; 100%

[0312] (Enantiomer 2)-5-((1-(1-(3-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-7-yl)ethyl)azetidin-3-yl)oxy)-6-fluoro-N-methylpicolinamide (Compound 50) (34 mg, 34% yield).

[0313] LC-MS(ES + )m / z:426.0(M+H) +

[0314] 1 H NMR (400MHz, DMSO-d6): δ11.92(s,1H),8.71(s,1H),8.48(q,J=4.7Hz,1H),7.85(d,J=8.2Hz, 1H),7.78(s,1H),7.56(dd,J=10.1,8.2Hz,1H),7.23(s,1H),5.01(q,J=5.4Hz,1H),3.79(t,J= 7.0Hz,1H),3.68(t,J=7.2Hz,1H),3.57(q,J=6.5Hz,1H),3.23(dd,J=8.2,4.8Hz,1H),3.11(dd ,J=8.1,4.8Hz,1H),2.77(d,J=4.7Hz,3H),2.52(s,1H),2.50–2.44(m,1H),1.26–1.12(m,6H).

[0315] SFC RT = 2.353 min; 100%

[0316] The absolute configuration of the chiral center of each isolated enantiomer is unknown.

[0317] Example 7 Bioassay

[0318] FP assay:

[0319] FP experiments were performed at room temperature using non-binding black 384-well microplates. In-house produced recombinant full-length PARP1 and PARP2 proteins were diluted to 20 nM and 60 nM, respectively, using assay buffer (50 mM Tris pH 8, 0.001% Triton X100, 10 mM MgCl2, and 150 mM NaCl) and incubated for 4 hours with an equal volume of 8 nM fluorescent probe diluted in assay buffer. The fluorescence anisotropy of the probe when bound to the protein was measured in the presence of the test compound or solvent control, and the effect on the anisotropy was determined. Polarization values ​​were read using an Envision plate reader using excitation and emission wavelengths of 590 and 630 nm, respectively. All FP values ​​are expressed in mP units. The inhibition rate was calculated using the reading (mP) according to the following formula:

[0320] Inhibition (%) = 100 × (mP HC -mP 样品 ) / (mP HC -mP LC ), where HC and LC represent high and low control wells, respectively.

[0321] The values ​​of % inhibition for different test compound concentrations were calculated and fitted to a four-parameter logistic plot to determine the IC using XLfit. 50 value.

[0322] The test results are summarized in Table 2.

[0323] Cell proliferation assay in DLD1-BRCA2- / - cell lines

[0324] BRCA2(- / -) cells cultured in RPMI 1640 + 10% FBS were harvested and diluted to 1×10 4 cells / mL and 2×10 4 Cells were inoculated into 384-well cell culture plates at a density of cells / mL. Cells (40 μL / well) were inoculated into 384-well cell culture plates. Before adding the test compound or vehicle, the plates were covered and incubated overnight at 37°C, 5% CO2. The plates were then incubated for 7 days at 37°C, 5% CO2. On the 8th day, the plates were removed from the incubator and equilibrated at room temperature for 15 minutes. CellTiter-Glo (40 μL, 1:1 with culture medium) was added to each well and the plates were placed at room temperature for 30 minutes. Luminescence was measured using an Envision plate reader. The resulting data were analyzed as follows; wherein LC is the culture medium without cells:

[0325] % Vehicle = 100 x (luminescence of test sample - luminescence of LC) / (luminescence of HC - luminescence of LC), where LC and HC are low and high control wells, respectively.

[0326] The values ​​of % inhibition for different test compound concentrations were calculated and fitted to a four-parameter logistic plot to determine the IC using XLfit. 50 value.

[0327] The test results are summarized in Table 2.

[0328] Caco2 (AB / BA) determination method

[0329] The transport of 5 μM test compounds in HBSS (10 mM HEPES, pH 7.4) was measured across Caco-2 cell monolayers (cells from the American Type Culture Collection, Manassas, VA) from the apical to the basolateral (AB) and basolateral to the apical (BA) directions. Two incubations were performed at approximately 37°C for 120 minutes, and the functionality of the test system was confirmed using 5 μM propranolol and digoxin as control compounds. Aliquots (50 μL) from the apical and basolateral wells were transferred to two new 96-well plates and quenched with an acetonitrile solution containing an analytical internal standard. The samples were vortexed, centrifuged, and a 100 μL aliquot of the resulting supernatant was mixed with an equal volume of ultrapure water and then analyzed by UPLC-MS / MS. The concentrations of test and control compounds in the incubation medium of the donor and receptor compartments at the beginning and end of the incubation period were used to calculate the apparent permeability (Papp) from the AB and BA directions. The efflux ratio (ER) is expressed as Papp B-A / Papp A-B The integrity of the cell monolayer was confirmed after 2 hours of incubation using the labeling reagent Lucifer yellow.

[0330] The test results are summarized in Table 2.

[0331] Table 2

[0332]

[0333]

[0334]

[0335] - indicates not tested

[0336] Example 8 Solubility Determination

[0337] Kinetic solubility, PBS, pH 7.4

[0338] Two kinetic solubility incubations (lasting 2 hours) were performed at 25°C, 1100 rpm in PBS pH 7.4 containing 300 μM test compounds or control compound progesterone prepared at a concentration of 10 mM in DMSO in 1.5 mL glass vials in an Eppendorf Thermomixer Comfort plate shaker. After incubation, the samples were filtered and the filtrate was diluted 1000-fold with water: acetonitrile 1:1 (v / v) for analysis by UPLC MS / MS to determine the concentration of the test compound. Solubility values ​​were calculated by quantification against standards of known concentrations.

[0339] The test results are summarized in Table 3.

[0340] Thermodynamic solubility, FaSSiF

[0341] All incubations were performed twice. The test compound or control compound diclofenac sodium (1.0 mg) was placed in a 1.5 mL glass vial. FaSSIF (1000 μL) was added to the vial. The sample was transferred to an Eppendorf Thermomixer Comfort plate shaker and shaken at 25°C, 1100 rpm for 24 hours. The sample was then filtered. The filtrate was diluted 1000 times with water: acetonitrile 1: 1 (v / v) for analysis by UPLC MS / MS to determine the concentration of the test compound. Solubility values ​​were calculated by quantification of standards for known concentrations.

[0342] The test results are summarized in Table 3.

[0343] Table 3

[0344]

[0345] *Test results from two different batches

[0346] #Test results of three different batches.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: in, X1, X2 or X3 are independently selected from N or CH; X5 is selected from N, CH or CF; A is -O-; B is selected from a substituted or unsubstituted and the substituent at any position of ring B is R5; R1 is selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C3-C5 cycloalkyl; R2 is One of X4 is N and one of X4 is CH; Each R3 is independently selected from H or unsubstituted or substituted -C1-C6 alkyl, the substituent of -C1-C6 alkyl is selected from H, -O-CH3, -CN, -OH, or two R3 are connected to form a C3-C5 cycloalkyl; Each R 4a Independently selected from H, CN, halogen, C1-C6 alkyl, -O-alkyl, C1-C6 haloalkyl, or -C1-C6 alkoxy; R5 is selected from H, C1-C6 alkyl, =O, -(CH2) 1-3 OH, or halogen.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the formula I is 3. The compound according to claim 1 or 2, wherein A is selected from -O-; B is selected from a substituted or unsubstituted and the substituent at any position of ring B is R5; R1 is selected from C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, C3-C5 cycloalkyl; R2 is One of X4 is N and one of X4 is CH; Each R3 is independently selected from H or unsubstituted or substituted -C1-C6 alkyl, the substituent of -C1-C6 alkyl is selected from H, -O-CH3, -CN, -OH, or two R3 are connected to form a C3-C6 cycloalkyl; R 4a is independently selected from H, CN, halogen, C1-C3 alkyl, -O-C1-C3 alkyl, C1-C3 haloalkyl, or -C1-C3 alkoxy; preferably, each R 4a Independently selected from H, -CH3, -CN, F; R5 is selected from -CH3, -CH2OH, or -F.

4. The compound according to claim 3, wherein the formula I is A is selected from -O-; R1 is selected from C1-C3 alkyl, C3-C5 cycloalkyl; R2 is One of X4 is N and one of X4 is CH; Each R3 is independently selected from H or unsubstituted or substituted -C1-C6 alkyl, the substituent of -C1-C6 alkyl is selected from H, -O-CH3, -CN, -OH, or two R3 are connected to form a C3-C5 cycloalkyl; Each R 4a Independently selected from H, -CH3, -CN, F. The compound according to claim 4 , wherein two R 3 are not H at the same time.

6. The compound according to any one of claims 5 or 6, wherein R2 is Each R 4a Independently selected from H, -CH3, -CN, F; Each R3 is independently selected from H or -C1-C3 alkyl, wherein two R3 are not H at the same time.

7. The compound according to claim 3, wherein the formula I is A is selected from -O-; R1 is selected from C1-C3 alkyl, C3-C5 cycloalkyl; R2 is Each R3 is independently selected from H or unsubstituted or substituted -C1-C6 alkyl, the substituent of -C1-C6 alkyl is selected from H, -O-CH3, -CN, -OH, or two R3 are connected to form a C3-C5 cycloalkyl; Each R 4a Independently selected from H, -CH3, -CN, F.

8. The compound according to claim 7, wherein R1 is selected from C1-C3 alkyl; Each R3 is independently selected from H or unsubstituted -C1-C3 alkyl; Each R 4a Independently selected from H, -CH3, F.

9. The compound according to any one of claims 1 to 8, wherein the compound is selected from or a pharmaceutically acceptable salt thereof.

10. The compound according to any one of claims 1 to 8, wherein the compound is selected from or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable diluent, excipient or inert carrier.

12. A compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof for use as a medicament; or a method of treatment comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof to a patient in need thereof; or use of a compound according to any one of claims 1 to 10 in the preparation of a medicament for treating cancer.

13. The compound or method or use according to claim 12, wherein the patient in need thereof suffers from cancer.

14. The compound or method or use according to claim 13, wherein the cancer is deficient in the HR-dependent DNA DSB repair pathway.

15. The compound or method or use of claim 14, wherein the cancer comprises one or more cancer cells having reduced or abolished ability to repair DNA DSBs by HR relative to normal cells.

16. The compound or method or use according to claim 14 or 15, wherein the cancer cells have a BRCA1 or BRCA2 deficient phenotype.

17. The compound or method or use according to claim 16, wherein the cancer cells lack BRCA1 or BRCA2.

18. The compound or method or use according to any one of claims 16 to 17, wherein the patient is heterozygous for a mutation in a gene encoding a component of the HR-dependent DNA DSB repair pathway.

19. The compound or method or use according to claim 18, wherein the patient is heterozygous for a mutation in BRCA1 and / or BRCA2.

20. The compound or method or use according to any one of claims 12 to 19, wherein the cancer is selected from any one of breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, blood cancer, gastrointestinal cancer and lung cancer.

21. The compound or method or use according to any one of claims 12 to 20, wherein inhibition of PARP1 is therapeutically beneficial.

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