Patent Application
20180273528
The present invention relates to new imidazo[4,5-b]pyridine derivatives, to a process for their preparation and to pharmaceutical compositions containing them.
The compounds of the present invention are new and have very valuable pharmacological characteristics in the field of oncology.
The present invention relates to the use of dual DYRK1/CLK1 inhibitors in the treatment of cancer, neurodegenerative disorders and metabolic disorders.
In cancer, the dual-specificity tyrosine-phosphorylation-regulated kinases DYRK1A and DYRK1B have been demonstrated to control several pathways that enhance cancer cell proliferation, migration and metastasis, induce resistance to cell death and repress responses to conventional and targeted anti-cancer therapies [Abbassi et al, Pharmacol Ther. 2015; 151:87-98; Ionescu et al, Mini Rev Med Chem. 2012; 12(13):1315-29; Friedman et al, J Cell Biochem. 2007; 102(2):274-9; Yoshida et al, Biochem Pharmacol. 2008; 76(11):1389-94]. Reported substrates of DYRK1A that are involved in this regulation of cancer progression and resistance to therapy include the transcription factors GLI1, STAT3 and FOXO1 [Mao et al, J Biol Chem. 2002; 277(38):35156-61; Matsuo et al, J Immunol Methods 2001; 247:141-51; Woods et al, Biochem J. 2001; 355(Pt 3): 597-607]. DYRK1A is also believed to stabilise cancer-associated tyrosine kinase receptors such as EGFR and FGFR via interaction with the protein Sprouty2 [Ferron et al, Cell Stem Cell. 2010; 7(3):367-79; Aranda et al, Mol Cell Biol. 2008; 28(19):5899-911]. DYRK1A, and also DYRK1B, have been shown to be required for the induction of cell quiescence in response to treatment of cancer cells by chemotherapeutic agents and targeted therapies. This is important since it is known that quiescent cancer cells are relatively insensitive to most anti-cancer drugs and radiation [Ewton et al, Mol Cancer Ther. 2011; 10(11):2104-14; Jin et al, J Biol Chem. 2009; 284(34):22916-25]. For example, DYRK1A activates the DREAM multisubunit protein complex, which maintains cells in quiescence and protects against apoptosis [Litovchick et al, Genes Dev. 2011; 25(8):801-13]. DYRK1B has been demonstrated to prevent cell-cycle exit in response to chemotherapy via phosphorylation of Cyclin D1 [Zou et al, J Biol Chem. 2004; 279(26):27790-8]. DYRK1B has also been shown to protect against chemotherapy through a reduction in reactive oxygen species content [Hu et al, Genes Cancer. 2010; 1(8):803-811].
It is thus clear that the use of DYRK1A/DYRK1B inhibitors would constitute a novel anti-cancer treatment in a wide variety of cancers when used either alone or in combination with conventional therapy, radiation or targeted therapies as a strategy to combat resistance.
The role of DYRK1A in neurological disorders is well established. DYRK1A is associated with neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases, as well as with Down's syndrome, mental retardation and motor defects and [Abbassi et al, Pharmacol Ther. 2015; 151:87-98; Beker et al, CNS Neurol Disord Drug Targets. 2014; 13(1):26-33; Dierssen, Nat Rev Neurosci. 2012 December; 13(12):844-58]. DYRK1A has been identified as a major kinase phosphorylating the microtubule-associated protein TAU, leading to the formation of neurotoxic neurofibrillary tangles and neurodegeneration as seen in Alzheimer's [Azorsa et al, BMC Genomics. 2010; 11:25]. DYRK1A also alters the splicing of TAU pre-mRNA leading to an imbalance between TAU isoforms which is sufficient to cause neurodegeneration and dementia [Liu et al, Mol Neurodegener. 2008; 3:8]. It is not surprising, therefore, that DYRK1A is believed to be causally involved in the development of Alzheimer-like neurodegenerative diseases in Down Syndrome patients, where three copies of the DYRK1A gene are present on chromosome 21. In these individuals, increased DYRK1A activity also causes premature neuronal differentiation and a decrease in mature neurones [Hämerle et al, Development. 2011; 138(12):2543-54].
It is thus clear that the use of DYRK1A inhibitors would offer a novel therapeutic approach for the treatment of neurodegenerative disorders, in particular Alzheimer's disease, as well as for other neurological conditions such as Down's syndrome.
The CDC2-like kinase (CLK) family contains four isoforms (CLK1-4) which are important in regulating the function of the spliceosome complex [Fedorov et al, Chem Biol. 2011; 18(1):67-76]. This complex, comprised of small nuclear RNAs (snRNA) and a large number of associated proteins, regulates the splicing of pre-mRNAs to give mature protein-encoding mRNAs. CLK1 is known to regulate the activity of the spliceosome via phosphorylation of the constituent serine-arginine-rich (SR) proteins [Bullock et at, Structure. 2009; 17(3):352-62]. By controlling the activity of the spliceosome in this way, many genes are able express more than one mRNA leading to diversity in the translated proteins. The alternative protein isoforms transcribed from the same gene will often have different activities and physiological functions. Deregulation of alternative splicing has been linked to cancer, where a number of cancer-related proteins are known to be alternatively spliced [Druillennec et al, J Nucleic Acids. 2012; 2012:639062]. An example of an alternatively spliced protein in cancer is Cyclin D1, important for the progression of cancer cells through the cell cycle [Wang et al, Cancer Res. 2008; 68(14):5628-38].
It is thus clear that the use of CLK1 inhibitors would constitute a novel anti-cancer treatment in a wide variety of cancers when used either alone or in combination with conventional therapy, radiation or targeted therapies.
Alternative splicing regulated by CLK1 has also been described to play a role in neurodegenerative diseases, including Alzheimer's and Parkinson's, via phosphorylation of the SR proteins of the spliceosome [Jain et al, Curr Drug Targets. 2014; 15(5):539-50]. In the case of Alzheimer's, CLK1 is known to regulate the alternative splicing of the microtubule-associated protein TAU leading to an imbalance between TAU isoforms which is sufficient to cause neurodegeneration and dementia [Liu et al, Mol Neurodegener. 2008; 3:8].
It is thus clear that the use of CLK1 inhibitors would offer a novel therapeutic approach for the treatment of neurodegenerative disorders, in particular Alzheimer's disease, as well as for other neurological conditions such as Parkinson's.
In the treatment of both cancer and neurological disease, there is thus undoubtedly an urgent need for compounds which potently inhibit the DYRK1 and CLK1 kinases whilst not affecting other closely-related kinases. The DYRK1 and CLK1 kinases are members of the CMGC group, which includes the CDK and the GSK kinases, the chronic inhibition of which is believed to be a cause of toxicity to the patient. For example, common toxicities observed in the clinic with CDK inhibition are similar to those observed with conventional cytotoxic therapy, and include hematologic toxicity (leukopenia and thrombocytopenia), gastrointestinal toxicity (nausea and diarrhea), and fatigue [Kumar et al, Blood. 2015; 125(3):443-8]. The present invention describes a new class of DYRK1/CLK1 inhibitors which are highly selective for DYRK1 and CLK1 over these other kinases and which would thus be suitable for use in the treatment of these pathologies.
Diabetes type 1 and type 2 both involve deficiency of functional pancreatic insulin-producing beta cells. Restoring functional beta-cell mass is thus an important therapeutic goal for these diseases which affect 380 million people worldwide. Recent studies have shown that DYRK1A inhibition promotes human beta-cell proliferation in vitro and in vivo and, following prolonged treatment, can increase glucose-dependent insulin secretion [Dirice et a, Diabetes. 2016; 65(6):1660-71; Wang et al, Nat Med. 2015; 21(4):383-8]. These observations clearly suggest that the use of potent and selective DYRK1A inhibitors would offer a novel therapeutic approach for the treatment and/or prevention of metabolic disorders including diabetes and obesity.
The present invention relates more especially to compounds of formula (I):
wherein.
Among the pharmaceutically acceptable acids there may be mentioned, without implying any limitation, hydrochloric acid, hydrobromic acid, sulphuric acid, phosphonic acid, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, oxalic acid, methanesulphonic acid, camphoric acid etc.
Among the pharmaceutically acceptable bases there may be mentioned, without implying any limitation, sodium hydroxide, potassium hydroxide, triethylamine, tert-butylamine etc.
Advantageously, R1 represents a methyl or a cyano group.
In another embodiment of the invention, R4 and R5 each represent a hydrogen atom
Preferably, R3 represents a NH2 group.
Alternatively, R3 represents a hydrogen atom.
In one embodiment, R2 represents a hydrogen, a linear or branched (C1-C6)alkyl group, a linear or branched (C2-C6)alkenyl group, a linear or branched (C2-C6)alkynyl group, —(C1-C6)alkylene-O-Cy1 group, —(C1-C6)alkenylene-[O]n-Cy1 group, —(C1-C6)alkylene-NR-Cy1 group, —(C1-C6)alkylene-S-Cy1 group, —(C0-C6)alkylene-Cy2-Cy1 group, or —Cy2-(C2-C6)alkylene-Cy1 group, it being understood that the alkyl and alkylene moieties defined hereinbefore may be linear or branched.
In another embodiment of the invention, R2 represents Cy1, a —(C1-C6)alkylene-Cy1 group, —(C0-C6)alkylene-Cy2-Cy1 group, or —Cy2-(C1-C6)alkylene-Cy1 group. More preferably, R2 represents:
In a third embodiment, R2 represents a linear or branched (C1-C6)alkyl group, wherein the alkyl group so defined can be optionally substituted according to the definitions mentioned previously. Halogens and CH3—S— are the preferred substituents for the alkyl group.
In a fourth embodiment, R2 represents —(C1-C6)alkylene-O-Cy1 group. More preferably, R2 represents a —(C1-C6)alkylene-O-pyridinyl group, wherein the pyridinyl group so defined can be optionally substituted according to the definitions mentioned previously. Halogens and linear or branched (C1-C6)polyhaloalkyl groups are the preferred substituents for the pyridinyl group.
Preferred compounds according to the invention are included in the following group:
The invention relates also to a process for the preparation of compounds of formula (I), which process is characterised in that there is used as starting material the compound of formula (II):
wherein A represents a halogen atom, or a linear or branched (C1-C6)alkyl group optionally substituted by from one to three halogen atoms. X represent a halogen atom, and R2 is as defined in formula (I),
which compound of formula (II) is subjected to coupling with a compound of formula (III):
wherein:
wherein A represents a halogen atom, or a linear or branched (C1-C6)alkyl group optionally substituted by from one to three halogen atoms, RB3 represents a hydrogen or group NH2, and R2, R4 and R5 are as defined in formula (I),
which compound of formula (IV):
The invention relates also to an alternative process for the preparation of compounds of formula (I), which process is characterised in that there is used as starting material the compound of formula (II′):
wherein A′ represents a linear or branched (C1-C6)alkyl group optionally substituted by from one to three halogen atoms, and X represents a halogen atom,
which compound of formula (II′) is subjected to coupling with a compound of formula (III):
wherein:
wherein.
The compounds of formulae (II), (II′), (III′) and the amine R2—NH2 are either commercially available or can be obtained by the person skilled in the art using conventional chemical reactions described in the literature.
Pharmacological study of the compounds of the invention has shown that they are powerful DYRK1/CLK1 inhibitors which are highly selective for DYRK1 and CLK1 over other kinases such as CDK9.
More especially, the compounds according to the invention will be useful in the treatment of chemo- or radio-resistant cancers.
Among the cancer treatments envisaged there may be mentioned, without implying any limitation, haematological cancer (lymphoma and leukemia) and solid tumors including carcinoma, sarcoma, or blastoma. There may be mentioned more preferably acute megakaryoblastic leukaemia (AMKL), acute lymphoblastic leukaemia (ALL), ovarian cancer, pancreatic cancer, gastrointestinal stromal tumours (GIST), osteosarcoma (OS), colorectal carcinoma (CRC), neuroblastoma and glioblastoma.
In another embodiment, the compounds of the invention will useful in the treatment of neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases, as well as with Down's syndrome, mental retardation and motor defects.
Alternatively, the compounds of the invention could be used in the treatment and/or prevention of metabolic disorders including diabetes and obesity.
The present invention relates also to pharmaceutical compositions comprising at least one compound of formula (I) in combination with one or more pharmaceutically acceptable excipients.
Among the pharmaceutical compositions according to the invention there may be mentioned more especially those that are suitable for oral, parenteral, nasal, per- or trans-cutaneous, rectal, perlingual, ocular or respiratory administration, especially tablets or dragées, sublingual tablets, sachets, paquets, capsules, glossettes, lozenges, suppositories, creams, ointments, dermal gels, and drinkable or injectable ampoules.
The dosage varies according to the sex, age and weight of the patient, the administration route, the nature of the therapeutic indication, or of any associated treatments, and ranges from 0.01 mg to 5 g per 24 hours in one or more administrations.
Furthermore, the present invention relates also to the combination of a compound of formula (I) with an anticancer agent selected from genotoxic agents, mitotic poisons, anti-metabolites, proteasome inhibitors, kinase inhibitors, signaling pathway inhibitors, phosphatase inhibitors, apoptosis inducers and antibodies, and also to pharmaceutical compositions comprising that type of combination and their use in the manufacture of medicaments for use in the treatment of cancer.
The combination of a compound of formula (I) with an anticancer agent may be administered simultaneously or sequentially. The administration route is preferably the oral route, and the corresponding pharmaceutical compositions may allow the instantaneous or delayed release of the active ingredients. The compounds of the combination may moreover be administered in the form of two separate pharmaceutical compositions, each containing one of the active ingredients, or in the form of a single pharmaceutical composition, in which the active ingredients are in admixture.
The compounds of the invention may also be used in combination with radiotherapy in the treatment of cancer.
nBu
nBuPAd2
tBu
The following Preparations and Examples illustrate the invention without limiting it in any way.
1 eq. of the appropriate halide derivative, 1.2 eq. tert-butyl N-[6-(tert-butoxycarbonylamino)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]carbamate (Preparation 1) and 3 eq. K2CO3 were dissolved in 1,2-dimethoxyethane-water 7:1 (8 mL/mmol). Then 0.05 eq. palladium acetate and 0.1 eq. nBuPAd2 were added and the mixture was heated at 100° C. under nitrogen in a microwave reactor until no further conversion was observed. Celite was added to the reaction mixture and the volatiles were evaporated under reduced pressure. The solid residue was purified via flash chromatography on silica gel using MeOH-containing 1% NH3— and DCM as eluents.
The product obtained in Step A was stirred in a mixture of DCM (5 mL/mmol) and TFA (5 mL/mmol) until no further conversion was observed. The volatiles were evaporated under reduced pressure, the solid residue was dissolved in ammonia solution (7N in methanol, 20 mL/mmol) and the volatiles were evaporated under reduced pressure again. The crude product was purified via preparative reversed phase chromatography using 5 mM aqueous NH4HCO3 solution and MeCN as eluents.
1.0 eq. of the appropriate amide (Preparation 2a, Preparation 2b, Preparation 2c or Preparation 2d) and 5.0 eq. 2,6-lutidine were dissolved in dry DCM (0.10 M solution for Preparation 2). The DCM solution was cooled to 0° C. under nitrogen and DCM solution of 5.0 eq. nonafluorobutanesulfonic anhydride (1.5 M) was added dropwise. The reaction mixture was allowed to warm up to room temperature over 1 hour then 5 eq. of the appropriate amine was added in one portion and the mixture was stirred until no further conversion was observed. The DCM mixture was washed with water, dried over Na2SO4, concentrated under reduced pressure and purified via flash chromatography using dichloromethane and methanolic ammonia as eluents to give the amidine intermediate.
1. eq. amidine intermediate from Step A was dissolved in 1,2-dimethoxyethane (0.15 M solution). 0.2 eq. Pd(OAc)2, 0.4 eq. PBuAd2, and 2 eq. K3PO4 were added and the reaction mixture was stirred under nitrogen at 115° C. in a microwave reactor until no further conversion was observed. The reaction mixture was concentrated under reduced pressure and purified via flash chromatography using dichloromethane and methanolic ammonia as eluents to yield the appropriate Boc-protected example.
Starting from the product of Step B following General procedure I Step B the appropriate example was obtained.
To the solution of 1 eq. of Preparation 3a or Preparation 3b in dry DMF (0.25 M) under nitrogen 3 eq. sodium hydride was added and the resulting mixture was stirred at 0° C. for 15 min. Following the addition of 2 eq. of the appropriate aryl halide the mixture was stirred at 50° C. for 5 hours. If formation of the expected product was not observed by HPLC-MS at this point the reaction temperature was raised to 120° C. and stirring continued until no further conversion was observed. After cooling water was added to the reaction mixture and the aqueous phase was extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous MgSO4 and solvent was removed under reduced pressure. The crude product was purified by flash chromatography using DCM and MeOH as eluents to give the Boc-protected example.
Starting from the product of Step A following General procedure I Step B the appropriate example was obtained.
1 eq. tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[3-(2-hydroxyethyl)-2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]carbamate (Preparation 3a), 2 eq. of the appropriate phenol derivative, 2 eq. PPh3, and 2 eq. ditertbutyl azodicarboxylate was dissolved in THF (10 mL/mmol of Preparation 3a). and the mixture was stirred at 60° C. until no further conversion was observed. Celite was added to the reaction mixture and the volatiles were evaporated under reduced pressure. The solid residue was purified via flash chromatography on silica gel using MeOH-containing 1% NH3— and DCM as eluents to give the appropriate Boc-protected example.
Starting from the product of Step A following General procedure I Step B the appropriate example was obtained.
The mixture of 1 eq. 2,6-dibromo-3-nitro-pyridine, 3.2 eq. K2CO3, and 1.05 eq. of the appropriate amine in 1,2-dichloroethane (0.17 M for the bromopyridine) was stirred at 50° C. until no further conversion was observed. Water was added to the mixture and the aqueous phase was separated and extracted three times with DCM. The combined organic layers were dried over MgSO4, solvent was removed under reduced pressure and the crude product was purified by flash chromatography using dichloromethane and methanol as eluents to give the appropriate 2-amino-3-nitro-6-bromopyridine.
1 eq. of the appropriate 2-amino-3-nitro-6-bromopyridine, 5 eq. Fe powder and 0.2 eq. NH4Cl were stirred in a mixture of EtOH and water (3:1, 0.1M for 2-amino-3-nitro-6-bromopyridine) at 90° C. until no further conversion was observed. The reaction mixture was filtered through celite, and the solvent was removed under reduced pressure to give the appropriate 2,3-diamino-6-bromopyridine that was used without further purification.
The mixture of 1 eq. of the appropriate N2-substituted 2,3-diamino-6-bromopyridine and 1.5 eq. CDI were stirred in dry THF (0.05M solution for 2,3-diamino-6-bromopyridine) until no further conversion was observed. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography using dichloromethane and methanol as eluents to give the appropriate 5-bromo-2-oxo-1H-imidazo[4,5-b]pyridine.
The mixture of 1 eq. 3-substituted-5-bromo-2-oxo-1H-imidazo[4,5-b]pyridine and POCl3 (5 ml) was stirred at 108° C. until no further conversion was observed. POCl3 was removed under reduced pressure. Dichloromethane and brine were added, organic phase was separated and the aqueous phase was extracted 2 times with dichloromethane. The combined organic layers were dried over MgSO4, the solvent was removed under reduced pressure and the crude product was purified by flash chromatography using dichloromethane and methanol as eluents to give the appropriate 3-substituted-5-bromo-2-chloro-imidazo[4,5-b]pyridine.
Starting from the appropriate 3-substituted-5-bromo-2-chloro-imidazo[4,5-b]pyridine and following the procedure described for Preparation 3a the appropriate 3-substituted-5-(2,6-bis(tert-butoxycarbamoyl)pyridin-4-yl)-2-chloro-imidazo[4,5-b]pyridine was obtained.
The mixture of 1 eq. of the appropriate 3-substituted-5-(2,6-bis(tert-butoxycarbamoyl)pyridin-4-yl)-2-chloro-imidazo[4,5-b]pyridine and 1.05 eq. tetraethylammonium cyanide was stirred in DMSO (0.03M solution for imidazopyridine) until no further conversion was observed. The reaction mixture was poured onto water, the solid was filtered off, and the aqueous phase was extracted with chloroform. The organic layers were combined, dried over anhydrous MgSO4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography using dichloromethane and ethyl acetate as eluents to give the appropriate 3-substituted-5-(2,6-bis(tert-butoxycarbamoyl)pyridin-4-yl)-2-cyano-imidazo[4,5-b]pyridine.
Starting from the product of Step F following General procedure I Step B the appropriate example was obtained.
1 eq. 6-chloro-2-methylamino-3-aminopyridine (Preparation 4) and 2.5 eq. of the appropriate acetic acid derivative were dissolved in toluene (1 mL/mmol) and the mixture was stirred at 85° C. until no further conversion was observed. The volatiles were evaporated under reduced pressure and the solid residue was purified via flash chromatography on silica gel using methanol and DCM as eluents to give 5-chloro-3-methyl-2-(trifluoromethyl)imidazo[4,5-b]pyridine
1H NMR (500 MHz, DMSO-d6) δ 8.39 (d, 1H), 7.57 (d, 1H), 3.95 (s, 3H) or 5-chloro-2-(difluoromethyl)-3-methyl-imidazo[4,5-b]pyridine—1H NMR (500 MHz, DMSO-d6) δ 8.27 (d, 1H), 7.46 (d, 1H), 7.44 (t, 1H), 3.9 (s, 3H).
1 eq. of the product obtained in Step A, 1.3 eq. tert-butyl N-[6-(tert-butoxycarbonylamino)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]carbamate (Preparation 1), and 2 eq. K3PO4 were dissolved in 1,2-dimethoxyethane (6 mL/mmol) then 0.1 eq Pd(PPh3)4 was added and the resulting mixture was heated at 100° C. under nitrogen using microwave irradiation until no further conversion was observed. Celite was added to the reaction mixture and the volatiles were evaporated under reduced pressure. The solid residue was purified via flash chromatography on silica gel using MeOH and DCM as eluents to give the Boc-protected example.
Starting from the product of Step B following General procedure I Step B the appropriate example was obtained.
1.05 eq. of the appropriate acid chloride derivative was added dropwise at −78° C. to the solution of 1 eq. 4-(3-butyl-2-methyl-3H-imidazo[4,5-b]pyridin-5-yl)pyridine-2,6-diamine (Example 148) and 3 eq. triethylamine in THF (16 mL/mmol of Example 148). The resulting mixture was allowed to warm up to room temperature and stirred until no further conversion was observed. The volatiles were evaporated under reduced pressure and the crude product was purified via preparative reversed phase chromatography using 5 mM aqueous NH4HCO3 solution and MeCN as eluents to give the appropriate example.
The mixture of 1 eq. 3-acetamino-2-fluoro-6-bromopyridine and 5 eq. of the appropriate amine in ethanol (2 M for the amine) was stirred at 50° C. until no further conversion was observed. Solvent and excess amine were removed under reduced pressure and the crude 3-acetamino-2-amino-6-bromopyridine derivative was used in the next step without further purification.
The solution of the crude 3-acetamino-2-amino-6-bromopyridine derivative in acetic acid (1.2 mL/mmol of starting 3-acetamino-2-fluoro-6-bromopyridine) was heated at 120° C. until no further conversion was observed. The solvent was removed under reduced pressure, the residue was taken up in EtOAc, the organic phase was washed with 10/o K2CO3, brine, it was dried over anhydrous MgSO4 and evaporated to dryness under reduced pressure. The crude product was purified by column chromatography using heptane and EtOAc as eluents to give the appropriate 3-substituted 5-bromo-2-methyl-imidazo[4,5-b]pyridine derivative.
To the solution of 1 eq. of the appropriate aryl halide derivative in 1,2-dimethoxyethane-water 7:1 (8 mL/mmol), 1.1 eq 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine, 3 eq. K3PO4, 0.05 eq. Pd(OAc)2, and 0.1 eq. nBuPAd2 were added, and the mixture was stirred at 90° C. under argon atmosphere until no further conversion was observed. The mixture was filtered through a pad of celite, the filtrate was concentrated under reduced pressure and purified via preparative reversed phase chromatography using 5 mM aqueous NH4HCO3 solution and MeCN as eluents to give the appropriate example.
A mixture of 1 eq. the appropriate 3-substituted 5-bromo-2-methyl-imidazo[4,5-b]pyridine derivative, 2.4 eq. 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane, 0.1 eq. Pd(OAc)2, 0.2 eq. bis(1-adamantyl)-butyl-phosphane, and 3 eq. K3PO4 was dispensed in 1,2-dimethoxyethane (0.25 M solution for the imidazopyridine derivative) and the resulting mixture was stirred at 90° C. under nitrogen atmosphere until no further conversion was observed. The reaction mixture was filtered through celite and the celite was washed with 1,2-dichloroethane. Organic layers were combined, dried over MgSO4, the solvent was removed under reduced pressure and the crude product was purified by flash chromatography using dichloromethane and methanol as eluents to give appropriate 3-substituted-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[4,5-b]pyridine.
A mixture of 1 eq. of the 3-substituted-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)imidazo[4,5-b]pyridine, 1.05 eq. of the 4-bromopyridine derivative, 0.1 eq. Pd(OAc)2, 0.2 eq. bis(1-adamantyl)-butyl-phosphane and 4 eq. K3PO4 was dispensed in 1,2-dimethoxyethane (0.17 M solution for the imidazopyridine derivative). The reaction mixture was stirred at 90° C. under nitrogen atmosphere until no further conversion was observed. The reaction mixture was filtered through celite and the celite was washed with 1,2-dichloroethane. Organic layers were combined, dried over MgSO4, the solvent was removed under reduced pressure and the crude product was purified by flash chromatography using dichloromethane and methanol as eluents to give the expected product.
109.7 g (4-bromo-6-tert-butoxycarbonylamino-pyridin-2-yl)-carbamicacid tert-butyl ester (283 mmol), prepared following J. Org. Chem. 2004, 69, 543-548, 107.7 g bis(pinacolato)diboron (424 mmol), 0.29 g Pd(OAc)2 (1.27 mmol), 0.70 g 1,1′-bis(diphenylphosphino)ferrocene (1.27 mmol) and 83.2 g KOAc (848 mmol) were added to 1100 mL previously degassed 1,4-dioxane, and the mixture was stirred at 80° C. under argon atmosphere until no further conversion was observed. Then the reaction mixture was filtered; the solid was washed with dioxane. 5.5 g charcoal was added to the filtrate, and it was-stirred for 2 minutes at reflux temperature. The mixture was filtered, washed with warm 1,4-dioxane and the volatiles were evaporated under reduced pressure. The residue was crystallised from tert-butyl-methyl-ether to give Preparation 1 as a white crystalline solid.
1H NMR (500 MHz, CDCl3) δ: 8.16 (brs, 2H), 7.92 (s, 2H), 1.54 (s, 18H), 1.34 (s, 12H).
31.4 g 6-bromo-2-chloro-pyridin-3-amine (151.3 mmol) was dissolved in 200 ml glacial acetic acid, 15 mL acetic anhydride (158.9 mmol) was added to this solution dropwise and the reaction mixture was stirred at room temperature until no further conversion was observed. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and the organic phase was washed with 10% aqueous K2CO3 and brine. Following drying over Na2SO4 removal of the solvents under reduced pressure gave N-(6-bromo-2-chloro-3-pyridyl)acetamide.
HPLC-MS: (M−H)=247.0; 249.0
13.1 g N-(6-bromo-2-chloro-3-pyridyl)acetamide (52.5 mmol), 24.0 g. tert-butyl N-[6-(tert-butoxycarbonylamino)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]carbamate (Preparation 1) (55.13 mmol) and 33.4 g K3PO4 (157.3 mmol) were dissolved in 1,2-dimethoxyethane-water 4:1 (250 mL). Then 304 mg tetrakis(triphenylphosphine)palladium(0) (0.26 mmol) was added and the mixture was heated under nitrogen at 90° C. until no further conversion was observed. Then the mixture was diluted with 250 mL water and extracted with EtOAc. The organic layer was dried over Na2SO4, the volatiles were removed under reduced pressure and the residue was recrystallized from EtOAc to obtain tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[6-chloro-5-(acetylamino)-2-pyridyl]-2-pyridyl]carbamate.
1H NMR (500 MHz, DMSO-d6) δ: 9.80 (s, 1H), 9.51 (s, 2H), 8.40 (d, 1H), 8.00 (s, 2H), 7.92 (d, 1H), 2.18 (s, 3H), 1.49 (s, 18H).
3.0 g 6-bromo-2-chloro-pyridin-3-amine (14.5 mmol) and 2.4 mL triethylamine (17.4 mmol) were dissolved in 60 mL DCM. The solution was cooled to 0° C. and 2.1 ml pentanoyl chloride (17.4 mmol) was added dropwise over 30 minutes. On completion of the addition the reaction mixture was allowed to warm up to room temperature where it was stirred until no further conversion was observed. The reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified via flash chromatography using DCM as eluent to give N-(6-bromo-2-chloro-3-pyridyl)pentanamide, a pale pink solid.
HPLC-MS: (M−H)=289.0; 291.0
Starting from N-(6-bromo-2-chloro-3-pyridyl)pentanamide following Step B of Preparation 2a, tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[6-chloro-5-(pentanoylamino)-2-pyridyl]-2-pyridyl]carbamate was obtained.
1H NMR (500 MHz, DMSO-d6) δ: 9.70 (s, 1H), 9.45 (s, 2H), 8.37 (d, 1H), 8.00 (s, 2H), 7.91 (d, 1H), 2.47 (t, 2H), 1.6 (m, 2H), 1.49 (s, 18H), 1.36 (m, 2H), 0.91 (t, 3H).
3.0 g 6-bromo-2-chloro-pyridin-3-amine (14.5 mmol) and 2.4 mL triethylamine (17.4 mmol), were dissolved in 60 ml DCM. The solution was cooled to 0° C. and 1.5 ml propanoyl chloride was added dropwise over 30 minutes. On completion of the addition the reaction mixture was allowed to warm up to room temperature where it was stirred until no further conversion was observed. The reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified via flash chromatography using DCM as eluent to give N-(6-bromo-2-chloro-3-pyridyl)propanamide.
HPLC-MS: (M−H)=261.0; 263.0
Starting from N-(6-bromo-2-chloro-3-pyridyl)propanamide following Step B of Preparation 2a, tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[6-chloro-5-(propanoylamino)-2-pyridyl]-2-pyridyl]carbamate was obtained.
1H NMR (500 MHz, DMSO-d6) δ: 9.68 (s, 1H), 9.45 (s, 2H), 8.40 (d, 1H), 8.00 (s, 2H), 7.91 (d, 1H), 2.48 (q, 2H), 1.49 (s, 18H), 1.11 (t, 3H).
3 g 6-bromo-2-chloro-pyridin-3-amine (14.46 mmol) and 2.4 ml triethylamine (17.35 mmol, 1.2 eq.), were dissolved in 60 ml DCM. This solution was cooled down to 0° C. and 1.8 ml (17.35 mmol, 1.2 eq.) butanoyl chloride was added dropwise over 30 minutes. On completion of the addition the reaction mixture was allowed to warm up to room temperature where it was stirred until no further conversion was observed. The reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The residue was purified via flash chromatography using DCM as eluent to give N-(6-bromo-2-chloro-3-pyridyl)butanamide.
HPLC-MS: (M−H)=275.0; 277.0
Starting from N-(6-bromo-2-chloro-3-pyridyl)butanamide following Step B of Preparation 2a, tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[6-chloro-5-(butiroylamino)-2-pyridyl]-2-pyridyl]carbamate was obtained.
1H NMR (500 MHz, DMSO-d6) δ: 9.70 (s, 1H), 9.46 (s, 2H), 8.37 (d, 1H), 8.00 (s, 2H), 7.91 (d, 1H), 2.45 (t, 2H), 1.64 (m, 2H), 1.49 (s, 18H), 0.95 (t, 3H).
1 eq. 5-chloro-3-(2-hydroxyethyl)-2-methyl-imidazo[4,5-b]pyridine, 1.1 eq. tert-butyl N-[6-(tert-butoxycarbonylamino)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]carbamate (Preparation 1), 0.1 eq. Pd(OAc)2, 0.2 eq. PBuAd2, and 3.0 eq. K2CO3 were suspended in DME (0.2 M) and the mixture was stirred under nitrogen at 100° C. in a microwave reactor until no further conversion was observed. The volatiles were removed under reduced pressure and the crude product was purified via flash chromatography using dichloromethane and methanolic ammonia as eluents to give tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[3-(2-hydroxyethyl)-2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]carbamate as a white solid.
1H NMR (400 MHz, DMSO-d6) δ: 9.34 (s, 2H), 8.03 (d, 1H), 8.02 (s, 2H), 7.68 (d, 1H), 4.97 (t, 1H), 4.34 (t, 2H), 3.81 (q, 2H), 2.63 (s, 3H), 1.49 (s, 18H).
1 eq. 3-amino-2-fluoropyridine was dissolved in DCM (0.6 M solution), 1.05 eq. N-bromosuccinimide was added and the reaction mixture was stirred at room temperature until no further conversion was observed. Water was added, the organic phase was separated, dried over anhydrous MgSO4 and concentrated under reduced pressure to give 3-amino-2-fluoro-6-bromopyridine.
1H NMR (400 MHz, DMSO-d6) δ: 7.22 (dd, 1H), 7.11 (dd, 1H), 5.62 (brs, 2H).
To a solution of 1 eq. 3-amino-2-fluoro-6-bromopyridine in acetic acid (0.9 M) 1.05 eq. acetic anhydride was added and the reaction mixture was stirred at room temperature until no further conversion was observed. The solvents were removed reduced pressure, the crude product was dissolved in DCM and washed with 10% K2CO3. The organic layer was dried over anhydrous MgSO4, and concentrated under reduced pressure to give 3-acetamino-2-fluoro-6-bromopyridine.
1H NMR (400 MHz, DMSO-d6) δ: 10.03 (brs, 1H), 8.42 (dd, 1H), 7.56 (d, 1H), 2.11 (s, 3H).
The mixture of 1 eq. 3-acetamino-2-fluoro-6-bromopyridine, 2.2 eq. 1-aminopropan-2-ol, and triethylamine (0.27 mL/mmol of the fluoropyridine) was stirred at 60° C. until no further conversion was observed. Solvent and excess amine were removed under reduced pressure and the crude 3-acetamino-2-(2-hydroxypropylamino)-6-bromopyridine was used in the next step without purification.
MS: (M+H)+=288.2
The solution of 1 eq. 3-acetamino-2-(2-hydroxypropylamino)-6-bromopyridine in acetic acid (13.6 mL/g of the crude amide) was heated at 130° C. until no further conversion was observed. The solvent was removed under reduced pressure, the residue was taken up in methanol:water (5:1, 7 mL/g of residue) containing LiOH*H2O (0.27 g/g of residue) and the mixture was stirred at ambient temperature for 2 hours than poured into water. The precipitate was filtered off, washed with water and dried to give 5-bromo-3-(2-hydroxypropyl)-2-methyl-imidazo[4,5-b]pyridine.
1H NMR (400 MHz, DMSO-d6) δ: 7.87 (d, 1H), 7.36 (d, 1H), 4.97 (d, 1H), 4.19-3.94 (m, 3H), 2.57 (s, 3H), 1.21 (d, 3H).
1 eq. S-bromo-3-(2-hydroxypropyl)-2-methyl-imidazo[4,5-b]pyridine, 1.0 eq. tert-butyl N-[6-(tert-butoxycarbonylamino)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]carbamate (Preparation 1), 0.05 eq. tetrakis(triphenylphosphine)palladium(0), and 3 eq. K3PO4 were suspended in DME (5 mL/mmol for the bromo compound) and the mixture was stirred under nitrogen at 100° C. in a microwave reactor until no further conversion was observed. The volatiles were removed under reduced pressure and the crude product was purified via flash chromatography using dichloromethane and methanolic ammonia as eluents to give tert-butyl N-[6-(tert-butoxycarbonylamino)-4-[3-(2-hydroxypropyl)-2-methyl-imidazo[4,5-b]pyridin-5-yl]-2-pyridyl]carbamate as a white solid.
1H NMR (500 MHz, DMSO-d6) δ: 9.42 (s, 2H), 8.10 (s, 2H), 8.03 (d, 11H), 7.70 (d, 1H), 5.03 (d, 1H), 4.28 (dd, 1H), 4.18 (m, 1H), 4.10 (dd, 1H), 2.64 (s, 3H), 1.50 (s, 18H), 1.19 (d, 3H).
3.86 g 2,6-dichloro-3-nitro-pyridine (20 mmol) was dissolved in 80 ml DCM, 6.9 g K2CO (50 mmol, 2.5 eq.) was added and the reaction mixture was cooled down to −20° C. At this temperature 3.1 ml of methylamine (33% solution in ethanol, 28.6 mmol, 1.43 eq.) was added dropwise then cooling was stopped and the reaction mixture was allowed to warm up to ambient temperature where it was stirred until no further conversion was observed. The reaction mixture was filtered, the filtrate was washed with water, the organic layer was dried on MgSO4 then concentrated under reduced pressure to give 6-chloro-N-methyl-3-nitro-pyridin-2-amine as a solid.
1H NMR (500 MHz, DMSO-d6) δ: 8.72 (d, 1H), 8.42 (d, 1H), 6.77 (d, 1H), 3.08 (d, 3H).
3.0 g 6-chloro-N-methyl-3-nitro-pyridin-2-amine (16 mmol) was dissolved in the mixture of 30 ml ethanol and 15 ml water then 4.47 g iron powder (80 mmol, 5 eq.) was added. To this mixture 1.2 ml glacial acetic acid was added dropwise then the reaction mixture was refluxed until no further conversion was observed. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure and the residue was purified via flash chromatography using DCM as eluent to give 6-chloro-2-methylamino-3-aminopyridine. MS (M+H)=158.2
8.35 mL triethylamine (6.07 g, 60.0 mmol) and 8.36 g triphenylmethyl chloride (30.0 mmol) were added at room temperature to a stirred solution of 2.96 g 4-(3-butyl-2-methyl-3H-imidazo[4,5-b]pyridin-5-yl)pyridine-2,6-diamine (Example 148) (10.0 mmol) in 100 mL THF and the mixture was stirred until no further conversion was observed. The volatiles were evaporated under reduced pressure and the crude product was purified via reversed phase flash chromatography using water and MeCN as eluents to obtain 4-(3-butyl-2-methyl-imidazo[4,5-b]pyridin-5-yl)-N2,N6-di(triphenylmethyl)-pyridine-2,6-diamine as an intermediate. This intermediate was dissolved in 400 mL methanol, 20 mL TFA was added at room temperature and the mixture was stirred at room temperature until the total amount of the bis-triphenylmethylated intermediate was converted to the desired product. Then 16.0 g NH4HCO3 (202.4 mmol) was added with stirring and the formed precipitate was removed by filtration to obtain the crude product, which was recrystallized from methanol to give 4-(3-butyl-2-methyl-imidazo[4,5-b]pyridin-5-yl)-N-triphenylmethyl-pyridine-2,6-diamine (Preparation 5).
1H NMR (500 MHz, CDCl3) δ: 7.84 (d, 1H), 7.42-7.35 (m, 6H), 7.33-7.25 (m, 6H), 7.23-7.16 (m, 3H), 7.20 (d, 1H), 6.33 (s, 1H), 6.26 (brs, 1H), 6.03 (s, 1H), 5.45 (brs, 2H), 4.12 (t, 2H), 2.58 (s, 3H), 1.64 (m, 2H), 1.16 (m, 2H), 0.85 (t, 3H).
Following General procedure VIII and using butylamine as the appropriate amine derivative 5-bromo-3-butyl-2-methyl-imidazo[4,5-b]pyridine was obtained.
1H NMR (500 MHz, DMSO-d6) δ: 7.89 (d, 1H), 7.38 (d, 1H), 4.18 (t, 2H), 2.58 (s, 3H), 1.71 (quint, 2H), 1.38-1.22 (m, 2H), 0.91 (t, 3H).
Following General procedure VIII and using (2-aminoethyl)-cyclohexane as the appropriate amine derivative 5-bromo-3-(2-cyclohexylethyl)-2-methyl-imidazo[4,5-b]pyridine was obtained.
1H NMR (500 MHz, DMSO-d6) δ: 7.88 (d, 1H), 7.38 (d, 1H), 4.19 (t, 2H), 2.57 (s, 3H), 1.79 (d, 2H), 1.66 (d, 2H), 1.62-1.57 (m, 1H), 1.59 (q, 2H), 1.29-1.08 (m, 4H), 0.94 (q, 2H).
Following General procedure VIII and using cyclopropyl-methylamine as the appropriate amine derivative 5-bromo-3-(cyclopropylmethyl)-2-methyl-imidazo[4,5-b]pyridine was obtained.
1H NMR (400 MHz, DMSO-d6) δ: 7.77 (d, 1H), 7.31 (d, 1H), 4.10 (d, 2H), 2.66 (s, 3H), m 1.33-1.19 (m, 1H), 0.64-0.43 (m, 4H).
Following General procedure VIII and using 1-amino-3-butene as the appropriate amine derivative 5-bromo-3-but-3-enyl-2-methyl-imidazo[4,5-b]pyridine was obtained.
1H NMR (500 MHz, DMSO-d6) δ: 7.88 (d, 1H), 7.38 (d, 1H), 5.86-5.71 (m, 1H), 5.01-4.94 (m, 2H), 4.26 (t, 2H), 2.57 (s, 3H), 2.52 (q, 2H).
Following General procedure VIII and using 3,3-difluoro-cyclobutanamine as the appropriate amine derivative 5-bromo-3-(3,3-difluorocyclobutyl)-2-methyl-imidazo[4,5-b]pyridine was obtained.
1H NMR (500 MHz, DMSO-d6) δ: 7.91 (d, 1H), 7.42 (d, 1H), 5.05-4.92 (m, 1H), 3.88-3.69 (m, 2H), 3.22-3.09 (m, 2H), 2.60 (s, 3H).
Following General procedure VIII and using cyclopropylamine as the appropriate amine derivative 5-bromo-3-cyclopropyl-2-methyl-imidazo[4,5-b]pyridine was obtained.
1H NMR (500 MHz, DMSO-d6) δ: 7.86 (d, 1H), 7.37 (d, 1H), 3.33-3.28 (m, 1H), 2.60 (s, 3H), 1.18-1.11 (m, 4H).
Starting from 6-chloro-3-cyclopentyl-2-methyl-imidazo[4,5-b]pyridine as the appropriate halide and following General procedure I Example 1 was obtained. HRMS (TOF, ESI) m/z: Calcd for C17H20N6 308.1749, Found: 309.1821 [M+H]+.
Starting from 3-propyl-6-chloro-2-methyl-imidazo[4,5-b]pyridine as the appropriate halide and following General procedure I Example 2 was obtained. HRMS (TOF, ESI) m/z: Calcd for C15H18N6 282.1593, Found: 283.1662 [M+H]+.
Starting from 6-chloro-3-(2-hydroxyethyl)-2-methyl-imidazo[4,5-b]pyridine as the appropriate halide and following General procedure I Example 3 was obtained. HRMS (TOF, ESI) m/z: Calcd for C14H16N6O 284.1386, Found: 285.1473 [M+H]+.
Starting from 6-chloro-2,3-dimethyl-imidazo[4,5-b]pyridine as the appropriate halide and following General procedure 1 Example 4 was obtained. HRMS (TOF, ESI) m/z: Calcd for C13H14N6 254.1280, Found: 255.1361 [M+H]+.
Starting from 6-chloro-3-(4-pyridylmethyl)-2-methyl-imidazo[4,5-b]pyridine as the appropriate halide and following General procedure I Example 5 was obtained. HRMS (TOF, ESI) m/z: Calcd for C18H17N7 331.1545, Found: 332.1623 [M+H]+.
Starting from 6-chloro-3-(2-pyridylmethyl)-2-methyl-imidazo[4,5-b]pyridine as the appropriate halide and following General procedure I Example 6 was obtained. HRMS (TOF, ESI) m/z: Calcd for C18H17N7 331.1545, Found: 332.1625 [M+H]+.
Starting from 6-chloro-3-(3-pyridylmethyl)-2-methyl-imidazo[4,5-b]pyridine as the appropriate halide and following General procedure I Example 7 was obtained. HRMS (TOF, ESI) m/z: Calcd for C18H17N7 331.1545, Found: 332.1625 [M+H]+.
Starting from 3-benzyl-6-chloro-2-methyl-imidazo[4,5-b]pyridine as the appropriate halide and following General procedure I Example 8 was obtained. HRMS (TOF, ESI) m/z: Calcd for C19H18N6 330.1593, Found: 331.1673 [M+H]+.
Starting from Preparation 2a following General procedure II and using cyclopropylamine as the appropriate amine Example 9 was obtained. HRMS (TOF, ESI) m/z: Calcd for C15H16N6 280.1436, Found: 281.1518 [M+H]+.
Starting from Preparation 2a following General procedure II and using 4-fluorobenzylamine as the appropriate amine Example 10 was obtained. HRMS (TOF, ESI) m/z: Calcd for C19H17N6F 348.1499, Found: 349.1565 [M+H]+.
Starting from Preparation 2a following General procedure II and using cyclopropylmethylamine as the appropriate amine Example 11 was obtained. HRMS (TOF, ESI) m/z: Calcd for C16H18N6 294.1593, Found: 295.1665 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2,3-dihydro-1H-inden-2-amine as the appropriate amine Example 12 was obtained. HRMS (TOF, ESI) m/z: Calcd for C21H20N6 356.1749, Found: 357.1822 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-(3-aminopropyl)pyrrolidin-2-one as the appropriate amine Example 13 was obtained. HRMS (TOF, ESI) m/z: Calcd for C19H23N7O 365.1964, Found: 366.2035 [M+H]+.
Starting from Preparation 2a following General procedure II and using 4-amino-1-butene as the appropriate amine Example 14 was obtained. HRMS (TOF, ESI) m/z: Calcd for C16H18N6 294.1593, Found: 295.1672 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using (2-aminoethyl)-cyclohexane as the appropriate amine Example 15 was obtained. HRMS (TOF, ESI) m/z: Calcd for C20H26N6 350.2219, Found: 351.2298 [M+H]+.
Starting from Preparation 2a following General procedure II and using (1S,2S,3S,5R)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-amine as the appropriate amine Example 16 was obtained. HRMS (TOF, ESI) m/z: Calcd for C22H28N6 376.2375, Found: 377.2456 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-cyclopropylethanamine as the appropriate amine Example 17 was obtained. HRMS (TOF, ESI) m/z: Calcd for C17H20N6 308.1749, Found: 309.1828 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using 2-ethylbutan-1-amine as the appropriate amine Example 18 was obtained. HRMS (TOF, ESI) m/z: Calcd for C18H24N6 324.2062, Found: 325.2139 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(2-furyl)ethanamine as the appropriate amine Example 19 was obtained. HRMS (TOF, ESI) m/z: Calcd for C18H18N6O 334.1542, Found: 335.1618 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-thienylmethanamine as the appropriate amine Example 20 was obtained. HRMS (TOF, ESI) m/z: Calcd for C17H16N6S 336.1157, Found: 337.1224 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(1-phenylpyrazol-4-yl)ethanamine as the appropriate amine Example 21 was obtained. HRMS (TOF, ESI) m/z: Calcd for C23H22N8 410.1967, Found: 411.2038 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-adamantylmethanamine as the appropriate amine Example 22 was obtained. HRMS (TOF, ESI) m/z: Calcd for C23H28N6 388.2375, Found: 389.2542 [M+H]+.
Starting from Preparation 2a following General procedure II and using cyclobutylamine as the appropriate amine Example 23 was obtained. HRMS (TOF, ESI) m/z: Calcd for C16H18N6 294.1593, Found: 295.1665 [M+H]+.
Starting from Preparation 2a following General procedure II and using N-[4-(2-aminoethyl)phenyl]acetamide as the appropriate amine Example 24 was obtained. HRMS (TOF, ESI) m/z: Calcd for C22H23N7O 401.1964, Found: 402.2039 [M+H]+.
Starting from Preparation 2a following General procedure II and using tert-butylamine as the appropriate amine Example 25 was obtained. HRMS (TOF, ESI) m/z: Calcd for C16H20N6 296.1479, Found: 240.1125 [M+H—C4H8]+. Fragment ion formula: C12H12N6 no molecular ion was detected due to extensive fragmentation.
Starting from Preparation 2a following General procedure II and using 2-thienylethaneamine as the appropriate amine Example 26 was obtained. HRMS (TOF, ESI) m/z: Calcd for C18H18N6S 350.1314, Found: 351.1385 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(naphthalen-1-yloxy)ethanamine as the appropriate amine Example 27 was obtained. HRMS (TOF, ESI) m/z: Calcd for C24H22N6O 410.1855, Found: 411.1923 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2,2,2-trifluoroethylamine as the appropriate amine Example 28 was obtained. HRMS (TOF, ESI) m/z: Calcd for C14H13N6F3 322.1154, Found: 323.1238 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3-thienylethaneamine as the appropriate amine Example 29 was obtained. HRMS (TOF, ESI) m/z: Calcd for C18H18N6S 350.1314, Found: 351.1379 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2,2-dimethylpropan-1-amine as the appropriate amine Example 30 was obtained. HRMS (TOF, ESI) m/z: Calcd for C17H22N6 310.1906, Found: 311.2010 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using 2-methylpropan-1-amine as the appropriate amine Example 31 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H20N6 296.1749, Found: 297.1824 [M+H]+.
Starting from Preparation 2a following General procedure II and using (1R)-1-(2-methyl-4-pyridyl)ethanamine as the appropriate amine Example 32 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C20H21N7 359.1858, Found: 360.1934 [M+H]+.
Starting from Preparation 2a following General procedure II and using butan-2-amine as the appropriate amine Example 33 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for Cl6H20N6 296.1749, Found: 297.1826 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-methylbutan-1-amine as the appropriate amine Example 34 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C17H22N6 310.1906, Found: 311.1983 [M+H]+.
Starting from Preparation 2a following General procedure II and using ethyl 4-aminopiperidine-1-carboxylate as the appropriate amine Example 35 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H25N7O2 395.2070, Found: 396.2152 [M+H]+.
Starting from Preparation 2a following General procedure II and using 5,6,7,8-tetrahydroquinolin-5-amine as the appropriate amine Example 36 was obtained. HRMS (IT-TOF, ESI) m/2: Calculated for C21H21N7 371.1858, Found: 372.1939 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3-aminopropane-1,2-diol as the appropriate amine Example 37 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C15H18N6O2 314.1491, Found: 315.1559 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-(4-amino-1-piperidyl)-2-methyl-propan-1-one as the appropriate amine Example 38 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C21H27N7O 393.2277, Found: 394.2356 [M+H]+.
Starting from Preparation 2a following General procedure II and using (4-chloro-2-methoxy-phenyl)methanamine as the appropriate amine Example 39 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C20H19N6OCl 394.1309, Found: 395.1387 [M+H]+.
Starting from Preparation 2a following General procedure II and using 4-(aminomethyl)benzonitrile as the appropriate amine Example 40 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C20H17N7 355.1545, Found: 356.1612 [M+H]+.
Starting from Preparation 2a following General procedure II and using tetrahydrofuran-3-ylmethanamine as the appropriate amine Example 41 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C17H20N6O 324.1699, Found: 325.1787 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using 3-furylmethanamine as the appropriate amine Example 42 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C17H16N6O 320.1386, Found: 321.1467 [M+H]+.
Starting from Preparation 2a following General procedure II and using [4-(difluoromethoxy)phenyl]methanamine as the appropriate amine Example 43 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C20H18N6OF2 396.1510, Found: 397.1581 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3-methylsulfanylpropan-1-amine as the appropriate amine Example 44 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C16H20N6S 328.1470, Found: 329.1551 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using (1,3,5-trimethyl-1H-pyrazol-4-yl)methanamine as the appropriate amine Example 45 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H22N8 362.1967, Found: 363.2037 [M+H]+.
Starting from Preparation 2a following General procedure II and using (2,5-difluorophenyl)methanamine as the appropriate amine Example 46 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H16N6F2 366.1405, Found: 367.1483 [M+H]+.
Starting from Preparation 2a following General procedure II and using (2-chlorophenyl)methanamine as the appropriate amine Example 47 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H17N6Cl 364.1203, Found: 365.1279 [M+H]+.
Starting from Preparation 2a following General procedure I and using (3-chlorophenyl)methanamine as the appropriate amine Example 48 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H17N6Cl 364.1203, Found: 365.1277 [M+H]+.
Starting from Preparation 2a following General procedure II and using tetrahydro-2H-pyran-4-amine as the appropriate amine Example 49 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H20N6O 324.12699, Found: 325.1790 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using [2-fluoro-5-(trifluoromethoxy)phenyl]methanamine as the appropriate amine Example 50 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H16N6OF4 432.1322, Found: 433.1416 [M+H]+.
Starting from Preparation 2a following General procedure II and using prop-2-en-1-amine as the appropriate amine Example 51 was obtained. HRMS (TOF, ESI) m/z: Calculated for C15H16N6 280.1436, Found: 281.1524 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3,3-dimethylbutan-1-amine as the appropriate amine Example 52 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H24N6 324.2062, Found: 325.2153 [M+H]+.
Starting from Preparation 2a following General procedure II and using propan-2-amine as the appropriate amine Example 53 was obtained. HRMS (TOF, ESI) m/z: Calculated for C15H18N6 282.1593, Found: 283.1675 [M+H]+.
Starting from Preparation 2a following General procedure II and using cyclohexanamine as the appropriate amine Example 54 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H22N6 323.1921, Found: 323.1994 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-cyclohexylmethanamine as the appropriate amine Example 55 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H24N6 336.2062, Found: 337.2151 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-adamantylamine as the appropriate amine Example 56 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H26N6 [M+H]+ 374.2219, Found: 375.2307.
Starting from Preparation 2a following General procedure II and using (2,5-dichlorophenyl)methanamine as the appropriate amine Example 57 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H16N6Cl2 398.0814, Found: 399.0896 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using 2-(3,4-dichlorophenyl)ethanamine as the appropriate amine Example 58 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H18N6Cl2 412.0970, Found: 413.1053 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(2,4-dichlorophenyl)ethanamine as the appropriate amine Example 59 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H18N6Cl2 412.097, Found: 413.1056 [M+H]+.
and
Starting from Preparation 2a following General procedure II and using 2-phenylpropan-2-amine as the appropriate amine Example 60 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N6 358.1906, Found: 359.1988 [M+H]+ From the same reaction Example 61 was also isolated. HRMS (TOF, ESI) m/z: Calculated for C12H12N6 240.1123, Found: 241.1206. [M+H]+.
Starting from Preparation 2a following General procedure II and using 1,2,3,4-tetrahydronaphthalen-1-amine as the appropriate amine Example 62 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1989 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using 2-(o-tolyl)ethanamine as the appropriate amine Example 63 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N6 358.1906, Found: 359.1985 [M+H]+.
Starting from Preparation 2a following General procedure II and using pentan-2-amine as the appropriate amine Example 64 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H22N6 310.1906, Found: 311.1980 [M+H]+.
Starting from Preparation 2a following General procedure II and using pentan-1-amine as the appropriate amine Example 65 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H22N6 310.1906, Found: 311.1983 [M+H]+.
Starting from Preparation 2a following General procedure II and using tetrahydro-2H-thiopyran-4-amine as the appropriate amine Example 66 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H20N6S 340.147, Found: 341.1545 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-phenylpropan-1-amine as the appropriate amine Example 67 was obtained. HRMS (TOF, ESI) m/z: Calculated for C211H22N6 358.1906, Found: 359.1979 [M+H]+.
Starting from Preparation 2a following General procedure II and using pentan-3-amine as the appropriate amine Example 68 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H22N6 310.1906, Found: 311.1985 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3-(2-methoxyphenyl)propan-1-amine as the appropriate amine Example 69 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H24N6O 388.2012, Found: 389.2095 [M+H]+.
Starting from Preparation 2a following General procedure II and using 4-aminobutan-1-ol as the appropriate amine Example 70 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H20N6O 312.1699, Found: 313.1767 [M+H]+.
Starting from Preparation 2a following General procedure II and using 4,4,4-trifluorobutan-1-amine as the appropriate amine Example 71 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H17N6F3 350.1467, Found: 351.1533 [M+H]+.
Starting from Preparation 2a following General procedure II and using (2-methoxy-4-pyridyl)methanamine as the appropriate amine Example 72 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H19N7O 361.1651, Found: 362.1726 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(1,3-benzodioxol-5-yl)ethanamine as the appropriate amine Example 73 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H20N6O2 388.1648, Found: 389.1728 [M+H]+.
Starting from Preparation 2a following General procedure II and using (2,2-dichlorocyclopropyl)methanamine as the appropriate amine Example 74 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H16N6Cl2 362.0814, Found: 363.0883 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3-methylbutan-1-amine as the appropriate amine Example 75 was obtained. HRMS (TOF, ESI) m/z: Calculated for C1H22N6 310.1906, Found: 311.1990 [M+H]+.
Starting from Preparation 2a following General procedure II and using tetrahydro-2H-pyran-3-ylmethanamine as the appropriate amine Example 76 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H22N6O 338.1855, Found: 339.1941 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(2,3-dihydro-1,4-benzodioxin-6-yl)ethanamine as the appropriate amine Example 77 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H22N6O2 402.1804, Found: 403.1888 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-tetrahydro-2H-pyran-4-ylethanamine as the appropriate amine Example 78 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H24N6O 352.2012, Found: 353.2080 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-[2-(trifluoromethyl)phenyl]ethanamine as the appropriate amine Example 79 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H19N6F3 412.1623, Found: 413.1708 [M+H]+.
Starting from Preparation 2a following General procedure II and using tetrahydro-2H-pyran-4-ylmethanamine as the appropriate amine Example 80 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H22N6O 338.1855, Found: 339.1929 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(2-methoxyphenyl)ethanamine as the appropriate amine Example 81 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O 374.1855, Found: 375.1924 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-(2-furyl)propan-2-amine as the appropriate amine Example 82 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H20N6O 348.1699, Found: 349.1772 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-phenyl-ethylamine as the appropriate amine Example 83 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H20N6 344.1749, Found: 345.1829 [M+H]+.
Starting from Preparation 2a following General procedure II and using (2-fluoro-4-pyridyl)methanamine as the appropriate amine Example 84 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H16N7F 349.1451, Found: 350.1531 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-tetrahydrofuran-2-ylethanamine as the appropriate amine Example 85 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H22N6O 338.1855, Found: 339.1930 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-(2-methylcyclopropyl)methanamine as the appropriate amine Example 86 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H20N6 308.1749, Found: 309.1822 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(3-isopropoxyphenyl)ethanamine as the appropriate amine Example 87 was obtained. HRMS (TOF, ESI) m/z: Calculated for C23H26N6O 402.2168, Found: 403.2153 [M+H]+.
Starting from Preparation 2a following General procedure I and using 1-(1-ethylcyclopropyl)methanamine as the appropriate amine Example 88 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H22N6 322.1906, Found: 323.1988 [M+H]+.
Starting from Preparation 2a following General procedure II and using cyclopentylmethanamine as the appropriate amine Example 89 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H22N6 322.1906, Found: 323.1987 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(3-ethoxyphenyl)ethanamine as the appropriate amine Example 90 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H24N6O 388.2012, Found: 389.1953 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-[3-(trifluoromethyl)phenyl]ethanamine as the appropriate amine Example 91 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H19N6F3 412.1623, Found: 413.1683 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-cyclopentylethanamine as the appropriate amine Example 92 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H24N6 336.2062, Found: 337.2012 [M+H]+.
Starting from Preparation 2a following General procedure II and using 5-methoxytetralin-2-amine as the appropriate amine Example 93 was obtained. HRMS (TOF, ESI) m/z: Calculated for C23H24N6O 400.2012, Found: 401.1963 [M+H]+.
Starting from Preparation 2a following General procedure II and using hexan-1-amine as the appropriate amine Example 94 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H24N6 324.2062, Found: 325.2014 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using 2-(2-methoxycyclohexyl)ethanamine as the appropriate amine Example 95 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H28N6O 380.2325, Found: 381.2378 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(4-fluorophenyl)ethanamine as the appropriate amine Example 96 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H19N6F 362.1655, Found: 363.1726 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-(2-phenylcyclopropyl)methanamine as the appropriate amine Example 97 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1976 [M+H]+.
Starting from Preparation 2a following General procedure II and using 5-methoxyindan-2-amine as the appropriate amine Example 98 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H22N6O 386.1855, Found: 387.1818 [M+H]+.
Starting from Preparation 2a following General procedure II and using 1-(2,2-dimethylcyclopropyl)methanamine as the appropriate amine Example 99 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H22N6 322.1906, Found: 323.1978 [M+H]+.
Starting from Preparation 2a following General procedure 11 and using 3-phenylcyclobutanamine as the appropriate amine Example 100 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1978 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3,3-difluorocyclobutanamine as the appropriate amine Example 101 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H16N6F2 330.1405, Found: 331.1463 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(2-methylcyclohexyl)ethanamine as the appropriate amine Example 102 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H28N6 364.2375, Found: 365.2447 and 365.2436 [M+H]+ for the two diastereoisomers.
Starting from Preparation 2a following General procedure II and using 3-fluorocyclobutanamine as the appropriate amine Example 103 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H17N6F 312.1499, Found: 313.1566 [M+H]+.
and
Starting from Preparation 2a following General procedure II and using 1-phenoxypropan-2-amine as the appropriate amine a mixture of Example 104 and Example 105 was obtained. The enantiomers were separated on CHIRALCEL OK column using MeOH+0.1% DEA as eluent to obtain Example 104 as the first eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O 374.1855, Found: 375.1913 [M+H]+ ee=99.8% (E1). Example 105 was obtained as the second eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O 374.1844, Found: 375.1917 [M+H]+ ee=98.4% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-phenoxypropan-1-amine as the appropriate amine a mixture of Example 106 and Example 107 was obtained. The enantiomers were separated on CHIRALCEL OK column using MeOH+0.1% DEA as eluent to obtain Example 106 as the first eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O 374.1855, Found: 375.1924. [M+H]+ ee=99.8% (E1). Example 107 was obtained as the second eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O 374.1855, Found: 375.1922 [M+H]+ ee=99.8% (E2).
Starting from Preparation 2a following General procedure II and using 3,3,3-trifluoropropan-1-amine as the appropriate amine Example 108 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C15H15N6F3 336.131, Found: 337.1381 [M+H]+.
and
Starting from Preparation 2a following General procedure II and using [1,2-trans-2-phenylcyclopropyl]methanamine as the appropriate amine a mixture of Example 109 and Example 110 was obtained. The enantiomers were separated on CHIRALCEL OD-H column using 40:60 l-PrOH/heptane+0.1% DEA as eluent to obtain Example 109 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1981 [M+H]+ ee=99.8% (E1). Example 110 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1984 [M+H]+ ee=99.8% (E2).
Starting from Preparation 2a following General procedure 11 and using (E)-3-phenylprop-2-en-1-amine as the appropriate amine Example 111 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C21H20N6 356.1749, Found: 357.1828 [M+H]+.
and
Starting from Preparation 2a following General procedure II and using 1-(bicyclo[4.2.0]octa-1,3,5-trien-7-ylmethanamine as the appropriate amine a mixture of Example 112 and Example 113 was obtained. The enantiomers were separated on CHIRALPAK AS-H column using 50:50 EtOH/heptane+0.1% DEA as eluent to obtain Example 112 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H20N6 356.1749, Found: 357.1818 [M+H]+ ee =99.8% (E1). Example 113 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H20N6 356.1749, Found: 357.1810 [M+H]+ ee 99.6% (E2).
Starting from Preparation 2a following General procedure II and using indan-2-ylmethanamine as the appropriate amine Example 114 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1962 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2,2-difluoroethanamine as the appropriate amine Example 115 was obtained. HRMS (TOF, ESI) m/z: Calculated for C14H14N6F2 304.1248, Found: 305.1318 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-methyl-2-phenoxy-propan-1-amine as the appropriate amine Example 116 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C22H24N6O 388.2012, Found: 389.2069 [M+H]+.
and
Starting from Preparation 2a following General procedure 11 and using 2-(2-chlorophenoxy)propan-1-amine as the appropriate amine a mixture of Example 0.117 and Example 118 was obtained. The enantiomers were separated on CHIRALPAK AS-H column using 50:50 EtOH/heptane+0.1% DEA as eluent to obtain Example 117 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H21N6OCl 408.1465, Found: 409.1558 [M+H]+ ee=99.8% (E1). Example 118 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H21N6OCl 408.1465, Found: 409.1538 [M+H]+ ee=99.8% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-phenoxybutan-1-amine as the appropriate amine a mixture of Example 119 and Example 120 was obtained. The enantiomers were separated on CHIRALPAK AS-V column using 40:60 EtOH/heptane+0.05% DEA as eluent to obtain Example 119 as the first eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C22H24N6O 388.2012, Found: 389.2084 [M+H]+ ee=99.8% (E1). Example 120 was obtained as the second eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C2H24N6O 388.2012, Found: 389.2093 [M+H]+ ee=99.2% (E2).
and
Starting from Preparation 2a following General procedure II and using N2-methyl-N2-phenyl-propane-1,2-diamine as the appropriate amine a mixture of Example 121 and Example 122 was obtained. The enantiomers were separated on CHIRALPAK LA column using 20:80 EtOH/heptane+0.1% DEA as eluent to obtain Example 122 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H25N7 387.2171, Found: 388.2253 [M+H]+ ee=99.8% (E1). Example 121 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H25N7 387.2171, Found: 388.2232 [M+H]+ ee=99.8% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-(3-fluorophenoxy)propan-1-amine as the appropriate amine a mixture of Example 123 and Example 124 was obtained. The enantiomers were separated on CHIRALCEL OJ-H column using EtOH+0.1% DEA as eluent to obtain Example 123 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H21N6OF 392.1761, Found: 393.1850 [M+H]+ ee=99.8% (E1). Example 124 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H21N6OF 392.1761, Found: 393.1828 [M+H]+ ee=99.8% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-(3-methoxyphenoxy)propan-1-amine as the appropriate amine a mixture of Example 125 and Example 126 was obtained. The enantiomers were separated on CHIRALPAK AS-H column to obtain Example 125 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H24N6O2 404.1961, Found: 405.2040 [M+H]+ ee=99.8% (E1). Example 126 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H24N6O2 404.1961, Found: 405.2048 [M+H]+=99.6% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-(3-methylphenoxy)propan-1-amine as the appropriate amine a mixture of Example 127 and Example 128 was obtained. The enantiomers were separated on CHIRALPAK AS-V column using 50:50 EtOH/heptane+0.05% DEA as eluent to obtain Example 127 as the 34) first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H24N6O 388.2012, Found: 389.2088. [M+H]+ ee=99.8% (E1). Example 128 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H24NO6O 388.2012, Found: 389.2079. [M+H]+ ee=99.2% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-(4-fluorophenoxy)propan-1-amine as the appropriate amine a mixture of Example 129 and Example 130 was obtained. The enantiomers were separated on CHIRALPAK AS-V column using 50:50 EtOH/heptane+0.05% DEA as eluent to obtain Example 129 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H21N6OF 392.1761, Found: 393.1832. [M+H]+ ee=99.8% (E1). Example 130 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H21N6OF 392.1761, Found: 393.1834. [M+H]+ ee=99.8% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-(2-methylphenoxy)propan-1-amine as the appropriate amine a mixture of Example 131 and Example 132 was obtained. The enantiomers were separated on OJ column using EtOH+0.05% DEA as eluent to obtain Example 131 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C2H24N6O 388.2012, Found: 389.2103 [M+H]+ ee=99.8% (E1). Example 132 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C22H24N6O 388.2012, Found: 389.2081 [M+H]+ ee=99.0% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-(2-fluorophenoxy)propan-1-amine as the appropriate amine a mixture of Example 133 and Example 134 was obtained. The enantiomers were separated on CHIRALPAK AS-V column using 70:30 EtOH/heptane+0.05% DEA as eluent to obtain Example 133 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H21N6OF 392.1761, Found: 393.1836 [M+H]V ee=99.8% (E1). Example 134 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H21NOF 392.1761, Found: 393.1852. [M+H]+ ee=99.6% (E2).
and
Starting from Preparation 2a following General procedure 11 and using 2-phenylsulfanylpropan-1-amine as the appropriate amine a mixture of Example 135 and Example 136 was obtained. The enantiomers were separated on CHIRALPAK AS-V column using 40:60 EtOH/heptane+0.05% DEA as eluent to obtain Example 135 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H22N6S 390.1627, Found: 391.1701 [M+H]+ ee=99.8% (E1). Example 136 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C21H22N6S 390.1627, Found: 391.1711 [M+H]+ ee=99.4% (E2).
and
and
and
Starting from Preparation 2a following General procedure II and using 2-benzylcyclopropanamine as the appropriate amine a mixture of the cis-products (Example 137, Example 138) and the mixture of the trans-products (Example 139, Example 140) were obtained. The enantiomers of these mixtures were separated on CHIRALCEL OD column using 50:50 EtOH/heptane+0.05% DEA as eluent.
We obtained Example 137 as the first eluting enantiomer of the cis-mixture. HRMS (IT-TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1966 [M+H]+ ee=99.8% (E1). Example 138 was obtained as the second eluting enantiomer of the cis-mixture. HRMS (IT-TOF, ESI) m/z: Calculated for C22H22N6[M+H]+ 370.1906, Found: 371.1981 ee=99.8% (E2).
We obtained Example 139 as the first eluting enantiomer of the trans-mixture. HRMS (IT-TOF, ESI) m/z: Calculated for C22H2N6 370.1906, Found: 371.1983 [M+H]+ ee=99.6% (E1). Example 140 was obtained as the second eluting enantiomer of the trans-mixture. HRMS (IT-TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1988 [M+H]+ ee=99.8% (E2).
and
Starting from Preparation 2a following General procedure II and using 2-(2-methoxyphenoxy)propan-1-amine as the appropriate amine a mixture of Example 141 and Example 142 was obtained. The enantiomers were separated on CHIRALPAK AS-H column using 50:50 1-PrOH/heptane+0.1% DEA as eluent to obtain Example 141 as the first eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C22H24N6O2 404.1961, Found: 405.2041 [M+H]+. Example 142 was obtained as the second eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C22H24N6O2 404.1961, Found: 405.2038 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3-phenoxypropan-1-amine as the appropriate amine Example 143 was obtained. HRMS (TOF, ESI) m/z: Calculated for C2, H22N6O 374.1855, Found: 375.1946 [M+H]+.
Starting from Preparation 2b following General procedure II and using butan-1-amine as the appropriate amine Example 144 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H26N6 338.2219, Found: 339.2289 [M+H]+.
Starting from Preparation 2b following General procedure II and using cyclopentanamine as the appropriate amine Example 145 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H26N6 350.2219 Found: 351.2270 [M+H]+.
Starting from Preparation 2b following General procedure 11 and using 2-phenoxyethanamine as the appropriate amine Example 146 was obtained. HRMS (TOF, ESI) m/z: Calculated for CH26N6O 402.2168 Found: 403.2235 [M+H]+.
Starting from Preparation 2b following General procedure II and using methanamine as the appropriate amine Example 0.147 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H20N6 296.1749, Found: 297.1824 [M+H]+.
Starting from Preparation 2a following General procedure II and using butan-1-amine as the appropriate amine Example 148 was obtained. HRMS (TOF, ESI) m/z: Calculated for Cl6H20N6 296.1749, Found: 297.1842 [M+H]+.
Starting from Preparation 2a following General procedure II and using (1R)-1-(2-fluoro-4-pyridyl)ethanamine as the appropriate amine Example 149 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H18N7F 363.1608, Found: 364.1674 [M+H]+.
Starting from Preparation 2a following General procedure II and using 3-methoxypropan-1-amine as the appropriate amine Example 150 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H20N6O 312.1699, Found: 313.1761 [M+H]+.
Starting from Preparation 2a following General procedure II and using 4-methoxybutan-1-amine as the appropriate amine Example 151 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H22N6O 326.1855, Found: 327.1919 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-(3-methoxyphenyl)ethanamine as the appropriate amine Example 152 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O 374.1855, Found: 375.1919 [M+H]+.
Starting from Preparation 2a following General procedure II and using 2-phenoxyethanamine as the appropriate amine Example 153 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H20N6O 360.1699, Found: 361.1774. [M+H]+.
Starting from Preparation 2a following General procedure II and using ethanamine as the appropriate amine Example 154 was obtained. HRMS (TOF, ESI) m/z: Calculated for C14H16N6 268.1436, Found: 269.1510 [M+H]+.
Starting from Preparation 2a following General procedure II and using norbornan-2-amine as the appropriate amine Example 155 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H22N6 334.1906, Found: 335.1981 [M+H]+.
Starting from Preparation 2c following General procedure II and using cyclopentanamine as the appropriate amine Example 156 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H22N6 322.1906, Found: 323.1993 [M+H]+.
Starting from Preparation 2c following General procedure II and using butan-1-amine as the appropriate amine Example 157 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H22N6 310.1906, Found: 311.1982 [M+H]+.
Starting from Preparation 2c following General procedure II and using indan-2-amine as the appropriate amine Example 158 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H22N6 370.1906, Found: 371.1992 [M+H]+.
Starting from Preparation 2c following General procedure 11 and using methanamine as the appropriate amine Example 159 was obtained. HRMS (TOF, ESI) m/z: Calculated for C14H16N6 268.1436, Found: 269.1512 [M+H]+.
Starting from Preparation 2d following General procedure II and using cyclopentanamine as the appropriate amine Example 160 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H24N6 336.2062 Found: 337.2150 [M+H]+.
Starting from Preparation 2d following General procedure II and using butan-1-amine as the appropriate amine Example 161 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H24N6 324.2062, Found: 325.2145 [M+H]+.
Starting from Preparation 2d following General procedure 11 and using 2-phenoxyethanamine as the appropriate amine Example 162 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C22H24N6O 388.2012 Found: 389.2081 [M+H]+.
Starting from Preparation 2d following General procedure 11 and using methanamine as the appropriate amine Example 163 was obtained. HRMS (TOF, ESI) m/z: Calculated for C15H18N6 282.1593, Found: 283.1663 [M+H]+.
Starting from Preparation 3a following General procedure III and using 2-fluoro-5-chloropyridine as the appropriate aryl halide Example 164 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H18N7OCl 395.1261, Found: 396.1326 [M+H]+.
Starting from Preparation 3a following General procedure II and using 2-fluoro-6-chloropyridine as the appropriate aryl halide Example 165 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H18N7OCl 395.1261, Found: 396.1331. [M+H]+.
Starting from Preparation 3a following General procedure III and using 2-fluoro-3-chloropyridine as the appropriate aryl halide Example 166 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H18N7OCl 395.1261, Found: 396.1319 [M+H]+.
Starting from Preparation 3a following General procedure III and using 2,6-difluoropyridine as the appropriate aryl halide Example 167 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H18N7OF 379.1557, Found: 380.1635 [M+H]+.
Starting from Preparation 3a following General procedure 111 and using 2-fluoro-6-bromopyridine as the appropriate aryl halide Example 168 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H18N7OBr 439.0756, Found: 440.0823 [M+H]+.
Starting from Preparation 3a following General procedure III and using 2-fluoro-3-bromopyridine as the appropriate aryl halide Example 169 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H18N7OBr 439.0756, Found: 440.0817 [M+H]+.
Starting from Preparation 3a following General procedure III and using 2-fluoro-5-bromopyridine as the appropriate aryl halide Example 170 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H18N7OBr 439.0756, Found: 440.0811 [M+H]+.
Starting from Preparation 3a following General procedure III and using 2,5-difluoropyridine as the appropriate aryl halide Example 171 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H18N7OF 379.1557, Found: 380.1617 [M+H]+.
Starting from Preparation 3a following General procedure III and using 2-fluoro-6-fluoromethylpyridine as the appropriate aryl halide Example 172 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H20FN7O 393.1713, Found: 394.1784 [M+H]+.
Starting from Preparation 3a following General procedure III and using 2-fluoro-6-difluoromethylpyridine as the appropriate aryl halide Example 173 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H19F2N7O 411.1619, Found: 412.1686 [M+H]+.
and
Starting from Preparation 3b following General procedure III and using 2-fluoropyridine as the appropriate aryl halide a mixture of Example 174 and Example 175 was obtained. The enantiomers were separated on CHIRALCEL OK column using 50:50 EtOH/heptane+0.05% DEA as eluent to obtain Example 174 as the first eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H21N7O 375.1808, Found: 376.1867 [M+H]+ ee=99.8% (E1). Example 175 was obtained as the second eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H21N7O 375.1808, Found: 376.1872 [M+H]+ ee=99.8% (E2).
and
Starting from Preparation 3b following General procedure III and using 2-fluoro-6-chloropyridine as the appropriate aryl halide a mixture of Example 176 and Example 177 was obtained. The enantiomers were separated on OJ column using EtOH+0.05% DEA as eluent to obtain Example 176 as the earlier eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OCl 409.1418, Found: 410.1469 [M+H]+ ee=99.8% (E1). Example 177 was obtained as the later eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OCl 409.1418, Found: 410.1482 [M+H]+ ee=98.8% (E2).
and
Starting from Preparation 3b following General procedure III and using 2,5-difluoropyridine as the appropriate aryl halide a mixture of Example 178 and Example 179 was obtained. The enantiomers were separated on AS column using 50:50 1-PrOH/heptane+0.1% DEA as eluent to obtain Example 178 as the earlier eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7O 393.1713, Found: 394.1780 [M+H]+ ee=99.4% (E1). Example 179 was obtained as the later eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7O 393.1713, Found: 394.1774 [M+H]+ ee=98.6% (E2).
and
Starting from Preparation 3b following General procedure III and using 2-fluoro-6-bromopyridine as the appropriate aryl halide a mixture of Example 180 and Example 181 34) was obtained. The enantiomers were separated on CHIRALPAK AS-H column using 40:60 EtOH/heptane+0.1% DEA as eluent to obtain Example 180 as the earlier eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OBr 453.0913, Found: 454.0970 [M+H]+ ee=99.8% (E1). Example 181 was obtained as the later eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OBr 453.0913, Found: 454.0967 [M+H]+ ee=99.0% (E2).
and
Starting from Preparation 3b following General procedure III and using 2-fluoro-3-bromopyridine as the appropriate aryl halide a mixture of Example 182 and Example 183 was obtained. The enantiomers were separated on CHIRALPAK AS-H column using 70:30 2-PrOH/heptane+0.1% DEA as eluent to obtain Example 182 as the earlier eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OBr 453.0913, Found: 454.0963 [M+H]+ ee=99.8% (E1). Example 183 was obtained as the later eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OBr 453.0913, Found: 454.0968 [M+H]+ ee=99.0% (E2).
and
Starting from Preparation 3b following General procedure III and using 2-fluoro-5-chloropyridine as the appropriate aryl halide a mixture of Example 184 and Example 185 was obtained. The enantiomers were separated on OJ column using 50:50 EtOH/heptane+0.05% DEA as eluent to obtain Example 184 as the earlier eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OCl 409.1418, Found: 410.1469. [M+H]+ ee=99.8% (E1). Example 185 was obtained as the later eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C20H20ClN7O 409.1418, Found: 410.1471 [M+H]+ ee 99.8% (E2).
and
Starting from Preparation 3b following General procedure III and using 2-fluoro-5-bromopyridine as the appropriate aryl halide a mixture of Example 186 and Example 187 was obtained. The enantiomers were separated on OJ column using 60:40 EtOH/heptane+0.05% DEA as eluent to obtain Example 186 as the earlier eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20NTOBr 453.0913 Found: 454.0967 [M+H]+. ee=99.8% (E1). Example 187 was obtained as the later eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OBr 453.0913 Found: 454.0983 [M+H]+ ee=99.2% (E2).
and
Starting from Preparation 3b following General procedure 1.11 and using 2,6-difluoropyridine as the appropriate aryl halide a mixture of Example 188 and Example 189 was obtained. The enantiomers were separated on CHIRALCEL OJ-H column using EtOH+0.1% DEA as eluent to obtain Example 188 as the earlier eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OF 393.1713, Found: 394.1779 [M+H]+ ee=99.8% (E1). Example 189 was obtained as the later eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OF 393.1713, Found: 394.1773 [M+H]+ ee=−99.6% (E2).
and
Starting from Preparation 3b following General procedure III and using 2-fluoro-3-chloropyridine as the appropriate aryl halide a mixture of Example 190 and Example 191 was obtained. The enantiomers were separated on CHIRALPAK AS-V column using 70:30 2-PrOH/heptane+0.05% DEA as eluent to obtain Example 190 as the earlier eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OCl 409.1418, Found: 410.1477 [M+H]+ ee=99.8% (E1). Example 191 was obtained as the later eluting enantiomer. HRMS (IT-TOF, ESI) m/z: Calculated for C20H20N7OCl 409.1418, Found: 410.1492 [M+H]+ ee=99.8% (E2).
and
Starting from Preparation 3b following General procedure III and using 2,3,6-trifluoropyridine as the appropriate aryl halide a mixture of Example 192 and Example 193 was obtained. The enantiomers were separated on CHIRALCEL OK column using 60:40 EtOH/heptane+0.05% DEA as eluent to obtain Example 192 as the earlier eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C20H19F2N7O 411.1699, Found: 412.1694 [M+H]+ ee=99.8% (E1). Example 193 was obtained as the later eluting enantiomer. HRMS (TOF, ESI) m/z: Calculated for C20H19F2N7O 411.1619, Found: 412.1700 [M+H]+ ee=99.8% (E2).
Starting from Preparation 3a following General procedure IV and using 6-trifluoromethyl-2-pyridone as the appropriate phenol analog Example 194 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C20H18N7OF3 429.1525, Found: 430.1608 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 6-methyl-2-pyridone as the appropriate phenol analog Example 195 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H21N7O 375.1808, Found: 376.1872 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 6-amino-2-pyridone as the appropriate phenol analog Example 196 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C19H20N8O 376.1760, Found: 377.1826 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 6-cyano-2-pyridone as the appropriate phenol analog Example 197 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C20H18N8O 386.1604, Found: 387.1655 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 4-fluorophenol as the appropriate phenol analog Example 198 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H1N6OF 378.1604, Found: 379.1683 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 3-fluorophenol as the appropriate phenol analog Example 0.199 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H19N6OF 378.1604, Found: 379.1621 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 3-chlorophenol as the appropriate phenol analog Example 200 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H19N6OCl 394.1309, Found: 395.1308 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 3-methylphenol as the appropriate phenol analog Example 201 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O 374.1855, Found: 375.1908 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 3-methoxyphenol as the appropriate phenol analog Example 202 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O2 390.1804, Found: 391.1810 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2-fluorophenol as the appropriate phenol analog Example 203 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H19N6OF 378.1604, Found: 379.1685 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2-chlorophenol as the appropriate phenol analog Example 204 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H19N6OCl 394.1309, Found: 395.1387 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2-methylphenol as the appropriate phenol analog Example 205 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O 374.1855, Found: 375.1929 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2-methoxyphenol as the appropriate phenol analog Example 206 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N6O2 390.1804, Found: 391.1888 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 4-chlorophenol as the appropriate phenol analog Example 207 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H19N6OCl 394.1309, Found: 395.1389 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 4-methylphenol as the appropriate phenol analog Example 208 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H22N4O 374.1855, Found: 375.1937 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 4-methoxyphenol as the appropriate phenol analog Example 209 was obtained. HRMS (TOF, ESI) m/z: Calculated for C21H2N6O2 390.1804, Found: 391.1883 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 1H-indol-5-ol as the appropriate phenol analog Example 210 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H21N7O 399.1808, Found: 400.1848 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2-methoxy-5-methylphenol as the appropriate phenol analog Example 211 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H24N6O2 404.1961, Found: 405.2022 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2,6-difluorophenol as the appropriate phenol analog Example 212 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H18N6OF2 396.1510, Found: 397.1570 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2,6-dimethoxyphenol as the appropriate phenol analog Example 213 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H24N6O3 420.1910, Found: 421.1979 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2-isopropoxyphenol as the appropriate phenol analog Example 214 was obtained. HRMS (TOF, ESI) m/z: Calculated for C23H26N6O2 418.2117, Found: 419.2195 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2-ethoxyphenol as the appropriate phenol analog Example 215 was obtained. HRMS (TOF, ESI) m/z: Calculated for C22H24N6O2 404.1961, Found: 405.2030 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 3-hydroxypyridine as the appropriate phenol analog Example 216 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H19N7O 361.1651, Found: 362.1724 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 2-pyridone as the appropriate phenol analog Example 217 was obtained. HRMS (TOF, ESI) m/z: Calculated for C19H19N7O 361.1651, Found: 362.1731 [M+H]+.
Starting from Preparation 3a following General procedure IV and using 1-methyl-1H-pyrazol-5-ol as the appropriate phenol analog Example 218 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H20N8O 364.1760, Found: 365.1822 [M+H]+.
Starting from Preparation 3a following General procedure IV and using pyrimidin-2(1H)-one as the appropriate phenol analog Example 219 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H18N8O 362.1604, Found: 363.1675 [M+H]+.
Following General procedure V using cyclopentanamine and as the appropriate amine and omitting Step F Example 220 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H17N6Cl 328.1203, Found: 329.1272 [M+H]+.
Following General procedure V and using cyclopentanamine as the appropriate amine Example 221 was obtained. HRMS (TOF, ESI) m/z: Calculated for C17H17N7 319.1545, Found: 320.1623 [M+H]+.
Following General procedure V and using 2-phenoxyethanamine as the appropriate amine Example 222 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H17N7O 371.1495, Found: 372.1575 [M+H]+.
Following General procedure V and using ethanamine as the appropriate amine Example 223 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C14H13N7 279.1232, Found: 280.1315 [M+H]+.
Following General procedure V and using cyclopropanamine as the appropriate amine Example 224 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C18H3N7 291.1232, Found: 292.1303 [M+H]+.
Following General procedure V and using prop-2-en-1-amine as the appropriate amine Example 225 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C18H13N7 291.1232, Found: 292.1300 [M+H]+.
Following General procedure V and using 4,4,4-trifluorobutan-1-amine as the appropriate amine Example 226 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C11H14N7F3 361.1263, Found: 362.1338 [M+H]+.
Following General procedure V and using methanamine as the appropriate amine Example 227 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C13H11N7 265.1076, Found: 266.1139 [M+H]+.
Following General procedure VI starting from Preparation 4 and using ethyl difluoroacetate as the appropriate acetic acid derivative Example 228 was obtained. HRMS (TOF, ESI) m/z: Calculated for C13H2N6F2 290.1092, Found: 291.1161 [M+H]+.
Following General procedure VI starting from Preparation 4 and using trifluoroacetic acid as the appropriate acetic acid derivative Example 229 was obtained. HRMS (TOF, ESI) m/z: Calculated for C13H11N6F3 308.0997, Found: 309.1078 [M+H]+.
To a solution of 200 mg of 4-(3-butyl-2-methyl-imidazo[4,5-b]pyridin-5-yl)-N-triphenylmethyl-pyridine-2,6-diamine (Preparation 5) (0.37 mmol, 1 eq.) and 155 μL triethyl amine (1.11 mmol, 3 eq.) in 4 mL dry THF, 41 μL 2-chloroacetyl chloride (0.51 mmol, 1.4 eq.) was added and the mixture was stirred until no further conversion was observed. The volatiles were removed under reduced pressure, the residue was dissolved in 5 mL dry DMF and then 70 my phenol (0.74 mmol, 2 eq.) and 154 mg potassium carbonate (1.11 mmol, 3 eq.) were added. The resulting mixture was stirred until no further conversion was observed. It was diluted with brine and extracted with DCM. The combined organic phases were dried over MgSO4, filtered and the filtrate was concentrated under reduced pressure. The crude product was first purified by flash chromatography on silica column using DCM/MeOH (1.2% NH3) as eluents during which the trityl group was also removed, followed by preparative reversed phase chromatography using 5 mM aqueous NH4HCO3 solution and MeCN as eluents to give Example 230. HRMS (TOF, ESI) m/z: Calculated for C24H26N6O2 430.2117, Found: 431.2195 [M+H]+.
To a solution of 269 mg of 4-(3-butyl-2-methyl-imidazo[4,5-b]pyridin-5-yl)-N2-triphenylmethyl-pyridine-2,6-diamine (Preparation 5) (0.5 mmol, 1 eq.) and 159 mg benzaldehyde (1.5 mmol, 3 eq.) in DMF/MeOH (3/2 mL) 113 mg sodium borohydride (3 mmol, 6 eq.) was added in small portions. The resulting mixture was stirred at 60° C. in the presence of 100 μL acetic acid until no further conversion was observed. The pH was set to 5 with 2 M aqueous HCl solution and the resulting mixture was stirred until no further conversion (detritylation) was observed. The mixture was neutralized with 10% aqueous K2CO3 solution, diluted with brine, and extracted with DCM. The combined organic phases were dried over MgSO4, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified via preparative reversed phase chromatography using 5 mM aqueous NH4HCO3 solution and MeCN as eluents to give Example 231. HRMS (TOF, ESI) m/z: Calculated for C23H26N6 386.2219, Found: 387.2295 [M+H]+.
Starting from Example 148 following General procedure VII using cyclohexylacetyl chloride as the appropriate acid chloride Example 232 was obtained. HRMS (TOF, ESI) m/z: Calculated for C24H2N6O 420.2638, Found: 421.2719 [M+H]+.
Starting from Example 148 following General procedure VII using 2-chlorobenzoyl chloride as the appropriate acid chloride Example 233 was obtained. HRMS (TOF, ESI) m/z: Calculated for C23H23N6OCl 434.1622, Found: 435.1702 [M+H]+.
Starting from Example 148 following General procedure VII using cyclohexanoyl chloride as the appropriate acid chloride Example 234 was obtained. HRMS (TOF, ESI) m/z: Calculated for C23H30N6O 406.2481, Found: 407.2557 [M+H]+.
Starting from Example 148 following General procedure VII using phenylacetyl chloride as the appropriate acid chloride Example 235 was obtained. HRMS (TOF, ESI) m/z: Calculated for C24H26N6O 414.2168, Found: 415.2246 [M+H]+.
Starting from Preparation 6a following General procedure IX Example 236 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C16H19N5 281.1640, Found: 282.1710 [M+H]+.
Starting from Preparation 6b following General procedure IX Example 237 was obtained. HRMS (TOF, ESI) m/z: Calculated for C20H25N5 335.2110, Found: 336.2184. [M+H]+.
Starting from Preparation 6c following General procedure IX Example 238 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H17N5 279.1484, Found: 280.1562 [M+H]+.
Starting from Preparation 6d following General procedure IX Example 239 was obtained. HRMS (IT-TOF, ESI) m/z: Calculated for C16H17N5 279.1484, Found: 280.1552 [M+H]+.
Starting from Preparation 7a following General procedure X and using 6-amino-4-bromo-2-fluoropyridine as the appropriate aryl halide Example 240 was obtained. HRMS (TOF, ESI) m/z: Calculated for C16H14F3N5 333.1201, Found: 334.1270 [M+H]+.
Starting from Preparation 6e following General procedure X and using 6-amino-4-bromo-2,5-difluoropyridine as the appropriate aryl halide Example 241 was obtained. HRMS (TOF, ESI) m/z: Calculated for C18H13F2N5 301.1139, Found: 302.1202 [M+H]+.
Starting from Preparation 6f following General procedure X and using 4-bromo-2,6-diamino-3,5-difluoropyridine as the appropriate aryl halide Example 242 was obtained. HRMS (TOF, ESI) m/z: Calculated for C15H4F2N 316.1248, Found: 317.1310 [M+H]+.
Inhibition of the enzymatic activity of human kinases was evaluated in a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay in 384-well reaction plates. In this assay, full-length human kinases from Carna Biosciences—DYRK1A (NM_001396, ref. 04-130; 2.0 ng/μl), DYRK1B (NM_004714, ref. 04-131; 1.2 ng/p), CLK1 (NM_001162407, ref. 04-126; 0.7 ng/μl), CDK9 (NM_001261, ref. 04-110; 0.9 ng/μl), or GSK3β (NM_001146156, ref. 04-141; 2.0 ng/μl—were incubated for 40 minutes (DYRK1A and DYRK1B) or 100 minutes (CLK1, CDK9 and GSK3β) at room temperature with ATP (Sigma A2383, 10 μM) and a ULight™-labelled human Myelin Basic Protein (MBP) peptide substrate (Perkin Elmer TRF0109, 100 nM) in a reaction buffer composed of 50 mM HEPES pH7.4, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT and 0.01% Tween20. Test compounds of the invention were added in reaction buffer at a range of concentrations from 0.1 nM to 30 μM. Following addition of EDTA (Sigma E7889, 10 mM) to stop the reaction, Europium-labelled mouse monoclonal antibody recognizing phospho-Thr232 in MBP (Perkin Elmer TRF0201, 1 nM) was added. After one hour, the reaction plates were read using a fluorescence reader (EnVision®, Perkin Elmer) at 620 nm and 665 nm (excitation at 340 nm); when the Europium donor fluorophore is excited by light at 340 nm, an energy transfer (620 nm) to the acceptor occurs, which will then emit light at 665 nm. The activity, and hence inhibition, of DYRK1A kinase activity is thus measured by the relative intensity of the emitted light. The IC50 was calculated from the concentration-activity curve as the concentration of the test compound required for 50% inhibition of kinase activity. The results are presented in Table 1.
The activity of His-TEV-DYRK1A Kinase domain (aa127-485) was measured using the accumulation of ADP produced during the phosphorylation of the peptide substrate Woodtide (Zinnsser Analytic) using ATP (Sigma Aldrich A7699). The enzyme reaction was conducted in assay buffer (pH 7.4), containing 15 mM Hepes; 20 mM NaCl; 1 mM EGTA; 10 mM MgCl2; 0.02% Tween20 and 0.1 mg/ml Bovine-y-globulin. Test compounds of the invention were added in reaction buffer in a range of concentrations for 10 minutes at 30° C. in the presence of 20 nM DYRK1A enzyme, 40 μM peptide substrate and 20 μM ATP. Detection reagents (DiscoveRx 90-0083), ADP Hunter Plus Reagent A and then ADP Hunter Plus Reagent B were added. After a following 20 minutes incubation at 30° C., ADP Hunter Plus Stop Solution was added. The fluorescence intensity was measured at 590 nm. The IC50 was calculated from the concentration-activity curve as the concentration of the test compound required for 50% inhibition of kinase activity. The results are presented in Table 1.
On day 0, human U2-OS osteosarcoma cells were seeded in 12-well culture plates (100,000 cells per well) and incubated at 37° C. in the presence of 5% CO2 in 1 ml McCoy's 5A (Modified) medium containing GlutaMAX™ (Gibco 36600), supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin, 10 mM Hepes buffer, pH=7.4, and 10% foetal calf serum (FCS, Sigma F7524). On day 1, medium was replaced with 500 μl Optimem medium containing GlutaMAX™ (Gibco 51985), 150 ng of a pcDNA3.1 plasmid (Invitrogen) containing a sequence coding for full-length, wild-type human DYRK1A (NM_001396) with an HA tag, 0.3% lipofectamine (Invitrogen 18324-020), and 0.6% Plus reagent (Invitrogen Cat No 11514-015). After 5 hours, medium was replaced with 900 μl McCoy's 5A (Modified) medium containing GlutaMAX™ (Gibco 36600). On day 2, cells were exposed to a range of concentrations of the test compounds of the invention for 5 hours. Cells were then washed in phosphate-buffered saline solution and cell lysed in lysis buffer comprised of 150 mM NaCl, 20 mM Tris-HCl pH 7.4, 1% triton X-100, 1 mM EGTA, 1 mM EDTA and protease (1% v/v; 539134; Calbiochem) and phosphatase (1% v/v; 524625; Calbiochem) inhibitor cocktails (50 μl lysis buffer/well). The relative levels of phospho-Ser520-DYRK1A were assayed using either western blotting or the Mesoscale ELISA platform. For analysis by western blot, lysates were diluted into Laemmli sample buffer (Bio-Rad) containing 5% v/v β-mercaptoethanol, heated for 5 min at 95° C., and resolved on Tris-glycine gels or NuPage Bis-Tris gels (Novex; Invitrogen). Biotinylated molecular weight standards (Cell Signaling Technology) were included in all gels. Proteins were transferred to nitrocellulose membranes (Hybond, ECL; Amersham), which were blocked in Tris-buffered saline/0.1% tween 20 (TBST) containing 5% milk, and probed at 4° C. overnight with anti-phospho-Ser520-DYRK1A antibody (Eurogentec SE6974-75; 0.23 μg/ml in 5% BSA) or anti DYRK1A antibody (Abnova H00001859; 0.5 μg/ml in 5% milk). Peroxidase-conjugated secondary antibodies were diluted into 5% milk and applied to membranes for 1 h at 20° C. Chemiluminescence detection was performed using the ECL plus western blotting detection kit (Amersham) and was recorded on ECL plus hyperfilm (Amersham). Blots were scanned using the Bio-Rad GS-800 calibrated densitometer and quantitative analysis of western blots was performed using TotalLab software (Amersham). IC50 values for inhibition of phospho-Ser520-DYRK1A were calculated from dose-response curves plotting the ratio between phospho-Ser520-DYRK1A and total DYRK1A signals at each concentration. For analysis by Mesoscale ELISA, lysates were transferred to BSA-blocked ELISA plates with pre-bound anti-HA capture antibodies (Novus biological NB600-364; 15 μg/ml) for 1 hour with shaking at RT. Anti-phospho-Ser520-DYRK1A antibody (Eurogentec SE6974-75; 2.3-3.0 mg/ml) and anti DYRK1A antibody (Abnova H00001859; 3 μg/ml) was then added for 1 hour at RT, followed by addition of Sulfa-TAG anti-rabbit detection antibody (ref MSD R32AB; 1 μg/ml) and Sulfa-TAG anti-mouse detection antibody (ref MSD R32-AC-1; 1 μg/ml). After a further 1 hour, Read Buffer was added and plates were read on the Sector Imager 2400 (Mesoscale). IC50 values for inhibition of phospho-Ser520-DYRK1A were calculated from dose-response curves. The results showed that the compounds of the invention are powerful inhibitors of cellular DYRK1A Ser520 autophosphorylation. The results are presented in Table 1.
For pharmacodynamics studies of inhibition of DYRK1A autophosphorylation, female SCID mice were injected subcutaneously with RS4; 11 human acute lymphoblastic leukemia cells. When tumors reached a size of 200-300 mm3, mice were randomized into homogeneous groups of 3 and given a single oral administration of the compounds of the invention at doses of up to 100 mg/kg. At various times after treatment, typically 2 hours and 6 hours, treated and control mice were sacrificed, tumors were excised and proteins were extracted in tissue lysis buffer comprised of 150 mM NaCl, 20 mM Tris-HCl pH 7.4, 1% triton X-100, 1 mM EGTA, 1 mM EDTA and protease (1% v/v; 539134; Calbiochem) and phosphatase (1% v/v; 524625; Calbiochem) inhibitor cocktails. The relative levels of phospho-Ser520-DYRK1A were assayed using western blotting. For this, lysates were diluted into Laemmli sample buffer (Bio-Rad) containing 5% v/v β-mercaptoethanol, heated for 5 min at 95° C., and resolved on Tris-glycine gels or NuPage Bis-Tris gels (Novex; Invitrogen). Biotinylated molecular weight standards (Cell Signaling Technology) were included in all gels. Proteins were transferred to nitrocellulose membranes (Hybond, ECL; Amersham), which were blocked in Tris-buffered saline/0.1% tween 20 (TBST) containing 5% milk, and probed at 4° C. overnight with anti-phospho-Ser520-DYRK1A antibody (Eurogentec SE6974-75; 0.23 μg/ml in 5% BSA) or anti DYRK1A antibody (Abnova H00001859; 0.5 μg/ml in 5% milk). Peroxidase-conjugated secondary antibodies were diluted into 5% milk and applied to membranes for 1 h at 20° C. Chemiluminescence detection was performed using the ECL plus western blotting detection kit (Amersham) and was recorded on ECL plus hyperfilm (Amersham). Blots were scanned using the Bio-Rad GS-800 calibrated densitometer and quantitative analysis of western blots was performed using TotalLab software (Amersham). The percentage inhibition of phospho-Ser520-DYRK1A as compared to the control tumors was calculated using the ratio between phospho-Ser520-DYRK1A and total DYRK1A signals at each dose. The results showed that the compounds of the invention are powerful inhibitors of tumor DYRK1A Ser520 autophosphorylation.
For anti-tumor efficacy studies, female nude balb/c nu/nu mice were injected subcutaneously with A2780 human ovarian carcinoma cells. When tumors reached a size of approximately 150 mm3, mice were randomized into homogeneous groups of 8 and treated orally with the compounds of the invention at doses of at doses of up to 75 mg/kg once daily for 2 weeks. Anti-tumor efficacy was monitored by at least twice-weekly measurement of tumor sizes using calipers, and body weights were recorded in order to document potential general toxicity. Percentage tumor growth inhibition (TGI) on a given day was calculated using the formula: (1-[RTV(treated)/RTV(untreated)])×100, where RTV=relative tumor volume on the given day versus start of treatment. The results showed that the compounds of the invention are powerful inhibitors of tumor growth.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15/59252 | Sep 2015 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/073395 | 9/30/2016 | WO | 00 |
