Patent Application
20240307362
This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 23, 2022, is named BHC183052-SL2.txt and is 36,099 bytes in size.
Field of application of the invention The invention relates to substituted 4H-pyrrolo[3,2-c]pyridin-4-one compounds, processes for their production and uses thereof.
The Epidermal Growth Factor Receptor (EGFR or EGF-receptor) receptor tyrosine kinase family consists of 4 members: EGFR (Erbb1, Her1), ERBB2 (Her2), ERBB3 (Her3), and ERBB4 (Her4). EGFR mediates activation of MAPK and PI3K signaling pathways and thereby regulates cell proliferation, differentiation, migration and survival (Pao et al., 2010). EGFR gene amplification, overexpression, and mutations are frequently observed in various cancer indications and are associated with a poor prognosis (Gridelli et al., 2015).
In lung adenocarcinoma, mutations of EGFR are prevalent in approximately 15% of Western patients and up to 50% of East Asian patients (Paez et al., 2004). These mutations typically occur in one of four exons, exons 18-21, in the kinase domain of EGFR (Paez et al., 2004). The most common activating mutations in EGFR are a point mutation in exon 21, substituting an arginine for a leucine (L858R), and a small in-frame deletion in exon 19 that removes four amino acids (del 19/L747_A750del) (Pao et al., 2010). The FDA-approved inhibitors gefitinib, erlotinib, and afatinib, targeting mutations in exons 18, 19, and 21 of EGFR, are effective in patients but the response is often not durable (Mok et al., 2009; Sequist et al., 2013). Resistance frequently occurs in these patients in response to acquisition of a second mutation, T790M (Pao et al., 2005). Second generation inhibitors, e.g. afatinib, irreversibly target this mutation but are still potent inhibitors of wild-type EGFR. A third-generation irreversible inhibitor, osimertinib, that maximizes activity towards T790M while minimizing activity towards wild-type EGFR, is also effective in T790M mutant patients and is currently the standard treatment for T790M positive patients (Mok et al., 2017). Osimertinib is also approved as a front-line therapy for patients with mutations of EGFR exons 19 or 21 (Soria et al., 2018).
However, patients also develop resistance to irreversible third-generation EGFR inhibitors, such as osimertinib. One of the major osimertinib resistance mechanisms identified is mutation of the cysteine in position 797 to a serine, resulting in loss of the covalently interacting cysteine and loss of sensitivity to irreversible EGFR inhibitors, at which point progressing patients have currently only limited treatment options (Thress et al., 2015; Oxnard et al., 2018). Such C797S mutations can also occur when osimertinib is used as a first-line therapy, in the absence of the T790M mutation (Ramalingham et al., 2018a; Ramalingham et al., 2018b). A novel targeted therapy that is able to specifically address the EGFR-C797S acquired resistance mutation would be highly beneficial for those patients.
By contrast, and with the exception of A763_Y764insFQEA, small in-frame insertions of EGFR exon20 are resistant to available EGFR inhibitors at doses achievable in lung cancer patients and comprise an unmet medical need (Yasuda et. al., 2013).
Patients with EGFR exon20 insertions, such as V769_D770insASV, D770_N771insSVD, D770_N771insNPG, N771_P772insH, H773_V774insH, H773_V774insNPH, V774_C775insHV show particular low response rates to all currently approved EGFR-targeted therapies, resulting in significantly reduced progression-free survival as well as overall survival (Chen et al., 2016). This has been shown for the first-generation inhibitors erlotinib and gefitinib as well as for the second-generation inhibitor afatinib (Chen et al., 2016; Yang et al., 2015).
Therefore, the standard treatment for EGFR exon20 insertion patients is currently chemotherapy.
The same resistance profile has been observed for exon20 insertion mutations in ERBB2 (e.g. ERBB2 A775_G776insYVMA with the highest prevalence), another member of the EGF-receptor family (Arcila et al., 2012) and some of the uncommon EGFR mutations like L681Q (Chiu et al., 2015).
Several irreversible inhibitors are currently in clinical trials for the treatment of EGFR exon20 insertion patients: Osimertinib, initially approved for the treatment of T790M mutant NSCLC patients (Floc'h et al., 2018); poziotinib (HM-781-36B), a non-approved pan-Her inhibitor targeting EGFR, Her2/neu, and Her4 (Robichaux et al., 2018); as well as TAK-788 (AP32788) (Doebele et al., ASCO 2018). Of these, the first clinical data have been published for poziotinib and TAK-788. Both compounds clearly show clinical efficacy in EGFR exon20 insertion patients. However, major adverse events, mediated by inhibition of wild-type EGFR, have been reported for both clinical trials and these adverse events may limit clinical utility.
More recently, new preclinical data has been published for two additional compounds showing activity on EGFR exon20 insertions: TAS6417 (TCP-064) and compound 1a (Hasako et al., 2018; Jang et al., 2018). No clinical results are yet available for these two compounds.
In summary, mutant EGFR is a promising drug target for cancer therapy. In particular, patients with primary resistance to approved anti-EGFR therapies, due to EGFR exon20 insertions, have only few treatment options to date and there is a great need for novel alternative and/or improved therapeutics to provide these patients with an efficacious, well-tolerable therapy (Oxnard et al., 2013). Therefore, potent inhibitors of mutant EGFR, particularly of mutant EGFR with exon20 insertion mutations that show improved selectivity versus wild-type EGFR, represent valuable compounds that should complement therapeutic options either as single agents or in combination with other drugs.
The invention provides compounds that inhibit a mutant EGFR; specifically, an EGFR comprising one or more exon 20 insertion mutations, an L858R mutation, or a small in-frame deletion of exon 19, in the presence or absence of a C797S mutation. These compounds furthermore have reduced activity towards the wild-type-EGFR.
It has now been found that the compounds of the present invention have surprising and advantageous properties.
In particular, said compounds of the present invention have surprisingly been found to effectively inhibit mutant EGFR with exon 20 insertion mutations, particularly those harboring a D770_N771ins SVD exon 20 insertion with an IC50 below 5 nM. Furthermore it has been found that these compounds additionally show cellular potency below 1 μM in EGFR V769_D770insASV, D770_N771insSVD, D770_N771insNPG, N771_P772insH, or H773_V774insNPH exon 20 insertion harboring BA/F3 cell lines. Furthermore, the here described compounds are active in BA/F3 cell lines harboring D770_N771insSVD C797S.
In addition, the here described compounds potently inhibit proliferation of BA/F3 cell lines carrying EGFR activating mutations with or without C797S acquired resistance mutations (EGFR E746_A750del, L858R, E746_A750del C797S, L858R C797S), uncommon EGFR mutations (EGFR L681Q) or ERBB2 exon20 insertion A775_G776insYVMA.
Surprisingly these compounds additionally show at least 5-fold selectivity in an antiproliferative assay of EGFR D770_N771ins SVD exon 20 insertion harboring BA/F3 cell lines versus wild-type EGFR harboring BA/F3 cells and may therefore be used for the treatment or prophylaxis of diseases of uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses or diseases which are accompanied with uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses mediated by mutant EGFR with exon 20 insertion mutations and/or reduce (or block) proliferation in cells harboring EGFR exon 20 insertion mutations, for example, haematological tumours, solid tumours, and/or metastases thereof, e.g. leukaemias and myelodysplastic syndrome, malignant lymphomas, head and neck tumours including brain tumours and brain metastases, tumours of the thorax including non-small cell and small cell lung tumours, gastrointestinal tumours, endocrine tumours, mammary and other gynaecological tumours, urological tumours including renal, bladder and prostate tumours, skin tumours, and sarcomas, and/or metastases thereof.
The invention provides compounds that inhibit a mutant EGFR; specifically, an EGFR comprising one or more exon 20 insertion mutations, an L858R mutation, or a small in-frame deletion of exon 19, in the presence or absence of a C797S mutation. These compounds furthermore have reduced activity towards the wild-type-EGFR.
In accordance with a first aspect, the invention relates to compounds of formula (I),
In a second aspect, the invention relates to compounds of formula (I) as described supra, wherein:
In a third aspect, the invention relates to compounds of formula (I) as described supra, wherein:
In a fourth aspect, the invention relates to compounds of formula (I) as described supra, which is selected from the group consisting of:
A further aspect of the invention relates to compounds of formula (I), which are present as their salts.
It is to be understood that the present invention relates to any sub-combination within any embodiment or aspect of the present invention of compounds of general formula (I), supra.
More particularly still, the present invention covers compounds of general formula (I) which are disclosed in the Example section of this text, infra.
In accordance with another aspect, the present invention covers methods of preparing compounds of the present invention, said methods comprising the steps as described in the Experimental Section herein.
Another embodiment of the invention are compounds as disclosed in the Claims section or disclosed analogs of the exemplified compounds and subcombinations thereof.
It is to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment or aspect of the invention are meant to be disclosed also in connection with other embodiments or aspects of the invention shown herein. If, in one case, a specific feature is not disclosed with one embodiment or aspect of the invention, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment or aspect of the invention. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment or aspect of the invention, but that just for purposes of clarity and to keep the length of this specification manageable. For example, it is to be understood that all aspects, embodiments, pharmaceutical compositions, combinations, uses and/or methods of the present invention defined herein for the compounds of formula (I) also relate to more specific embodiments of the compounds of formula (I), such as, but not limited to, the compounds of formula (Ia) and vice-versa, for example.
It is further to be understood that the content of the documents referred to herein is incorporated by reference in their entirety, e.g., for enablement purposes, namely when e.g. a method is discussed details of which are described in said document. This approach serves to keep the length of this specification manageable.
Constituents, which are optionally substituted as stated herein, may be substituted, unless otherwise noted, one or more times, independently of one another at any possible position. When any variable occurs more than one time in any constituent, each definition is independent. For example, when R1, R1a, R1b, R1c, R2, R3 and/or R4 occur more than one time in any compound of formula (I) each definition of R1, R1a, R1b, R1c, R2, R3 and R4 is independent.
Should a constituent be composed of more than one part, e.g. C1-C4-alkoxy-C2-C4-alkyl, the position of a possible substituent can be at any of these parts at any suitable position. A hyphen at the beginning or at the end of the constituent marks the point of attachment to the rest of the molecule. Should a ring be substituted the substitutent could be at any suitable position of the ring, also on a ring nitrogen atom, if suitable.
The term “comprising” when used in the specification includes “consisting of”.
If it is referred to “as mentioned above” or “mentioned above”, “supra” within the description it is referred to any of the disclosures made within the specification in any of the preceding pages.
If it is referred to “as mentioned herein”, “described herein”, “provided herein,” or “as mentioned in the present text,” or “stated herein” within the description it is referred to any of the disclosures made within the specification in any of the preceding or subsequent pages. “Suitable” within the sense of the invention means chemically possible to be made by methods within the knowledge of a skilled person.
The terms as mentioned in the present text may have the following meanings: The term “halogen atom”, “halo-” or “Hal-” is to be understood as meaning a fluorine, chlorine, bromine or iodine atom.
The term “C1-C6-alkyl” is to be understood as meaning a linear or branched, saturated, monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms, e.g. a methyl, ethyl, propyl, butyl, pentyl, hexyl, iso-propyl, iso-butyl, sec-butyl, tert-butyl, iso-pentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neo-pentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl group, or an isomer thereof. Particularly, said group has 1, 2, 3 or 4 carbon atoms (“C1-C4-alkyl”), e.g. a methyl, ethyl, propyl, butyl, iso-propyl, iso-butyl, sec-butyl, tert-butyl group, more particularly 1, 2 or 3 carbon atoms (“C1-C3-alkyl”), e.g. a methyl, ethyl, n-propyl or iso-propyl group.
The term “C1-C4-haloalkyl” is to be understood as meaning a linear or branched, saturated, monovalent hydrocarbon group in which the term “C1-C4-alkyl” is defined supra, and in which one or more hydrogen atoms is replaced by a halogen atom, in identically or differently, i.e. one halogen atom being independent from another. Particularly, said halogen atom is F. Said C1-C4-haloalkyl group is, for example, —CF3, —CHF2, —CH2F, —CF2CF3, —CH2CH2F, —CH2CHF2, —CH2CF3, —CH2CH2CF3, or —CH(CH2F)2.
The term “C1-C4-alkoxy” is to be understood as meaning a linear or branched, saturated, monovalent, hydrocarbon group of formula —O-alkyl, in which the term “alkyl” is defined supra, e.g. a methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, tert-butoxy or sec-butoxy group, or an isomer thereof.
Unless defined otherwise, the term “5- to 6-membered heterocycloalkyl” or “5- to 6-membered heterocyclic ring”, is to be understood as meaning a saturated, monovalent, monocyclic hydrocarbon ring which contains 4 or 5 carbon atoms, and one heteroatom-containing group selected from O and NR, wherein R means a hydrogen atom, a C1-C3-alkyl or a C1-C3-haloalkyl group, it being possible for said heterocycloalkyl group to be attached to the rest of the molecule via any one of the carbon atoms.
Particularly, without being limited thereto, said heterocycloalkyl can be a 5-membered ring, such as tetrahydrofuranyl, pyrazolidinyl, or a 6-membered ring, such as tetrahydropyranyl, piperidinyl, for example.
The term “C1-C6”, as used throughout this text, e.g. in the context of the definition of “C1-C6-alkyl” or “C1-C6-haloalkyl” is to be understood as meaning an alkyl group having a finite number of carbon atoms of 1 to 6, i.e. 1, 2, 3, 4, 5 or 6 carbon atoms. It is to be understood further that said term “C1-C6” is to be interpreted as any sub-range comprised therein, e.g. C1-C6, C2-C6, C3-C6, C1-C2, C1-C3, particularly C1-C2, C1-C3, C1-C4,
The term “C1-C4”, as used throughout this text, e.g. in the context of the definition of “C1-C4-alkyl”, “C1-C4-haloalkyl”, “C1-C4-alkoxy”, or “C1-C4-haloalkoxy” is to be understood as meaning an alkyl group having a finite number of carbon atoms of 1 to 4, i.e. 1, 2, 3 or 4 carbon atoms. It is to be understood further that said term “C1-C4” is to be interpreted as any sub-range comprised therein, e.g. C1-C4, C2-C4, C3-C4, C1-C2, C1-C3, particularly C1-C2, C1-C3, C1-C4, in the case of “C1-C6-haloalkyl” or “C1-C4-haloalkoxy” even more particularly C1-C2.
Further, as used herein, the term “C3-C6”, as used throughout this text, e.g. in the context of the definition of “C3-C6-cycloalkyl”, is to be understood as meaning a cycloalkyl group having a finite number of carbon atoms of 3 to 6, i.e. 3, 4, 5 or 6 carbon atoms. It is to be understood further that said term “C3-C6” is to be interpreted as any sub-range comprised therein, e.g. C3-C6, C4-C5, C3-C5, C3-C4, C4-C6, C5—C; particularly C3-C6.
The term “substituted” means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties.
Ring system substituent means a substituent attached to an aromatic or nonaromatic ring system which, for example, replaces an available hydrogen on the ring system.
As used herein, the term “one or more”, e.g. in the definition of the substituents of the compounds of the general formulae of the present invention, is understood as meaning “one, two, three, four, five, etc., particularly one, two, three or four, more particularly one, two or three, even more particularly one or two”.
The compounds of general formula (I) may exist as isotopic variants. The invention therefore includes one or more isotopic variant(s) of the compounds of general formula (I), particularly deuterium-containing compounds of general formula (I).
The term “isotopic variant” of a compound or a reagent is defined as a compound exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound.
The term “isotopic variant of the compound of general formula (I)” is defined as a compound of general formula (I) exhibiting an unnatural proportion of one or more of the isotopes that constitute such a compound.
The expression “unnatural proportion” is to be understood as meaning a proportion of such isotope which is higher than its natural abundance. The natural abundances of isotopes to be applied in this context are described in “Isotopic Compositions of the Elements 1997”, Pure Appl. Chem., 70(1), 217-235, 1998. Examples of such isotopes include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as 2H (deuterium), 3H (tritium), 11C, 13C, 14C, 15N, 17O, 18O, 32P, 33P, 33S, 34S, 35S, 36S, 18F, 36Cl, 82Br, 123I, 124I, 125I, 129I, and 131I, respectively.
With respect to the treatment and/or prophylaxis of the disorders specified herein the isotopic variant(s) of the compounds of general formula (I) in one embodiment contain deuterium (“deuterium-containing compounds of general formula (I)”). Isotopic variants of the compounds of general formula (I) in which one or more radioactive isotopes, such as 3H or 14C, are incorporated are useful e.g. in drug and/or substrate tissue distribution studies. These isotopes are particularly preferred for the ease of their incorporation and detectability. Positron emitting isotopes such as 18F or 11C may be incorporated into a compound of general formula (I). These isotopic variants of the compounds of general formula (I) are useful for in vivo imaging applications. Deuterium-containing and 13C-containing compounds of general formula (I) can be used in mass spectrometry analyses (H. J. Leis et al., Curr. Org. Chem., 1998, 2, 131) in the context of preclinical or clinical studies.
Isotopic variants of the compounds of general formula (I) can generally be prepared by methods known to a person skilled in the art, such as those described in the schemes and/or examples herein, by substituting a reagent for an isotopic variant of said reagent, in one embodiment for a deuterium-containing reagent. Depending on the desired sites of deuteration, in some cases deuterium from D2O can be incorporated either directly into the compounds or into reagents that are useful for synthesizing such compounds (Esaki et al., Tetrahedron, 2006, 62, 10954; Esaki et al., Chem. Eur. J., 2007, 13, 4052). Deuterium gas is also a useful reagent for incorporating deuterium into molecules. Catalytic deuteration of olefinic bonds (H. J. Leis et al., Curr. Org. Chem., 1998, 2, 131; J. R. Morandi et al., J. Org. Chem., 1969, 34 (6), 1889) and acetylenic bonds (N. H. Khan, J. Am. Chem. Soc., 1952, 74 (12), 3018; S. Chandrasekhar et al., Tetrahedron, 2011, 52, 3865) is a rapid route for incorporation of deuterium. Metal catalysts (i.e. Pd, Pt, and Rh) in the presence of deuterium gas can be used to directly exchange deuterium for hydrogen in functional groups containing hydrocarbons (J. G. Atkinson et al., U.S. Pat. No. 3,966,781). A variety of deuterated reagents and synthetic building blocks are commercially available from companies such as for example C/D/N Isotopes, Quebec, Canada; Cambridge Isotope Laboratories Inc., Andover, MA, USA; and CombiPhos Catalysts, Inc., Princeton, NJ, USA. Further information on the state of the art with respect to deuterium-hydrogen exchange is given for example in Hanzlik et al., J. Org. Chem. 55, 3992-3997, 1990; R. P. Hanzlik et al., Biochem. Biophys. Res. Commun. 160, 844, 1989; P. J. Reider et al., J. Org. Chem. 52, 3326-3334, 1987; M. Jarman et al., Carcinogenesis 16(4), 683-688, 1993; J. Atzrodt et al., Angew. Chem., Int. Ed. 2007, 46, 7744; K. Matoishi et al., J. Chem. Soc, Chem. Commun. 2000, 1519-1520; K. Kassahun et al., WO2012/112363.
The term “deuterium-containing compound of general formula (I)” is defined as a compound of general formula (I), in which one or more hydrogen atom(s) is/are replaced by one or more deuterium atom(s) and in which the abundance of deuterium at each deuterated position of the compound of general formula (I) is higher than the natural abundance of deuterium, which is about 0.015%. Particularly, in a deuterium-containing compound of general formula (I) the abundance of deuterium at each deuterated position of the compound of general formula (I) is higher than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, in one embodiment higher than 90%, 95%, 96% or 97%, in other embodiments higher than 98% or 99% at said position(s). It is understood that the abundance of deuterium at each deuterated position is independent of the abundance of deuterium at other deuterated position(s).
The selective incorporation of one or more deuterium atom(s) into a compound of general formula (I) may alter the physicochemical properties (such as for example acidity [A. Streitwieser et al., J. Am. Chem. Soc., 1963, 85, 2759; C. L. Perrin, et al., J. Am. Chem. Soc., 2007, 129, 4490], basicity [C. L. Perrin, et al., J. Am. Chem. Soc., 2003, 125, 15008; C. L. Perrin in Advances in Physical Organic Chemistry, 44, 144; C. L. Perrin et al., J. Am. Chem. Soc., 2005, 127, 9641], lipophilicity [B. Testa et al., Int. J. Pharm., 1984, 19(3), 271]) and/or the metabolic profile of the molecule and may result in changes in the ratio of parent compound to metabolites or in the amounts of metabolites formed. Such changes may result in certain therapeutic advantages and hence may be preferred in some circumstances. Reduced rates of metabolism and metabolic switching, where the ratio of metabolites is changed, have been reported (D. J. Kushner et al., Can. J. Physiol. Pharmacol., 1999, 77, 79; A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). These changes in the exposure to parent drug and metabolites can have important consequences with respect to the pharmacodynamics, tolerability and efficacy of a deuterium-containing compound of general formula (I). In some cases deuterium substitution reduces or eliminates the formation of an undesired or toxic metabolite and enhances the formation of a desired metabolite (e.g. Nevirapine: A. M. Sharma et al., Chem. Res.Toxicol., 2013, 26, 410; Uetrecht et al., Chemical Research in Toxicology, 2008, 21, 9, 1862; Efavirenz: A. E. Mutlib et al., Toxicol. Appl. Pharmacol., 2000, 169, 102). In other cases the major effect of deuteration is to reduce the rate of systemic clearance. As a result, the biological half-life of the compound is increased. The potential clinical benefits would include the ability to maintain similar systemic exposure with decreased peak levels and increased trough levels. This could result in lower side effects and enhanced efficacy, depending on the particular compound's pharmacokinetic/pharmacodynamic relationship. Indiplon (A. J. Morales et al., Abstract 285, The 15th North American Meeting of the International Society of Xenobiotics, San Diego, CA, Oct. 12-16, 2008), ML-337 (C. J. Wenthur et al., J. Med. Chem., 2013, 56, 5208), and Odanacatib (K. Kassahun et al., WO2012/112363) are examples for this deuterium effect. Still other cases have been reported in which reduced rates of metabolism result in an increase in exposure of the drug without changing the rate of systemic clearance (e.g. Rofecoxib: F. Schneider et al., Arzneim. Forsch. Drug. Res., 2006, 56, 295; Telaprevir: F. Maltais et al., J. Med. Chem., 2009, 52, 7993). Deuterated drugs showing this effect may have reduced dosing requirements (e.g. lower number of doses or lower dosage to achieve the desired effect) and/or may produce lower metabolite loads.
A compound of general formula (I) may have multiple potential sites of attack for metabolism. To optimize the above-described effects on physicochemical properties and metabolic profile, deuterium-containing compounds of general formula (I) having a certain pattern of one or more deuterium-hydrogen exchange(s) can be selected. Particularly, the deuterium atom(s) of deuterium-containing compound(s) of general formula (I) is/are attached to a carbon atom and/or is/are located at those positions of the compound of general formula (I), which are sites of attack for metabolizing enzymes such as e.g. cytochrome P450.
Where the plural form of the word compounds, salts, polymorphs, hydrates, solvates and the like, is used herein, this is taken to mean also a single compound, salt, polymorph, isomer, hydrate, solvate or the like.
By “stable compound” or “stable structure” is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
The compounds of this invention may contain one or more asymmetric centre, depending upon the location and nature of the various substituents desired. Asymmetric carbon atoms may be present in the (R) or (S) configuration, resulting in racemic mixtures in the case of a single asymmetric centre, and diastereomeric mixtures in the case of multiple asymmetric centres. In certain instances, asymmetry may also be present due to restricted rotation about a given bond, for example, the central bond adjoining two substituted aromatic rings of the specified compounds.
Substituents on a ring may also be present in either cis or trans form. It is intended that all such configurations (including enantiomers and diastereomers), are included within the scope of the present invention.
Preferred compounds are those which produce the more desirable biological activity. Separated, pure or partially purified isomers and stereoisomers or racemic or diastereomeric mixtures of the compounds of this invention are also included within the scope of the present invention. The purification and the separation of such materials can be accomplished by standard techniques known in the art.
The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example, by the formation of diastereoisomeric salts using an optically active acid or base or formation of covalent diastereomers. Examples of appropriate acids are tartaric, diacetyltartaric, ditoluoyltartaric and camphorsulfonic acid. Mixtures of diastereoisomers can be separated into their individual diastereomers on the basis of their physical and/or chemical differences by methods known in the art, for example, by chromatography or fractional crystallisation. The optically active bases or acids are then liberated from the separated diastereomeric salts. A different process for separation of optical isomers involves the use of chiral chromatography (e.g., chiral HPLC columns), with or without conventional derivatisation, optimally chosen to maximise the separation of the enantiomers. Suitable chiral HPLC columns are manufactured by Daicel, e.g., Chiracel OD and Chiracel OJ among many others, all routinely selectable. Enzymatic separations, with or without derivatisation, are also useful. The optically active compounds of this invention can likewise be obtained by chiral syntheses utilizing optically active starting materials.
In order to limit different types of isomers from each other reference is made to IUPAC Rules Section E (Pure Appl Chem 45, 11-30, 1976).
The present invention includes all possible stereoisomers of the compounds of the present invention as single stereoisomers, or as any mixture of said stereoisomers, e.g. R- or S-isomers, or E- or Z-isomers, in any ratio. Isolation of a single stereoisomer, e.g. a single enantiomer or a single diastereomer, of a compound of the present invention may be achieved by any suitable state of the art method, such as chromatography, especially chiral chromatography, for example.
Further, the compounds of the present invention may exist as tautomers. For example, any compound of the present invention which contains a pyrazole moiety as a heteroaryl group for example can exist as a 1H tautomer, or a 2H tautomer, or even a mixture in any amount of the two tautomers, or a triazole moiety for example can exist as a 1H tautomer, a 2H tautomer, or a 4H tautomer, or even a mixture in any amount of said 1H, 2H and 4H tautomers, namely:
The present invention includes all possible tautomers of the compounds of the present invention as single tautomers, or as any mixture of said tautomers, in any ratio.
Further, the compounds of the present invention can exist as N-oxides, which are defined in that at least one nitrogen of the compounds of the present invention is oxidised. The present invention includes all such possible N-oxides.
The present invention also relates to useful forms of the compounds as disclosed herein, such as metabolites, hydrates, solvates, prodrugs, salts, in particular pharmaceutically acceptable salts, and co-precipitates.
The compounds of the present invention can exist as a hydrate, or as a solvate, wherein the compounds of the present invention contain polar solvents, in particular water, methanol or ethanol for example as structural element of the crystal lattice of the compounds. The amount of polar solvents, in particular water, may exist in a stoichiometric or non-stoichiometric ratio. In the case of stoichiometric solvates, e.g. a hydrate, hemi-, (semi-), mono-, sesqui-, di-, tri-, tetra-, penta- etc. solvates or hydrates, respectively, are possible. The present invention includes all such hydrates or solvates.
Further, the compounds of the present invention can exist in free form, e.g. as a free base, or as a free acid, or as a zwitterion, or can exist in the form of a salt. Said salt may be any salt, either an organic or inorganic addition salt, particularly any pharmaceutically acceptable organic or inorganic addition salt, customarily used in pharmacy.
The term “pharmaceutically acceptable salt” refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present invention. For example, see S. M. Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci. 1977, 66, 1-19.
A suitable pharmaceutically acceptable salt of the compounds of the present invention may be, for example, an acid-addition salt of a compound of the present invention bearing a nitrogen atom, in a chain or in a ring, for example, which is sufficiently basic, such as an acid-addition salt with an inorganic acid, such as hydrochloric, hydrobromic, hydroiodic, sulfuric, bisulfuric, phosphoric or nitric acid, for example, or with an organic acid, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic, cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic, pamoic, pectinic, persulfuric, 3-phenylpropionic, picric, pivalic, 2-hydroxyethanesulfonate, itaconic, sulfamic, trifluoromethanesulfonic, dodecylsulfuric, ethansulfonic, benzenesulfonic, para-toluenesulfonic, methansulfonic, 2-naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid, citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic, adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic, glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, hemisulfuric or thiocyanic acid, for example.
Further, another suitably pharmaceutically acceptable salt of a compound of the present invention which is sufficiently acidic, is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically acceptable cation, for example a salt with N-methyl-glucamine, dimethyl-glucamine, ethyl-glucamine, lysine, dicyclohexylamine, 1,6-hexadiamine, ethanolamine, glucosamine, sarcosine, serinol, tris-hydroxy-methyl-aminomethane, aminopropandiol, sovak-base, 1-amino-2,3,4-butantriol. Additionally, basic nitrogen containing groups may be quaternised with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, and dibutyl sulfate; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and strearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others.
Those skilled in the art will further recognise that acid addition salts of the claimed compounds may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods. Alternatively, alkali and alkaline earth metal salts of acidic compounds of the invention are prepared by reacting the compounds of the invention with the appropriate base via a variety of known methods.
The present invention includes all possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio.
In the present text, in particular in the Experimental Section, for the synthesis of intermediates and of examples of the present invention, when a compound is mentioned as a salt form with the corresponding base or acid, the exact stoichiometric composition of said salt form, as obtained by the respective preparation and/or purification process, is, in most cases, unknown.
Unless specified otherwise, suffixes to chemical names or structural formulae such as “hydrochloride”, “trifluoroacetate”, “sodium salt”, or “x HCl”, “x CF3COOH”, “x Na+”, for example, are to be understood as not a stoichiometric specification, but solely as a salt form.
This applies analogously to cases in which synthesis intermediates or example compounds or salts thereof have been obtained, by the preparation and/or purification processes described, as solvates, such as hydrates with (if defined) unknown stoichiometric composition.
The salts include water-insoluble and, particularly, water-soluble salts.
Furthermore, derivatives of the compounds of formula (I) and the salts thereof which are converted into a compound of formula (I) or a salt thereof in a biological system (bioprecursors or pro-drugs) are covered by the invention. Said biological system is e.g. a mammalian organism, particularly a human subject. The bioprecursor is, for example, converted into the compound of formula (I) or a salt thereof by metabolic processes.
As used herein, the term “in vivo hydrolysable ester” is understood as meaning an in vivo hydrolysable ester of a compound of the present invention containing a carboxy or hydroxy group, for example, a pharmaceutically acceptable ester which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters for carboxy include for example alkyl, cycloalkyl and optionally substituted phenylalkyl, in particular benzyl esters, C1-C6 alkoxymethyl esters, e.g. methoxymethyl, C1-C6 alkanoyloxymethyl esters, e.g. pivaloyloxymethyl, phthalidyl esters, C3-C8 cycloalkoxy-carbonyloxy-C1-C6 alkyl esters, e.g. 1-cyclohexylcarbonyloxyethyl, 1,3-dioxolen-2-onylmethyl esters, e.g. 5-methyl-1,3-dioxolen-2-onylmethyl, and C1-C6-alkoxycarbonyloxyethyl esters, e.g. 1-methoxycarbonyloxyethyl, and may be formed at any carboxy group in the compounds of this invention.
An in vivo hydrolysable ester of a compound of the present invention containing a hydroxy group includes inorganic esters such as phosphate esters and [alpha]-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group. Examples of [alpha]-acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. A selection of in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl. The present invention covers all such esters.
Furthermore, the present invention includes all possible crystalline forms, or polymorphs, of the compounds of the present invention, either as single polymorphs, or as a mixture of more than one polymorphs, in any ratio.
In the context of the properties of the compounds of the present invention the term “pharmacokinetic profile” means one single parameter or a combination thereof including permeability, bioavailability, exposure, and pharmacodynamic parameters such as duration, or magnitude of pharmacological effect, as measured in a suitable experiment. Compounds with improved pharmacokinetic profiles can, for example, be used in lower doses to achieve the same effect, may achieve a longer duration of action, or a may achieve a combination of both effects.
The term “combination” in the present invention is used as known to persons skilled in the art and may be present as a fixed combination, a non-fixed combination or kit-of-parts.
A “fixed combination” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein the said first active ingredient and the said second active ingredient are present together in one unit dosage or in a single entity. One example of a “fixed combination” is a pharmaceutical composition wherein the said first active ingredient and the said second active ingredient are present in admixture for simultaneous administration, such as in a formulation. Another example of a “fixed combination” is a pharmaceutical combination wherein the said first active ingredient and the said second active ingredient are present in one unit without being in admixture.
A non-fixed combination or “kit-of-parts” in the present invention is used as known to persons skilled in the art and is defined as a combination wherein the said first active ingredient and the said second active ingredient are present in more than one unit. One example of a non-fixed combination or kit-of-parts is a combination wherein the said first active ingredient and the said second active ingredient are present separately. The components of the non-fixed combination or kit-of-parts may be administered separately, sequentially, simultaneously, concurrently or chronologically staggered. Any such combination of a compound of formula (I) of the present invention with an anti-cancer agent as defined below is an embodiment of the invention.
The term “(chemotherapeutic) anti-cancer agents” relates to any agent that reduces the survival or proliferation of a cancer cell, and includes but is not limited to 131I-chTNT, abarelix, abiraterone, aclarubicin, ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alemtuzumab, Alendronic acid, alitretinoin, altretamine, amifostine, aminoglutethimide, Hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione, angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide, asparaginase, axitinib, azacitidine, basiliximab, belotecan, bendamustine, belinostat, bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, bortezomib, buserelin, bosutinib, brentuximab vedotin, busulfan, cabazitaxel, cabozantinib, calcium folinate, calcium levofolinate, capecitabine, capromab, carboplatin, carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, copanlisib, crisantaspase, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin+estrone, dronabinol, eculizumab, edrecolomab, elliptinium acetate, eltrombopag, endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine dihydrochloride, histrelin, hydroxycarbamide, 1-125 seeds, Iansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, interferon alfa, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole, ixabepilone, Ianreotide, lapatinib, lasocholine, lenalidomide, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, Ionidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate, methoxsalen, methylaminolevulinate, methylprednisolone, methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphine hydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin, naloxone+pentazocine, naltrexone, nartograstim, nedaplatin, nelarabine, neridronic acid, nivolumabpentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, omacetaxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palifermin, palladium-103 seed, palonosetron, pamidronic acid, panitumumab, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane, perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate, polyvinylpyrrolidone+sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, poziotinib, pralatrexate, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, razoxane, refametinib, regorafenib, risedronic acid, rhenium-186 etidronate, rituximab, romidepsin, romiplostim, romurtide, roniciclib, samarium (153Sm) lexidronam, sargramostim, satumomab, secretin, sipuleucel-T, sizofiran, sobuzoxane, sodium glycididazole, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, tamibarotene, tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur, tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa, thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine+tipiracil, trilostane, triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.
By “Epidermal Growth Factor Receptor (EGFR) Polypeptide” is meant a polypeptide having at least about 95% amino acid sequence identity to the sequence provided at UniProt Accession No. P00533-1 or a fragment thereof. In some embodiments, the EGFR fragment binds an EFGR ligand and/or has kinase activity. Mutant EGFR polypeptides include those having an insertion between, for example, amino acids V769 and D770 or between D770 and N771. In other embodiments, the amino acid sequence identity is 96, 97, 98, 99, or 100% to UniProt Accession No. P00533-1.
An exemplary full length sequence of human EGFR, which indicates V769, D770, and N771 in bold, is provided at UniProt Accession No. P00533-1, which is reproduced below:
An exemplary polynucleotide encoding EGFR is provided at NCBI Reference Sequence: NM_001346897.1, which is reproduced below:
The intermediates used for the synthesis of the compounds of claims 1-4 as described below, as well as their use for the synthesis of the compounds of claims 1-4, are one further aspect of the present invention. Preferred intermediates are the Intermediate Examples as disclosed below.
The compounds according to the invention can be prepared according to the following schemes 1-5.
The schemes and procedures described below illustrate synthetic routes to the compounds of general formula (I) of the invention and are not intended to be limiting. It is obvious to the person skilled in the art that the order of transformations as exemplified in the schemes can be modified in various ways. The order of transformations exemplified in the schemes is therefore not intended to be limiting. In addition, interconversion of any of the substituents R1, R2, R3, R4, R5 and PG can be achieved before and/or after the exemplified transformations. These modifications can be such as the introduction of protecting groups, cleavage of protecting groups, reduction or oxidation of functional groups, halogenation, metallation, substitution or other reactions known to the person skilled in the art. These transformations include those which introduce a functionality which allows for further interconversion of substituents. Appropriate protecting groups and their introduction and cleavage are well-known to the person skilled in the art. Specific examples are described in the subsequent paragraphs.
Scheme 1: Route for the preparation of compounds of general formula (I), wherein R1, R2, R3, R4 and R5 have the meaning as given for general formula (I) and PG can be hydrogen or optionally a suitable protecting group, e.g. tert-butoxycarbonyl (Boc).
Compound of formula 1, 2, and 4 are either commercially available or can be prepared according to procedures available from the public domain, as understandable to the person skilled in the art. Specific examples are described in the subsequent paragraphs.
A suitably substituted piperadine-2,4-diones of general formula (Compound of formula 1), such as, for example, 2,4-piperadinedione, can be reacted with a suitably substituted isothiocyanate (Compound of formula 2), such as, for example, 3-fluorophenylisothiocyanate, in a suitable solvent system, such as, for example, acetonitrile, in the presence of a suitable base, such as, for example, triethylamine or DBU, at temperatures ranging from −78° C. to +100° C., in some embodiments the reaction is carried out at 0° C. or +100° C., to furnish general formula (3). Similar reactions have been performed in the literature (D. E. Worrall, J. Am. Chem. Soc., 1940, 62, 675).
Intermediates of general formula (3) can be converted to Intermediates of general formula (5) by reaction with a suitable amine (compounds of general formula 4), such as, for example 4-(aminomethyl)pyridine, in a suitable solvent system, such as, for example, ethanol and ethyl acetate, at a temperature between room temperature and the boiling point of the respective solvents, in some embodiments the reaction is carried out at the boiling point of the respective solvents, whereby the water formed in the reaction is removed from the reaction by methods known to those skilled in the art, such as, for example, azeotropic removal of water (Dean-Stark conditions) or with molecular sieves, to furnish general formula (5).
Intermediates of general formula (3) and intermediates of general formula (5) in which PG represents a protecting group can be converted to Intermediates in which PG represents a hydrogen atom using standard deprotection conditions known to those skilled in the art. When PG is a protecting group such as, for example, tert-butoxycarbonyl (Boc), the deprotection can be carried out using acids, such as, for example, hydrochloric acid and trifluoroacetic acid, in a suitable solvent system, such as, for example, dichloromethane and dioxane, at a temperature between 0° C. and the boiling point of the respective solvents, in one embodiment the reaction is carried out at the room temperature, to furnish compounds of general formula (3) and intermediates of general formula (5) whereby PG is hydrogen atom.
Intermediates of general formula (5) are reacted with a base and/or oxidizing reagent, in one embodiment an oxidizing agent, such as, for example hydrogen peroxide or SIBX (stabilized iodoxybenoic acid, in a suitable solvent system, such as, for example, methanol, in a temperature range from −30° C. to the boiling point of the respective solvent, in one embodiment the reaction is carried out at the boiling point of the respective solvent, to furnish compounds of general formula (I). Optionally, these types of reactions can be carried on with an additive, such as, for example, an acid or base, such as, for example, acetic acid or trifluoroacetic acid (not-limiting), and triethylamine or diispropylethylamine (not-limiting).
Intermediates of general formula (5) could be converted to compounds of general formula (I) by thermal heating them in a suitable solvent at elevated temperatures, which could be above the boiling point of the said solvent, such as, for example, RT to +250° C. These reactions could optionally be carried out in vessel whereby the pressure can be increased, such as, for example, in an autoclave. Intermediates of general formula (5) can also be converted to compounds of general formula (I) by thermal heating in the presence of a metal catalyst, such as, for example, palladium on activated charcoal, in a suitable solvent, such as, for example, DMF, DMA, EtOH, MeOH, NMP (not-limiting) at elevated temperatures, such as, for example, RT to +150° C. Optionally, these types of reactions can be carried on with an additive, such as, for example, an acid or base, such as, for example, acetic acid or trifluoroacetic acid (not-limiting), and triethylamine or diispropylethylamine (not-limiting), to furnish compounds of general formula (I).
Scheme 2: Process for the preparation of compounds of general formula (4), wherein R4 and R5 have the meaning as given for general formula (I). Compounds of general formula 6, whereby LG is a leaving group, such as, for example, F, Cl, Br, I or aryl sulfonate such as for example p-toluene sulfonate, or alkyl sulfonate such as for example methane sulfonate or trifluoromethane sulfonate, are commercially available or can be synthesized by those skilled in the art.
Compounds of general formula (6) can be converted to compounds of general formula (7) by treatment with a suitable nucleophile, such as for example, amines, alcohols, metal alkoxides, azides, thiols or metal thiolates, under either basic, neutral, acidic, catalytic conditions, in one embodiment basic conditions, in a suitable solvent or using the nucleophile as solvent, such as, for example, DMF, tetrahydrofuran (THF), in a temperature range from −78° C. to the boiling point of the respective solvent, in one embodiment the reaction is carried out −10° C. to the boiling point of the respective solvent, to furnish general formula (7). Such substitution reactions have been previously reported (Clark et al., J. Med. Chem., 2008, 51, 6631-6634; Guo et al., Tetrahedron Letts., 2013, 54, 3233-3237; Watterson et al., J. Med. Chem., 2007, 50, 3730-3742; Bellale et al., J. Med. Chem., 2014, 57, 6572-6582; Klimesova et al., Eur. J. Med. Chem., 1996, 31, 389-395; Leroy et al., Synth. Commun., 1997, 27, 2905-2916; LaMattina et al., J. Org. Chem., 1981, 46, 4179-4182; Beugelmans et al., Tetrahedron, 1983, 39, 4153-4162).
Compounds of general formula (7) can be converted to compounds of general formula (4) by many reducing methods known to those skilled in the art, using numerous different reagents and reaction conditions; such methods and reagents can be carried out with metal hydrides, such as, for example, lithium aluminum hydride in THF (Bullock et al., J. Am. Chem. Soc., 1956, 78, 490, Wang et al., J. Org. Chem., 2006, 71, 4021-3160), or using zinc in acetic acid (Rabe, Chem. Ber., 1913, 46, 1024), or using diborane (De Munno et al., Heterocycles, 1996, 43, 1893-1900), or using catalytic hydrogenation methods, for example, hydrogen and palladium on carbon under acidic conditions (Stokker et al., J. Med. Chem., 1981, 24, 115-117; Bertini et al., J. Med. Chem., 2005, 48, 664-670), hydrogen and nickel under basic conditions (Walpole et al., J. Med. Chem., 1993, 36, 2362-2372, Kuramochi et al., Bioorg. Med. Chem., 2005, 13, 4022-4036.)
Scheme 3: Process for the preparation of compounds of general formula 2, wherein R1a represents methyl or difluoromethyl corresponding to R1 in the general formula (I) with the meening of methoxy and difluoromethoxy. The synthesis of compounds 9 and 10 relates to alkoxy substitution of the phenyl ring. However, the isothiocyanate containing product 2 and the synthesis thereof (i.e., 10→2 or 11→2) is general to R1 groups according to general formula (I).
Compounds of general formula (8), can be converted to compounds of general formula (9), using various methods which are known to those skilled in the art. Such transformations could be, for example, to alkylate the phenolic alcohol with alkylating reagents, such as, for example, alkyl halides, alkyl sulfonates, in which these alkyl groups can optionally contain fluorides, alkoxyl groups. These alkylation reactions are known to those skilled in the art using a variety of methods: i) K2CO3 in a solvent such as, DMF, acetone, DMFA (see the teachings of Muro et al., J. Med. Chem., 2009, 52, 7974 and WO2009/20990 A1); ii) KOH in EtOH (see the teachings of Macias et al., J. Agric. Food Chem., 2006, 54, 9843); iii) Mitsunobu reaction (see the teachings of US2006/122168 A1 and EP2151431 A1) to furnish intermediates of general formula (9).
Compounds of general formula (9) can be converted to compounds of general formula (10) by reduction methods and these methods are known to those skilled in the art. These reductions can be carried using: i) hydrogen gas and a catalyst (for Pd/C as catalyst see the teachings of Chan et al., J. Am. Chem. Soc., 2011, 133, 2989; for platinum see the teachings of Niemann et al., J. Am. Chem Soc., 1941, 63, 2204; for Raney-Nickel see the teachings of US2009/253767 A1); ii) iron and ammonium chloride (see the teachings of Sweeney et al., Bioorg. Med. Chem. Lett., 2008, 18, 4348); iii) sodium dithionite (see the teachings of Chong et al., J. Med. Chem., 2012, 55, 10601); iv) zinc and ammonium chloride (see the teachings of WO2010/42699 A1) to furnish intermediates of general formula (10).
Compounds of general formula (10) can be converted to compounds of general formula (2) by using reagents such as, for example, thiophosgene, carbon disulphide, 1,1″-thiocarbonyldi-2(1H)-pyridone or 1,1′-thiocarbonyldiimidazole, in one embodiment thiophosgene, under basic conditions, in a suitable solvent, such as, for example, dichloromethane, chloroform, acetone, or biphasic mixtures, such as, for example, dichloromethane, chloroform with aqueous basic solutions, in another embodiment, dichloromethane with an aqueous saturated solution of sodium hydrogen carbonate or sodium carbonate, in a temperature range from −78° C. to the boiling point of the respective solvent, in another embodiment the reaction is carried out 0° C. to room temperature, to furnish compounds of general formula (2). Such transformations reactions have been previously reported (Harris et al., J. Med. Chem., 2005, 48, 1610; Degorce et al., Tetrahedron Lett., 2011, 52, 6719; WO2016/91845 A1; Fairhurst et al., Org. Lett., 2005, 7, 4697; Chaskar et al., Synth. Commun., 2008, 38, 16940; US2004/122237 A1).
Scheme 4: Route for the preparation of compounds of general formula (I), wherein R1, R2, R3, R4 and R5 have the meaning as given for general formula (I) and PG represents hydrogen or a suitable protecting group, e.g. tert-butoxycarbonyl (Boc).
Compounds similar to those of general formula 12 are known to those skilled in the art and their syntheses have been reported in the literature (see the teachings of Voss et al., WO2015/22073 A1; Hart et al., WO2016/100166 A1; Anderson et al., J. Med. Chem., 2007, 50, 2647; Vanotti et al., J. Med. Chem., 2008, 51, 487).
Compounds of general formula (12) could be converted to compounds of general formula (13) using standard bromination methods which are known to those skilled in the art (WO2016/100166 A1). Such brominations could be carried out using a brominating agent, such as, for example, N-bromosuccinimide, in a suitable solvent, such as, for example, DMF, in a temperature range from −78° C. to the boiling point of said solvent, in one embodiment the temperature range is 0° C. to RT.
Intermediates of general formula (13) can be reacted with suitable anilines, such as, for example, 2-difluoromethoxyaniline, in the presence of a base, such as, for example, lithium bis(trimethylsilyl)amide (LHMDS), in the presence of a catalyst, such as, for example a suitable ligand, in one embodiment 2-(di-tert-butylphosphino)-2′,4′,6′-triisopropyl-3,6-dimethoxy-1,1′-biphenyl (tBuBrettPhos) and in the presence of a pre-catalyst, such as, for example a palladium pre-catalyst, in another embodiment chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2′,4′,6′-triisopropyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl]palladium(II) (BrettPhos-PreCat MTBE ether adduct) in a suitable solvent system, such as, for example, tetrahydrofuran (THF), at a temperature range of 0° C. to 200° C. In one embodiment, the reaction is carried out at 80° C., to furnish compounds of general formula (I). Similar transformations have been carried out and have been reported (WO2015/193339 A1).
Scheme 5: Route for the preparation of compounds of general formula (I), wherein R1, R2, R3, R4 and R5 have the meaning as given for general formula (I) and PG represents hydrogen or a suitable protecting group, e.g. tert-butoxycarbonyl (Boc).
Compounds similar to those of general formula (14) can be prepared according to the procedure described in Scheme 1 by using 4-(aminomethyl)-3-hydroxypyridine instead of intermediate (4). Intermediates of general formula (14) can be converted to compounds of general formula (I) by reaction with a suitable alcohol under Mitsunobu conditions such as, for example, oxetan-3-ylmethanol, in the presence of (tributylphosphoranylidene)acetonitrile or triphenylphosphin together with diisopropyl azodicarboxylate in a suitable solvent system, such as, for example, dioxane or THF, at a temperature between room temperature and the boiling point of the respective solvents.
It is known to the person skilled in the art that, if there are a number of reactive centers on a starting or intermediate compound, it may be necessary to block one or more reactive centers temporarily by protective groups in order to allow a reaction to proceed specifically at the desired reaction center.
The compounds according to the invention are isolated and purified in a manner known per se, e.g. by distilling off the solvent in vacuo and recrystallizing the residue obtained from a suitable solvent or subjecting it to one of the customary purification methods, such as chromatography on a suitable support material. Furthermore, reverse phase preparative HPLC may be applied. The compounds of the present invention which possess a sufficiently basic or acidic functionality, may result as a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. Salts of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. Additionally, the drying process during the isolation of the compounds of the present invention may not fully remove traces of cosolvents, especially such as formic acid ortrifluoroacetic acid, to give solvates or inclusion complexes. The person skilled in the art will recognise which solvates or inclusion complexes are acceptable to be used in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base, free acid, solvate, inclusion complex) of a compound of the present invention as isolated and described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity.
Salts of the compounds of formula (I) according to the invention can be obtained by dissolving the free compound in a suitable solvent (for example a ketone such as acetone, methylethylketone or methylisobutylketone, an ether such as diethyl ether, tetrahydrofuran or dioxane, a chlorinated hydrocarbon such as methylene chloride or chloroform, or a low molecular weight aliphatic alcohol such as methanol, ethanol or isopropanol) which contains the desired acid or base, or to which the desired acid or base is then added. The acid or base can be employed in salt preparation, depending on whether a mono- or polybasic acid or base is concerned and depending on which salt is desired, in an equimolar ratio or one differing therefrom. The salts are obtained by filtering, reprecipitating, precipitating with a non-solvent for the salt or by evaporating the solvent. Salts obtained can be converted into the free compounds which, in turn, can be converted into salts. In this manner, pharmaceutically unacceptable salts, which can be obtained, for example, as process products in the manufacturing on an industrial scale, can be converted into pharmaceutically acceptable salts by processes known to the person skilled in the art. Especially preferred are hydrochlorides and the process used in the example section.
Pure diastereomers and pure enantiomers of the compounds and salts according to the invention can be obtained e.g. by asymmetric synthesis, by using chiral starting compounds in synthesis or by splitting up enantiomeric and diasteriomeric mixtures obtained in synthesis.
Enantiomeric and diastereomeric mixtures can be split up into the pure enantiomers and pure diastereomers by methods known to the person skilled in the art. In one embodiment, diastereomeric mixtures are separated by crystallization, in particular fractional crystallization, or chromatography. Enantiomeric mixtures can be separated e.g. by forming diastereomers with a chiral auxillary agent, resolving the diastereomers obtained and removing the chiral auxillary agent. As chiral auxillary agents, for example, chiral acids can be used to separate enantiomeric bases such as e.g. mandelic acid and chiral bases can be used to separate enantiomeric acids by formation of diastereomeric salts. Furthermore, diastereomeric derivatives such as diastereomeric esters can be formed from enantiomeric mixtures of alcohols or enantiomeric mixtures of acids, respectively, using chiral acids or chiral alcohols, respectively, as chiral auxillary agents. Additionally, diastereomeric complexes or diastereomeric clathrates may be used for separating enantiomeric mixtures. Alternatively, enantiomeric mixtures can be split up using chiral separating columns in chromatography. Another suitable method for the isolation of enantiomers is the enzymatic separation.
One preferred aspect of the invention is the process for the preparation of the compounds of claims 1-4 according to the examples as well as the intermediates used for their preparation.
Optionally, compounds of the formula (I) can be converted into their salts, or, optionally, salts of the compounds of the formula (I) can be converted into the free compounds. Corresponding processes are customary for the skilled person.
As mentioned supra, the compounds of the present invention have surprisingly been found to effectively inhibit mutant EGFR in a cell (e.g., a cancer cell) contacted with the compound, thereby inducing cell death (e.g., apoptosis) and may therefore be used for the treatment or prophylaxis of diseases of uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses, or diseases which are accompanied with uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses, particularly in which the uncontrolled cell growth, proliferation and/or survival, inappropriate cellular immune responses, or inappropriate cellular inflammatory responses is mediated by mutant EGFR, such as, for example, benign and malignant neoplasia, more specifically haematological tumours, solid tumours, and/or metastases thereof, e.g. leukaemias and myelodysplastic syndrome, malignant lymphomas, head and neck tumours including brain tumours and brain metastases, tumours of the thorax including non-small cell and small cell lung tumours, gastrointestinal tumours, endocrine tumours, mammary and other gynaecological tumours, urological tumours including renal, bladder and prostate tumours, skin tumours, and sarcomas, and/or metastases thereof, especially haematological tumours, solid tumours, and/or metastases of breast, bladder, bone, brain, central and peripheral nervous system, cervix, colon, endocrine glands (e.g., thyroid and adrenal cortex), endocrine tumours, endometrium, esophagus, gastrointestinal tumours, germ cells, kidney, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, stomach, skin, testis, ureter, vagina and vulva as well as malignant neoplasias including primary tumours in said organs and corresponding secondary tumours in distant organs (“tumour metastases”). Haematological tumours can, e.g., be exemplified by aggressive and indolent forms of leukemia and lymphoma, namely non-Hodgkins disease, chronic and acute myeloid leukemia (CML/AML), acute lymphoblastic leukemia (ALL), Hodgkins disease, multiple myeloma and T-cell lymphoma. Also included are myelodysplastic syndrome, plasma cell neoplasia, paraneoplastic syndromes, and cancers of unknown primary site, as well as AIDS related malignancies.
A further aspect of the invention is the use of the compounds according to formula (I) for the treatment of lung cancer, particularly lung cancer harboring mutant EGFR with exon 20 insertion mutations, more particularly lung cancer harboring V769_770ins ASV and/or D770_N771ins SVD exon 20 insertions, and/or metastases thereof, comprising administering an effective amount of a compound of formula (I).
In accordance with an aspect of the present invention therefore the invention relates to a compound of general formula I, or an N-oxide, a salt, a tautomer or a stereoisomer of said compound, or a salt of said N-oxide, tautomer or stereoisomer particularly a pharmaceutically acceptable salt thereof, or a mixture of same, as described and defined herein, for use in the treatment or prophylaxis of a disease, especially for use in the treatment of a disease.
Another particular aspect of the present invention is therefore the use of a compound of general formula I, described supra, or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, for the prophylaxis or treatment of hyperproliferative disorders or disorders responsive to induction of cell death, i.e., apoptosis.
By “hyperproliferative disease” is meant a disease, such as cancer, associated with inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. The term “inappropriate” within the context of the present invention, in particular in the context of “inappropriate cellular immune responses, or inappropriate cellular inflammatory responses”, as used herein, is to be understood as generally meaning a response, which is less than, or greater than normal, and which is associated with, responsible for, or results in, the pathology of said diseases.
In particular embodiments, the use is in the treatment or prophylaxis of diseases, especially the treatment, wherein the diseases are haematological tumours, solid tumours and/or metastases thereof.
Another aspect is the use of a compound of formula (I) for the prophylaxis and/or treatment of lung cancer, particularly lung cancer harboring mutant EGFR with exon 20 insertion mutations, more particularly lung cancer harboring V769_770ins ASV and/or D770_N771ins SVD exon 20 insertions, and/or metastases thereof, especially preferred for the treatment thereof.
A further aspect of the invention is the use of the compounds according to formula (I) for the treatment of lung cancer, particularly lung cancer harboring a mutant EGFR with in-frame deletions in exon 19 (such as EGFR E746_A750del) or point mutations in exon 21 (e.g. L858R), and/or metastases thereof.
A further aspect of the invention is the use of the compounds according to formula (I) for the treatment of lung cancer, particularly lung cancer harboring a mutant EGFR with a D770_N771insSVD C797S, E746_A750del C797S, or L858R C797S acquired resistance mutation, and/or metastases thereof.
A further aspect of the invention is the use of the compounds according to formula (I) for the treatment of lung cancer, particularly lung cancer harboring a mutant ERBB2 with exon 20 insertion mutations (such as ERBB2 A775_G776insYVMA), and/or metastases thereof.
A further aspect of the invention is the use of the compounds according to formula (I) for the treatment of lung cancer, particularly lung cancer harboring a mutant EGFR with in-frame deletions in exon 19 (such as EGFR E746_A750del) or point mutations in exon 21 (e.g. L858R), and/or metastases thereof.
A further aspect of the invention is the use of the compounds according to formula (I) for the treatment of lung cancer, particularly lung cancer harboring a mutant EGFR with a D770_N771insSVD C797S, E746_A750del C797S, or L858R C797S acquired resistance mutation, and/or metastases thereof.
A further aspect of the invention is the use of the compounds according to formula (I) for the treatment of lung cancer, particularly lung cancer harboring a mutant ERBB2 with exon 20 insertion mutations (such as ERBB2 A775_G776insYVMA), and/or metastases thereof.
Another aspect of the present invention is the use of a compound of formula (I) or a stereoisomer, a tautomer, an N-oxide, a hydrate, a solvate, or a salt thereof, particularly a pharmaceutically acceptable salt thereof, or a mixture of same, as described herein, in the manufacture of a medicament for the treatment or prophylaxis of a disease, wherein such disease is a hyperproliferative disorder or a disorder responsive to induction of cell death e.g., apoptosis. In an embodiment the disease is a haematological tumour, a solid tumour and/or metastases thereof. In another embodiment the disease is lung cancer, particularly lung cancer harboring mutant EGFR with exon 20 insertion mutations, more particularly lung cancer harboring V769_770ins ASV and/or D770_N771ins SVD exon 20 insertions, and/or metastases thereof.
The present invention relates to a method for using the compounds of the present invention and compositions thereof, to treat mammalian hyper-proliferative disorders. Compounds can be utilized to inhibit, block, reduce, decrease, etc., cell proliferation and/or cell division, and/or produce cell death e.g. apoptosis. This method comprises administering to a mammal in need thereof, including a human, an amount of a compound of this invention, or a pharmaceutically acceptable salt, isomer, polymorph, metabolite, hydrate, solvate or ester thereof; etc. which is effective to treat the disorder. Hyper-proliferative disorders include but are not limited, e.g., psoriasis, keloids, and other hyperplasias affecting the skin, benign prostate hyperplasia (BPH), solid tumours, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases. Those disorders also include lymphomas, sarcomas, and leukaemias.
Examples of breast cancer include, but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
Examples of cancers of the respiratory tract include, but are not limited to small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.
Examples of brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumour.
Tumours of the male reproductive organs include, but are not limited to prostate and testicular cancer. Tumours of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus.
Tumours of the digestive tract include, but are not limited to anal, colon, colorectal, oesophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.
Tumours of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, urethral and human papillary renal cancers.
Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.
Examples of liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, inverted sinonasal papilloma, inverted sinonasal papilloma-associated sinonasal squamous cell carcinoma, Merkel cell skin cancer, and non-melanoma skin cancer.
Head-and-neck cancers include, but are not limited to laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, inverted sinonasal papilloma, inverted sinonasal papilloma-associated sinonasal squamous cell carcinoma, lip and oral cavity cancer and squamous cell. Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of the central nervous system.
Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.
Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
These disorders have been well characterized in humans, but also exist with a similar etiology in other mammals, and can be treated by administering pharmaceutical compositions of the present invention.
The term “treating” or “treatment” as stated throughout this document is used conventionally, e.g., the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of, etc., of a disease or disorder, such as a carcinoma.
The present invention relates to a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a compound of general formula (I) as defined herein.
The present invention relates to a method of treating cancer in a subject, wherein the cancer is or has acquired resistance to an anti-EGF receptor therapy, the method comprising administering to the subject an effective amount of a compound of general formula (I) as defined herein.
The present invention relates to a method of enhancing the efficacy of an anti-EGF-receptor therapy, the method comprising administering to the subject an anti-EGF receptor therapy in combination with a a compound of general formula (I) as defined herein.
In a further embodiment, the present invention relates to a method of treating cancer in a subject, wherein the cancer is selected from the group consisting of leukemia, myelodysplastic syndrome, malignant lymphoma, head and neck tumours, tumours of the thorax, gastrointestinal tumours, endocrine tumours, mammary and other gynaecological tumours, urological tumours, skin tumours, and sarcomas, the method comprising administering to the subject an effective amount of a compound of general formula (I) as defined herein.
In a further embodiment, the present invention relates to a method of treating cancer in a subject, wherein the cancer is selected from the group consisting of inverted sinonasal papilloma or inverted sinonasal papilloma associated sinanonasal squamous cell carcinoma, the method comprising administering to the subject an effective amount of a compound of general formula (I) as defined herein.
In a further embodiment, the present invention relates to a method of treating cancer in a subject, wherein the tumour of the thorax is non-small cell lung cancer, the method comprising administering to the subject an effective amount of a compound of general formula (I) as defined herein.
In a further embodiment, the present invention relates to a method of treating cancer in a subject, wherein the cancer is lung cancer, particularly lung cancer harboring a mutant EGFR with in-frame deletions in exon 19 (such as EGFR E746_A750del) or point mutations in exon 21 (e.g. L858R), and/or metastases thereof, the method comprising administering to the subject an effective amount of a compound of general formula (I) as defined herein.
In a further embodiment, the present invention relates to a method of treating cancer in a subject, wherein the cancer is lung cancer, particularly lung cancer harboring a mutant EGFR with a D770_N771 insSVD C797S, E746_A750del C797S, or L858R C797S acquired resistance mutation, and/or metastases thereof, the method comprising administering to the subject an effective amount of a compound of general formula (I) as defined herein.
In a further embodiment, the present invention relates to a method of treating cancer in a subject, wherein the cancer is lung cancer, particularly lung cancer harboring a mutant ERBB2 with exon 20 insertion mutations (such as ERBB2 A775_G776insYVMA), and/or metastases thereof, the method comprising administering to the subject an effective amount of a compound of general formula (I) as defined herein.
In a further embodiment, the present invention relates to a method of inhibiting or reducing proliferation or survival of a cancer cell (e.g., lung cancer cell) harboring a mutant EGFR with a D770_N771insSVD C797S, E746_A750del C797S, or L858R C797S acquired resistance mutation, and/or metastases thereof, the method comprising contacting the cell with an effective amount of a compound of general formula (I) as defined herein.
The present disclosure is also related to method of selecting a patient for cancer treatment with a compound of general formula (I) comprising detecting the presence of a mutation in exon 20 of the gene encoding the EGF-receptor in a biological sample of the subject, thereby determining that the patient should be treated with said compound. In some embodiments, the EGFR comprises aD770_N771insSVD C797S, E746_A750del C797S, or L858R C797S acquired resistance mutation, and/or metastases thereof. In some embodiments, the method of selecting a patient for cancer treatment with a compound of general formula (I) may comprise detecting the presence of in-frame deletions in exon 19 or point mutations in exon 21 of the gene encoding EGF-receptor in a biological sample of the subject, thereby determining that the patient should be treated with said compound. For example, the in-frame deletion in exon 19 may be EGFR E746_A750del or the point mutation in exon 21 may be L858R. In some embodiments, the method of selecting a patient for cancer treatment with a compound of general formula (I) may comprise detecting the presence of a mutation in exon 20 of the gene encoding ERBB2 in a biological sample of the subject, thereby determining that the patient should be treated with said compound. In some embodiments, the ERBB2 comprises an ERBB2 A775 or_G776insYVMA insertion mutation, and/or metastases thereof. Furthermore, methods of treating a patient with cancer may comprise administering to the subject a compound of general formula (I) (e.g., in combination with anti-EGF receptor therapy), wherein the subject is selected for therapy by detecting the presence of a mutation in EGFR in a biological sample of the subject. Detection of the presence of a mutation in exon 20 is within the skill of one of the art.
In some embodiments, the detection of a mutation (e.g., in an EGFR or a mutaton in exon 20 of the gene encoding EGFR) may be performed by sequencing (e.g., Sanger, Next Generation Sequencing) or a method selected from the group consisting of immunoblotting, mass spectrometry, immunoprecipitation quantitative PCR, Northern Blot, microarray, enzyme-linked immunosorbent assay (ELISA), in situ hybridization, and combinations thereof.
The present invention also provides methods for the treatment of disorders associated with aberrant mitogen extracellular kinase activity, including, but not limited to stroke, heart failure, hepatomegaly, cardiomegaly, diabetes, Alzheimer's disease, cystic fibrosis, symptoms of xenograft rejections, septic shock or asthma.
Effective amounts of compounds of the present invention can be used to treat such disorders, including those diseases (e.g., cancer) mentioned in the Background section above. Nonetheless, such cancers and other diseases can be treated with compounds of the present invention, regardless of the mechanism of action and/or the relationship between the kinase and the disorder.
The phrase “aberrant kinase activity” or “aberrant tyrosine kinase activity,” includes any abnormal expression or activity of the gene encoding the kinase or of the polypeptide it encodes. Examples of such aberrant activity, include, but are not limited to, over-expression of the gene or polypeptide; gene amplification; mutations which produce constitutively-active or hyperactive kinase activity; gene mutations, deletions, substitutions, additions, etc.
The present invention also provides for methods of inhibiting kinase activity, especially of mitogen extracellular kinase, comprising administering an effective amount of a compound of the present invention, including salts, polymorphs, metabolites, hydrates, solvates, prodrugs (e.g.: esters) thereof, and diastereoisomeric forms thereof. Kinase activity can be inhibited in cells (e.g., in vitro), or in the cells of a mammalian subject, especially a human patient in need of treatment.
The present invention also provides methods of treating disorders and diseases associated with excessive and/or abnormal angiogenesis.
Inappropriate and ectopic expression of angiogenesis can be deleterious to an organism. A number of pathological conditions are associated with the growth of extraneous blood vessels. These include, e.g., diabetic retinopathy, ischemic retinal-vein occlusion, and retinopathy of prematurity [Aiello et al. New Engl. J. Med. 1994, 331, 1480; Peer et al. Lab. Invest. 1995, 72, 638], age-related macular degeneration [AMD; see, Lopez et al. Invest. Opththalmol. Vis. Sci. 1996, 37, 855], neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma, inflammation, rheumatoid arthritis (RA), restenosis, in-stent restenosis, vascular graft restenosis, etc. In addition, the increased blood supply associated with cancerous and neoplastic tissue, encourages growth, leading to rapid tumour enlargement and metastasis. Moreover, the growth of new blood and lymph vessels in a tumour provides an escape route for renegade cells, encouraging metastasis and the consequence spread of the cancer. Thus, compounds of the present invention can be utilized to treat and/or prevent any of the aforementioned angiogenesis disorders, e.g., by inhibiting and/or reducing blood vessel formation; by inhibiting, blocking, reducing, decreasing, etc. endothelial cell proliferation or other types involved in angiogenesis, as well as causing cell death e.g. apoptosis of such cell types.
In various embodiments, the diseases of said method are haematological tumours, solid tumour and/or metastases thereof.
The compounds of the present invention can be used in particular in therapy and prevention i.e. prophylaxis, especially in therapy of tumour growth and metastases, especially in solid tumours of all indications and stages with or without pre-treatment of the tumour growth.
This invention also relates to pharmaceutical compositions containing one or more compounds of the present invention. These compositions can be utilised to achieve the desired pharmacological effect by administration to a patient in need thereof. A patient, for the purpose of this invention, is a mammal, including a human, in need of treatment for the particular condition, disorder, or disease.
Therefore, the present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier or auxiliary and a pharmaceutically effective amount of a compound, or salt thereof, of the present invention.
Another aspect of the invention is a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) and a pharmaceutically acceptable auxiliary for the treatment of a disease mentioned supra, especially for the treatment of haematological tumours, solid tumours and/or metastases thereof.
A pharmaceutically acceptable carrier or auxiliary may be a carrier that is non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Carriers and auxiliaries are all kinds of additives assisting to the composition to be suitable for administration.
A pharmaceutically effective amount of compound may be in that amount which produces a result or exerts the intended influence on the particular condition being treated.
The compounds of the present invention can be administered with pharmaceutically-acceptable carriers or auxiliaries well known in the art using any effective conventional dosage unit forms, including immediate, slow and timed release preparations, orally, parenterally, topically, nasally, ophthalmically, optically, sublingually, rectally, vaginally, and the like.
For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions, and may be prepared according to methods known to the art for the manufacture of pharmaceutical compositions. The solid unit dosage forms can be a capsule that can be of the ordinary hard- or soft-shelled gelatine type containing auxiliaries, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.
In another embodiment, the compounds of this invention may be tableted with conventional tablet bases such as lactose, sucrose and cornstarch in combination with binders such as acacia, corn starch or gelatine, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration, such as potato starch, alginic acid, corn starch, and guar gum, gum tragacanth, acacia, lubricants intended to improve the flow of tablet granulation and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example talc, stearic acid, or magnesium, calcium or zinc stearate, dyes, colouring agents, and flavouring agents such as peppermint, oil of wintergreen, or cherry flavouring, intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable excipients for use in oral liquid dosage forms include dicalcium phosphate and diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent or emulsifying agent. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance tablets, pills or capsules may be coated with shellac, sugar or both.
Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example those sweetening, flavouring and colouring agents described above, may also be present.
The pharmaceutical compositions of this invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil such as liquid paraffin or a mixture of vegetable oils. Suitable emulsifying agents may be (1) naturally occurring gums such as gum acacia and gum tragacanth, (2) naturally occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived form fatty acids and hexitol anhydrides, for example, sorbitan monooleate, (4) condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil such as, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent such as, for example, beeswax, hard paraffin, or cetyl alcohol. The suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more colouring agents; one or more flavouring agents; and one or more sweetening agents such as sucrose or saccharin.
Syrups and elixirs may be formulated with sweetening agents such as, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, and preservative, such as methyl and propyl parabens and flavouring and colouring agents.
The compounds of this invention may also be administered parenterally, that is, subcutaneously, intravenously, intraocularly, intrasynovially, intramuscularly, or interperitoneally, as injectable dosages of the compound in, for example, a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-1,1-dioxolane-4-methanol, ethers such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or, a fatty acid glyceride, or an acetylated fatty acid glyceride, with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pectin, carbomers, methycellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agent and other pharmaceutical adjuvants.
Illustrative of oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum and mineral oil. Suitable fatty acids include oleic acid, stearic acid, isostearic acid and myristic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate. Suitable soaps include fatty acid alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamine acetates; anionic detergents, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; non-ionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and poly(oxyethylene-oxypropylene)s or ethylene oxide or propylene oxide copolymers; and amphoteric detergents, for example, alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures.
The parenteral compositions of this invention will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may also be used advantageously. In order to minimise or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) in one embodiment of from about 12 to about 17. The quantity of surfactant in such formulation in one embodiment ranges from about 5% to about 15% by weight. The surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB.
Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
The pharmaceutical compositions may be in the form of sterile injectable aqueous suspensions. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, dispersing or wetting agents which may be a naturally occurring phosphatide such as lecithin, a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate, a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadeca-ethyleneoxycetanol, a condensation product of ethylene oxide with a partial ester derived form a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensation product of an ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, for example polyoxyethylene sorbitan monooleate.
The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Diluents and solvents that may be employed are, for example, water, Ringer's solution, isotonic sodium chloride solutions and isotonic glucose solutions. In addition, sterile fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland, fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables.
A composition of the invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritation excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are, for example, cocoa butter and polyethylene glycol.
Controlled release formulations for parenteral administration include liposomal, polymeric microsphere and polymeric gel formulations that are known in the art.
It may be desirable or necessary to introduce the pharmaceutical composition to the patient via a mechanical delivery device. The construction and use of mechanical delivery devices for the delivery of pharmaceutical agents is well known in the art. Direct techniques for administration, for example, administering a drug directly to the brain usually involve placement of a drug delivery catheter into the patient's ventricular system to bypass the blood-brain barrier. One such implantable delivery system, used for the transport of agents to specific anatomical regions of the body, is described in U.S. Pat. No. 5,011,472, issued Apr. 30, 1991.
The compositions of the invention can also contain other conventional pharmaceutically acceptable compounding ingredients, generally referred to as carriers or diluents, as necessary or desired. Conventional procedures for preparing such compositions in appropriate dosage forms can be utilized.
Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M. F. et al., “Compendium of Excipients for Parenteral Formulations” PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311; Strickley, R. G “Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1” PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349; and Nema, S. et al., “Excipients and Their Use in Injectable Products” PDA Journal of Pharmaceutical Science & Technology 1997, 51(4), 166-171.
Commonly used pharmaceutical ingredients that can be used as appropriate to formulate the composition for its intended route of administration include:
Pharmaceutical compositions according to the present invention can be illustrated as follows:
Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of hyper-proliferative disorders and angiogenic disorders, by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known medicaments that are used to treat these conditions, the effective dosage of the compounds of this invention can readily be determined for treatment of each desired indication. The amount of the active ingredient to be administered in the treatment of one of these conditions can vary widely according to such considerations as the particular compound and dosage unit employed, the mode of administration, the period of treatment, the age and sex of the patient treated, and the nature and extent of the condition treated.
The total amount of the active ingredient to be administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, and in particular embodiments from about 0.01 mg/kg to about 20 mg/kg body weight per day. Clinically useful dosing schedules will range from one to three times a day dosing to once every four weeks dosing. In addition, “drug holidays” in which a patient is not dosed with a drug for a certain period of time, may be beneficial to the overall balance between pharmacological effect and tolerability. A unit dosage may contain from about 0.5 mg to about 1500 mg of active ingredient, and can be administered one or more times per day or less than once a day. The average daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will in other embodiments be from 0.01 to 200 mg/kg of total body weight. The average daily rectal dosage regimen will in particular embodiments be from 0.01 to 200 mg/kg of total body weight. The average daily vaginal dosage regimen will in other embodiments be from 0.01 to 200 mg/kg of total body weight. The average daily topical dosage regimen will in still other embodiments be from 0.1 to 200 mg administered between one to four times daily. The transdermal concentration will in other embodiments be that required to maintain a daily dose of from 0.01 to 200 mg/kg. The average daily inhalation dosage regimen will in other embodiments be from 0.01 to 100 mg/kg of total body weight.
Of course the specific initial and continuing dosage regimen for each patient will vary according to the nature and severity of the condition as determined by the attending diagnostician, the activity of the specific compound employed, the age and general condition of the patient, time of administration, route of administration, rate of excretion of the drug, drug combinations, and the like. The desired mode of treatment and number of doses of a compound of the present invention or a pharmaceutically acceptable salt or ester or composition thereof can be ascertained by those skilled in the art using conventional treatment tests.
The compounds of this invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutical agents where the combination causes no unacceptable adverse effects. Those combined pharmaceutical agents can be other agents having antiproliferative effects such as for example for the treatment of haematological tumours, solid tumours and/or metastases thereof and/or agents for the treatment of undesired side effects. The present invention relates also to such combinations.
Other anti-hyper-proliferative agents suitable for use with the composition of the invention include but are not limited to those compounds acknowledged to be used in the treatment of neoplastic diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics (Ninth Edition), editor Molinoff et al., publ. by McGraw-Hill, pages 1225-1287, (1996), which is hereby incorporated by reference, especially (chemotherapeutic) anti-cancer agents as defined supra. The combination can be a non-fixed combination or a fixed-dose combination as the case may be.
Methods of testing for a particular pharmacological or pharmaceutical property are well known to persons skilled in the art.
The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.
As will be appreciated by persons skilled in the art, the invention is not limited to the particular embodiments described herein, but covers all modifications of said embodiments that are within the spirit and scope of the invention as defined by the appended claims.
The following examples illustrate the invention in greater detail, without restricting it. Further compounds according to the invention, of which the preparation is not explicitly described, can be prepared in an analogous way.
The compounds, which are mentioned in the examples and the salts thereof represent preferred embodiments of the invention as well as a claim covering all subcombinations of the residues of the compound of formula (I) as disclosed by the specific examples.
The term “according to” within the experimental section is used in the sense that the procedure referred to is to be used “analogously to”.
Chemical names were generated using the ACD/Name software from ACD/Labs. In some cases generally accepted names of commercially available reagents were used in place of ACD/Name generated names.
The following table 1 lists the abbreviations used in this paragraph and in the Examples section as far as they are not explained within the text body. Other abbreviations have their meanings customary per se to the skilled person.
Other abbreviations have their meanings customary per se to the skilled person.
The various aspects of the invention described in this application are illustrated by the following examples which are not meant to limit the invention in any way.
The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.
All reagents, for which the synthesis is not described in the experimental part, are either commercially available, or are known compounds or may be formed from known compounds by known methods by a person skilled in the art.
The compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. In some cases, the compounds may be purified by crystallization. In some cases, impurities may be removed by trituration using a suitable solvent. In some cases, the compounds may be purified by chromatography, particularly flash column chromatography, using for example prepacked silica gel cartridges, e.g. Biotage SNAP cartridges KP-Sil® or KP-NH® in combination with a Biotage autopurifier system (SP4® or Isolera Four®) and eluents such as gradients of hexane/ethyl acetate or DCM/methanol. In flash column chromatography, unmodified (“regular”) silica gel may be used as well as aminophase functionalized silica gel. If reference is made to flash column chromatography or to flash chromatography in the experimental section without specification of a stationary phase, regular silica gel was used.
In some cases, the compounds may be purified by preparative HPLC using for example a Waters autopurifier equipped with a diode array detector and/or on-line electrospray ionization mass spectrometer in combination with a suitable prepacked reverse phase column and eluents such as gradients of water and acetonitrile which may contain additives such as trifluoroacetic acid, formic acid or aqueous ammonia.
In some cases, purification methods as described above can provide those compounds of the present invention which possess a sufficiently basic or acidic functionality in the form of a salt, such as, in the case of a compound of the present invention which is sufficiently basic, a trifluoroacetate or formate salt for example, or, in the case of a compound of the present invention which is sufficiently acidic, an ammonium salt for example. A salt of this type can either be transformed into its free base or free acid form, respectively, by various methods known to the person skilled in the art, or be used as salts in subsequent biological assays. It is to be understood that the specific form (e.g. salt, free base etc.) of a compound of the present invention as isolated and as described herein is not necessarily the only form in which said compound can be applied to a biological assay in order to quantify the specific biological activity.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 μm, 50×2.1 mm; eluent A: water+0.1 vol. % formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min. 1-99% B, 1.6-2.0 min. 99% B; flow 0.8 ml/min; temperature: 60° C.; DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 μm, 50×2.1 mm; eluent A: water+0.2 vol. % aqueous ammonia (32%), eluent B: acetonitrile; gradient: 0-1.6 min. 1-99% B, 1.6-2.0 min. 99% B; flow 0.8 ml/min; temperature: 60° C.; DAD scan: 210-400 nm.
Instrument: Waters Acquity UPLC H-Class system; Column: Acquity CSH C18 1.7 μm 2.1×50 mm; eluent A: water+0.1 vol. % formic acid, eluent B: acetonitrile, eluent C: 2 vol. % ammonia (28%) in water, eluent D: 2 vol. % formic acid in water; gradient: 0-1.2 min 2-95% B with A and 5% D throughout, 1.2-1.4 min. 95% B; flow 0.8 ml/min; temperature: 40° C.; PDA: 215-350 nm.
Instrument: Waters Acquity UPLC H-Class system; Column: XBridge BEH C18 2.5 μm 2.1×50 mm; eluent A: water+0.1 vol % formic acid, eluent B: acetonitrile, eluent C: 2 vol % ammonia (28%) in water, eluent D: 2 vol % formic acid in water; gradient: 0-1.2 min 2-95% B with A and 5% C throughout, 1.2-1.4 min 95% B; flow 0.8 ml/min; temperature: 40° C.; PDA: 215-350 nm.
MS instrument: SHIMADZU LCMS-2020; HPLC instrument: LabSolution Version 5.72; Column: Kinetex@5 um EVO C18 30×2.1 mm; eluent A: 0.0375% TFA in water (v/v), eluent B: 0.01875% TFA in acetonitrile: gradient: 0.0 min 0% B→3.00 min 60% B→3.50 min 60% B→3.51 min 0% B→4.00 min 0% B; flow rate: 0.8 mL/mix; oven temperature: 50° C.; UV detection: 220 nm & 254 nm.
Instrument: Agilent 1290 UPLCMS 6230 TOF; Saule: BEH C 18 1.7 μm, 50×2.1 mm; Eluent A: Wasser+0.05% Ameisensaure (99%); Eluent B: Acetonitril+0.05% Ameisensaure (99%); Gradient: 0-1.7 2-90% B, 1.7-2.0 90% B; Fluss 1.2 ml/min; Temperatur: 60° C.; DAD scan: 190-400 nm.
Instrument: Waters Acquity UPLCMS Single Quad; column: Kinetex 2.6 μm, 50×2.1 mm; Eluent A: water+0.05% formic acid (99%); Eluent B: acetonitrile+0.05% formic acid (99%); gradient: 0-1.9 1-99% B, 1.9-2.1 99% B; flow 1.3 ml/min; temperature: 60° C.; DAD scan: 200-400 nm.
Instrument: Waters Autopurification MS SingleQuad; Column: Waters Xbridge C18 5μ 100×30 mm; eluent A: water+0.2 vol. % aqueous ammonia (32%), eluent B: acetonitrile; gradient: 0-5.5 min. 5-100% B; flow 70 ml/min; temperature: 25° C.; DAD scan: 210-400 nm
Instrument: Waters Autopurification MS SingleQuad; Column: Waters XBrigde C18 5μ 50×50 mm; eluent A: water+0.1 vol % formic acid, eluent B: methanol; gradient: 0-0.50 min. 20% B; flow 50 to 100 ml/min, 0.50-8.00 min. 20-60% B; flow 100 ml/min, temperature: 25° C.; DAD scan: 210-400 nm
Instrument: Labomatic HD-5000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 2000, Knauer UV detector Azura UVD 2.1S, Prepcon 5 software. Column: Chromatorex C18 10 μM 120×30 mm; Eluent A: water+0.1% formic acid; Eluent B: acetonitrile; gradient: given for intermediates and examples, rate 150 mL/min, temperature 25° C.; UV 220 nm
Instrument: Labomatic HD-5000, pump head HDK-280, gradient module NDB-1000, fraction collector Labomatic Labocol Vario 2000, Knauer UV detector Azura UVD 2.1S, Prepcon 5 software. Column: Chromatorex C18 10 μM 120×30 mm; Eluent A: 0.1% ammonia in water; Eluent B: acetonitrile; gradient: given for intermediates and examples, rate 150 mL/min, temperature 25° C.; UV 220 nm
The multiplicities of proton signals in 1H NMR spectra given in the following paragraphs reflect the observed signal form and do not take into account any higher-order signal phenomena. As a rule, the chemical shift data refers to the center of the signal in question. In the case of wide multiplets, a range is specified. Signals hidden by solvent or water were either assigned tentatively or are not listed. Strongly broadened signals—e.g. caused by rapid rotation of molecular moieties or by interchanging protons—have also been assigned tentatively (often referred to as a broad multiplet or broad singlet) or are not shown.
The 1H-NMR data of selected compounds are listed in the form of 1H-NMR peaklists. Therein, for each signal peak the δ value in ppm is given, followed by the signal intensity, reported in round brackets. The δ value-signal intensity pairs from different peaks are separated by commas. Therefore, a peaklist is described by the general form: δ1 (intensity1), δ2 (intensity2), . . . , δi (intensityi), . . . , δn (intensityn).
The intensity of a sharp signal correlates with the height (in cm) of the signal in a printed NMR spectrum. When compared with other signals, this data can be correlated to the real ratios of the signal intensities. In the case of broad signals, more than one peak, or the center of the signal along with their relative intensity, compared to the most intense signal displayed in the spectrum, are shown. A 1H-NMR peaklist is similar to a classical 1H-NMR readout, and thus usually contains all the peaks listed in a classical NMR interpretation. Moreover, similar to classical 1H-NMR printouts, peaklists can show solvent signals, signals derived from stereoisomers of the particular target compound, peaks of impurities, 13C satellite peaks, and/or spinning sidebands. The peaks of stereoisomers, and/or peaks of impurities are typically displayed with a lower intensity compared to the peaks of the target compound (e.g., with a purity of >90%). Such stereoisomers and/or impurities may be typical for the particular manufacturing process, and therefore their peaks may help to identify a reproduction of the manufacturing process on the basis of “by-product fingerprints”. An expert who calculates the peaks of the target compound by known methods (MestReC, ACD simulation, or by use of empirically evaluated expectation values), can isolate the peaks of the target compound as required, optionally using additional intensity filters. Such an operation would be similar to peak-picking in classical 1H-NMR interpretation. A detailed description of the reporting of NMR data in the form of peaklists can be found in the publication “Citation of NMR Peaklist Data within Patent Applications” (cf. http://www.researchdisclosure.com/searching-disclosures, Research Disclosure Database Number 605005, 2014, 1 Aug. 2014). In the peak picking routine, as described in the Research Disclosure Database Number 605005, the parameter “MinimumHeight” can be adjusted between 1% and 4%. However, depending on the chemical structure and/or depending on the concentration of the measured compound it may be reasonable to set the parameter “MinimumHeight”<1%.
3-Chloropyridine-4-carbonitrile (CAS 68325-15-5, 1.00 g, 7.22 mmol) and (oxetan-2-yl)methanol (CAS 61266-70-4, 699 mg, 7.94 mmol) were dissolved in THF (50 ml). Potassium tert-butoxide (972 mg, 8.66 mmol) was added and the mixture was stirred for 1.5 h at RT. The reaction mixture was diluted slowly with sat. ammonium chloride solution and extracted with EtOAc (3×). The organic phase was washed with brine and filtered over a water-repellent filter, concentrated under reduced pressure and purified by flash chromatography (silica, EtOAc/EtOH gradient 0-35%) to give 1.00 g of the title compound (66% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.75 (s, 1H), 8.40 (d, 1H), 7.75-7.85 (m, 1H), 5.05 (ddt, 1H), 4.50-4.59 (m, 2H), 4.46 (d, 2H), 2.57-2.82 (m, 2H).
LC-MS (method 2): Rt=0.68 min; MS (ESIpos): m/z=191 [M+H]+
To a solution of [(2R)-oxetan-2-yl]methanol (CAS 1932342-97-6, 1.00 g, 10.8 mmol) in THF (20 ml) at 0° C. was slowly added sodium hydride (518 mg, 12.9 mmol, 60% purity). The reaction mixture was stirred for 3 h at RT. 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 1.49 g, 10.8 mmol) in THF (8 ml) was added and the mixture was stirred overnight. The reaction mixture was quenched with 1N HCl until pH=6. The suspension was filtered through a hydrophobic filter paper and the filter cake was washed with EtOAc. The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography (basic silica, hexane/EtOAc gradient 0-100%) to give 1.41 g of the title compound (69% yield).
LC-MS (method 2): Rt=0.68 min; MS (ESIpos): m/z=191.5 [M+H]+
To a solution of 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 715 mg, 5.16 mmol) and (oxetan-3-yl)methanol (CAS 6246-06-6, 500 mg, 5.67 mmol) in 2-methyl-THF (20 ml) was added potassium tert-butoxide (695 mg, 6.19 mmol) and the mixture stirred at RT for 4 h. The mixture was diluted slowly with water and extracted with EtOAc, the organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, methanol/EtOAc gradient 5/95) to give 890 mg of the title compound (77% yield).
1H NMR (400 MHz, CDCl3): 5 [ppm]=3.50-3.66 (m, 1H), 4.50 (d, 2H), 4.60 (dd, 2H), 4.96 (dd, 2H), 7.48 (d, 1H), 8.43 (d, 1H), 8.55 (s, 1H).
According to the method described for intermediate 1-2 with 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 2.39 g, 17.2 mmol) and (3-ethyloxetan-3-yl)methanol (CAS 3047-32-3, 2.00 g, 17.2 mmol) as the starting materials, 3.07 g of the title compound were prepared (80% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.78 (s, 1H), 8.41 (d, 1H), 7.79 (d, 1H), 4.40-4.51 (m, 4H), 4.36 (d, 2H), 1.82 (q, 2H), 0.92 (t, 3H).
LC-MS (method 2): Rt=0.85 min; MS (ESIpos): m/z=219.1 [M+H]+
According to the method described for intermediate 1-2 with 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 673 mg, 4.86 mmol) and tert-butyl (2S)-2-(hydroxymethyl)azetidine-1-carboxylate (CAS 161511-85-9, 1.00 g, 5.34 mmol) as the starting materials 830 mg of the title compound were prepared (56% yield).
Optical rotation:[α]D=−98.3°+/−0.11° (c=14.9 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=1.30 (s, 9H), 2.17-2.26 (m, 1H), 2.31-2.40 (m, 1H), 3.76 (m, 2H), 4.32-4.40 (m, 1H), 4.49 (td, 1H), 4.59-4.72 (m, 1H), 7.80 (d, 1H), 8.40 (d, 1H), 8.76 (s, 1H).
LC-MS (method 2): Rt=1.05 min; MS (ESIpos): m/z=290.5 [M+H]+
According to the method described for intermediate 1-2 with 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 1.48 g, 10.7 mmol) and tert-butyl (2R)-2-(hydroxymethyl)azetidine-1-carboxylate (CAS 161511-90-6, 2.00 g, 10.7 mmol) as the starting materials 1.97 g of the title compound were prepared (63% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.76 (s, 1H), 8.40 (d, 1H), 7.80 (d, 1H), 4.66 (br d, 1H), 4.49 (tt, 1H), 4.31-4.41 (m, 1H), 3.70-3.90 (m, 2H), 2.31-2.41 (m, 1H), 2.21 (ddt, 1H), 1.30 (s, 9H).
LC-MS (method 2): Rt=1.04 min; MS (ESIpos): m/z=290.2 [M+H]+
According to the method described for intermediate 1-2 with 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 626 mg, 4.52 mmol) and tert-butyl 2-(hydroxymethyl)-2-methylazetidine-1-carboxylate (CAS 850789-22-9, 1.00 g, 4.97 mmol) as the starting materials, 355 mg of the title compound were prepared (25% yield) after flash chromatography (silica, hexane/EtOAc gradient 0-100%, EtOAc/EtOH gradient 10-35%).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=8.69-8.86 (m, 1H), 8.40 (s, 1H), 7.81 (dd, 1H), 4.49-4.72 (m, 1H), 4.15-4.22 (m, 1H), 3.59-3.97 (m, 2H), 2.34-2.45 (m, 1H), 1.98-2.08 (m, 1H), 1.38-1.45 (m, 3H), 1.21-1.31 (m, 9H).
LC-MS (method 2): Rt=1.09 min; MS (ESIpos): m/z=304.3 [M+H]+
According to the method described for intermediate 1-2 with 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 1.38 g, 9.79 mmol) and 2-(oxetan-2-yl)ethan-1-ol (CAS 362604-33-9, 1.00 g, 9.79 mmol) as the starting materials, 1.62 g of the title compound were prepared (71% yield) without further purification.
1H-NMR (400 MHz, DMSO-d6) 5 [ppm]: 0.850 (0.49), 1.166 (0.45), 1.231 (1.32), 2.133 (0.41), 2.153 (2.36), 2.159 (2.42), 2.169 (5.28), 2.173 (6.33), 2.188 (7.07), 2.202 (2.86), 2.204 (2.94), 2.322 (0.47), 2.326 (0.59), 2.331 (0.42), 2.402 (1.14), 2.420 (1.85), 2.425 (1.62), 2.430 (1.63), 2.438 (1.63), 2.443 (2.22), 2.447 (2.50), 2.452 (1.92), 2.461 (1.60), 2.465 (2.18), 2.470 (2.61), 2.522 (1.78), 2.659 (1.66), 2.664 (0.64), 2.669 (0.82), 2.674 (2.11), 2.678 (2.33), 2.680 (2.32), 2.686 (1.55), 2.693 (2.28), 2.699 (2.36), 2.701 (2.06), 2.705 (1.86), 2.707 (1.89), 2.713 (1.79), 2.720 (1.83), 2.722 (1.78), 2.726 (1.52), 2.741 (1.26), 3.159 (2.86), 3.172 (2.76), 4.094 (0.66), 4.108 (0.70), 4.288 (1.56), 4.304 (2.15), 4.306 (2.11), 4.313 (3.31), 4.322 (1.90), 4.328 (4.16), 4.331 (3.82), 4.346 (3.12), 4.353 (0.86), 4.359 (3.06), 4.373 (6.16), 4.384 (2.30), 4.387 (3.55), 4.398 (3.22), 4.414 (3.38), 4.429 (6.11), 4.437 (3.09), 4.443 (3.97), 4.451 (5.85), 4.466 (3.25), 4.515 (3.25), 4.530 (2.86), 4.536 (4.42), 4.549 (3.42), 4.551 (3.42), 4.555 (3.37), 4.570 (2.28), 4.896 (1.13), 4.913 (3.57), 4.930 (4.70), 4.947 (3.43), 4.964 (1.11), 7.459 (1.17), 7.471 (1.22), 7.772 (8.97), 7.774 (8.59), 7.784 (9.37), 7.786 (9.02), 8.377 (11.46), 8.389 (11.04), 8.570 (1.60), 8.582 (1.58), 8.693 (2.34), 8.705 (16.00).
LC-MS (method 2): Rt=0.77 min; MS (ESIpos): m/z=205.3 [M+H]+
According to the method described for intermediate 1-1 with 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 1.23 g, 8.90 mmol) and (2-methyloxetan-2-yl)methanol (CAS 61266-71-5, 1.00 g, 9.79 mmol) as the starting materials, 1.30 g of the title compound were prepared (68% yield) after flash chromatography (silica, hexane/EtOAc gradient 0-100%, EtOAc/EtOH gradient 0-35%).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.71-8.82 (m, 1H), 8.40 (d, 1H), 7.75-7.82 (m, 1H), 4.35-4.50 (m, 1H), 4.33-4.49 (m, 1H), 4.24-4.50 (m, 2H), 2.68-2.77 (m, 1H), 2.40-2.47 (m, 1H), 1.41-1.47 (m, 3H).
LC-MS (method 2): Rt=0.76 min; MS (ESIpos): m/z=205.1 [M+H]+
According to the method described for intermediate 1-2 with 3-chloropyridine-4-carbonitrile (CAS 68325-15-5, 1.22 g, 8.61 mmol) and (3,3-dimethyloxetan-2-yl)methanol (CAS 1346262-59-6, 1.00 g, 8.61 mmol) as the starting materials 1.06 g of the title compound were prepared (56% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.78 (s, 1H), 8.40 (d, 1H), 7.78 (d, 1H), 4.47-4.64 (m, 3H), 4.21-4.25 (m, 2H), 1.31 (s, 3H), 1.20-1.26 (m, 3H).
LC-MS (method 1): Rt=0.91 min; MS (ESIpos): m/z=219.3 [M+H]+
An autoclave was charged with 3-[(oxetan-2-yl)methoxy]pyridine-4-carbonitrile (intermediate 1-1, 5.78 g, 30.4 mmol), ammonia (110 ml, 7.0 M in methanol, 4.9 mol) and Raney-Nickel (CAS 7440-02-0, 4.46 g, 50% wetted) and the mixture was stirred under 25 bar hydrogen atmosphere at RT for 17 h. The mixture was filtered through a pad of celite, eluted with methanol and the combined filtrates were concentrated under reduced pressure. The residue was used directly in the next step without further purification (4.88 g, 74% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.27 (s, 1H), 8.19 (br d, 1H), 7.35-7.48 (m, 1H), 4.95-5.08 (m, 1H), 4.45-4.57 (m, 2H), 4.18-4.29 (m, 2H), 3.69-3.83 (m, 2H), 3.34 (br s, 2H), 2.65-2.76 (m, 1H), 2.52-2.65 (m, 1H).
LC-MS (method 1): Rt=0.52 min; MS (ESIpos): m/z=195.1 [M+H]+
According to the method described for intermediate 2-1 with 3-{[(2R)-oxetan-2-yl]methoxy}pyridine-4-carbonitrile (intermediate 1-2, 1.41 g, 7.41 mmol) as the starting material, 1.16 g of the title compound were prepared (76% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.27 (s, 1H), 8.19 (d, 1H), 7.40 (d, 1H), 5.02 (dddd, 1H), 4.45-4.58 (m, 2H), 4.20-4.27 (m, 2H), 3.74 (s, 2H), 2.57-2.77 (m, 2H), 1.52-2.08 (m, 2H).
To a solution of 3-[(oxetan-3-yl)methoxy]pyridine-4-carbonitrile (intermediate 1-3, 890 mg, 4.68 mmol) in methanol (40 ml) was added palladium on activated carbon (10%, 120.7 mg, 1.13 mmol, slurried in 100 μl toluene) and the mixture was stirred under hydrogen atmosphere for 4 days at RT. The mixture was filtered through celite, washed with ethanol and concentrated under reduced pressure. The residue was used directly in the next step without further purification (790 mg, 66% yield).
1H NMR (400 MHz, CDCl3): d [ppm]=1.60 (br s, 2H), 3.44-3.56 (m, 1H), 3.90 (s, 2H), 4.35 (d, 2H), 4.62 (t, 2H), 4.92 (t, 2H), 7.29 (d, 1H), 8.26 (s, 1H), 8.28 (d, 1H).
LC-MS (method 4): Rt=0.37 min., 76%. MS (ESIpos): m/z=(M+H)+ 195.
According to the method described for intermediate 2-1 with 3-[(3-ethyloxetan-3-yl)methoxy]pyridine-4-carbonitrile (intermediate 1-4, 3.07 g, 14.1 mmol) as the starting material 3.0 g of the title compound were prepared (96% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.31 (s, 1H), 8.19 (d, 1H), 7.41 (d, 1H), 4.48 (d, 2H), 4.35 (d, 2H), 4.16-4.23 (m, 2H), 3.70 (br s, 2H), 1.79 (br d, 4H), 0.90 (t, 3H).
According to the method described for intermediate 2-1 with tert-butyl (2S)-2-{[(4-cyanopyridin-3-yl)oxy]methyl}azetidine-1-carboxylate (intermediate 1-5, 1.48 g, 5.12 mmol) as the starting material 1.41 g of the title compound were prepared (85% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.24-8.33 (m, 1H), 8.14-8.22 (m, 1H), 7.31-7.48 (m, 1H), 4.45-4.53 (m, 1H), 4.37-4.44 (m, 1H), 4.08-4.21 (m, 1H), 3.64-3.91 (m, 4H), 1.69-2.42 (m, 4H), 1.19-1.32 (m, 9H).
LC-MS (method 1): Rt=0.69 min; MS (ESIpos): m/z=294.6 [M+H]+
According to the method described for intermediate with 2-1; tert-butyl (2R)-2-{[(4-cyanopyridin-3-yl)oxy]methyl}azetidine-1-carboxylate (intermediate 1-6, 1.97 g, 6.81 mmol) as the starting material 1.84 g of the title compound were prepared (88% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.23-8.31 (m, 1H), 8.18 (d, 1H), 7.40 (d, 1H), 4.36-4.51 (m, 2H), 4.16 (dd, 1H), 3.67-3.85 (m, 4H), 2.30-2.40 (m, 1H), 2.19 (ddt, 1H), 1.75 (s, 2H), 1.23-1.37 (m, 9H).
According to the method described for intermediate 2-1 with tert-butyl 2-{[(4-cyanopyridin-3-yl)oxy]methyl}-2-methylazetidine-1-carboxylate (intermediate 1-7, 355 mg, 1.17 mmol) as the starting material, 270 mg of the title compound were prepared (68% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.07-8.42 (m, 2H), 7.27-7.44 (m, 1H), 4.27 (br d, 1H), 3.95-4.04 (m, 1H), 3.58-3.83 (m, 4H), 2.31-2.41 (m, 1H), 1.97-2.10 (m, 1H), 1.61-1.95 (m, 1H), 1.11-1.47 (m, 13H).
LC-MS (method 2): Rt=0.88 min; MS (ESIpos): m/z=308.2 [M+H]+
According to the method described for intermediate 2-1 with 3-[2-(oxetan-2-yl)ethoxy]pyridine-4-carbonitrile (intermediate 1-8, 1.62 g, 6.98 mmol) as the starting material 1.44 g of the title compound were prepared (69% yield).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.806 (1.14), 0.825 (1.39), 0.833 (1.55), 0.851 (2.04), 1.234 (5.96), 2.074 (1.47), 2.089 (2.86), 2.109 (4.49), 2.124 (7.59), 2.143 (8.65), 2.148 (9.31), 2.165 (8.08), 2.180 (3.84), 2.201 (2.12), 2.214 (1.06), 2.323 (3.43), 2.327 (5.06), 2.331 (4.33), 2.369 (2.20), 2.387 (3.92), 2.395 (3.84), 2.414 (5.96), 2.437 (5.22), 2.454 (3.02), 2.583 (0.98), 2.601 (1.06), 2.627 (0.98), 2.644 (2.86), 2.665 (8.08), 2.670 (8.00), 2.678 (7.18), 2.684 (5.88), 2.691 (4.98), 2.698 (3.84), 2.706 (4.33), 2.725 (2.37), 3.671 (4.49), 3.707 (16.00), 4.096 (2.20), 4.120 (6.45), 4.139 (11.10), 4.154 (13.71), 4.169 (6.45), 4.179 (3.92), 4.194 (1.71), 4.381 (1.39), 4.396 (1.47), 4.404 (5.55), 4.418 (10.61), 4.426 (5.88), 4.432 (7.18), 4.441 (9.96), 4.455 (5.39), 4.468 (0.90), 4.488 (1.31), 4.509 (5.80), 4.529 (9.96), 4.544 (7.84), 4.549 (6.69), 4.564 (3.92), 4.856 (0.90), 4.902 (1.88), 4.920 (6.20), 4.937 (8.57), 4.953 (6.12), 4.970 (2.04), 7.378 (7.18), 7.388 (7.67), 7.416 (1.71), 7.729 (1.06), 7.755 (3.35), 7.769 (3.67), 8.170 (8.16), 8.180 (8.41), 8.245 (10.94), 8.263 (4.41), 8.708 (2.78), 8.721 (2.86).
According to the method described for intermediate 2-1 with 3-[(2-methyloxetan-2-yl)methoxy]pyridine-4-carbonitrile (intermediate 1-9, 1.30 g, 6.37 mmol) as the starting material, 1.31 g of the title compound were prepared (89% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.16-8.31 (m, 2H), 7.41 (d, 1H), 4.27-4.46 (m, 2H), 4.02-4.20 (m, 2H), 3.69-3.83 (m, 2H), 2.64-2.73 (m, 1H), 2.42 (ddd, 1H), 1.82 (br s, 2H), 1.35-1.45 (m, 3H).
LC-MS (method 2): Rt=0.60 min; MS (ESIpos): m/z=209.1 [M+H]+
According to the method described for intermediate 2-1 with 3-[(3,3-dimethyloxetan-2-yl)methoxy]pyridine-4-carbonitrile (intermediate 1-10, 1.06 g, 4.86 mmol) as the starting material 1.01 g of the title compound were prepared (89% yield).
1H-NMR (400 MHz, DMSO-d6): 5 [ppm]=8.24-8.36 (m, 1H), 8.18 (d, 1H), 7.39 (d, 1H), 4.60 (dd, 1H), 4.13-4.41 (m, 4H), 3.33 (br s, 2H), 1.85 (br d, 2H), 1.25-1.32 (m, 3H), 1.20 (s, 3H).
3-chloro-2-methoxyaniline (CAS 51114-68-2, 8.4 ml, 63 mmol) was solved in DCM (100 ml) and sat. sodium bicarbonate solution (100 ml) was added. To the ice cooled mixture was slowly added thiophosgene (5.4 ml, 70 mmol). The reaction was stirred at 0° C. for 2 h. At RT the DCM layer was separated and washed with sat. sodium bicarbonate solution, filtered through a hydrophobic filter and concentrated under reduced pressure to give the title compound (12.97 g, 100% yield) which was used directly in the next step.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=7.51 (dd, 1H), 7.35 (dd, 1H), 7.20 (t, 1H), 3.85-3.91 (m, 3H).
Using an analogous method as described for intermediate 3-1, 3-fluoro-2-methoxyaniline (CAS 437-83-2, 5.00 g, 35.4 mmol) as the starting material, 6.24 g of the title compound was prepared (96% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=7.32 (m, 1H), 7.10-7.19 (m, 2H), 3.96 (d, 3H).
According to the method described for intermediate 3-1; 3-chloro-4-fluoro-2-methoxyaniline (intermediate C-13, 4.59 g, 26.1 mmol) as the starting material, 5.42 g of the title compound were prepared (91% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=3.93 (s, 3H), 7.28-7.35 (m, 1H), 7.44 (dd, 1H).
According to the method described for intermediate 3-1, 2-chloro-3-fluoroaniline (CAS 21397-08-0, 10.0 g, 68.7 mmol) as the starting material, 10.24 g of the title compound were prepared (64% yield).
LC-MS (method 4): Rt=1.06 min, 95.56%, No ionisation
According to the method described for intermediate 3-1; 3-chloro-2-(difluoromethoxy)aniline (intermediate C-17, 2.00 g, 9.81 mmol) as the starting material, 2.4 g of the title compound were prepared (83% yield).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.912 (0.72), 2.326 (0.43), 2.517 (1.73), 2.522 (1.15), 3.158 (0.58), 3.396 (0.43), 3.418 (0.58), 3.715 (0.72), 6.639 (1.30), 6.643 (1.44), 6.659 (1.59), 6.663 (1.59), 6.690 (1.44), 6.713 (1.30), 6.717 (1.30), 6.734 (1.73), 6.737 (1.44), 6.876 (2.74), 6.934 (0.72), 6.947 (1.87), 6.967 (2.45), 6.987 (1.15), 7.030 (7.21), 7.060 (1.30), 7.211 (15.14), 7.327 (0.43), 7.347 (1.01), 7.367 (7.50), 7.388 (16.00), 7.391 (8.22), 7.409 (11.39), 7.488 (0.72), 7.494 (9.80), 7.497 (10.52), 7.508 (0.86), 7.514 (7.35), 7.518 (6.63), 7.597 (10.09), 7.601 (9.80), 7.618 (8.79), 7.622 (7.78), 9.798 (0.58).
According to the method described for intermediate 3-1; 3-bromo-2-methoxyaniline (CAS 116557-46-1, 4.7 g, 23.3 mmol) as the starting material, 4.18 g of the title compound were prepared (72% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=7.64 (dd, 1H), 7.38 (dd, 1H), 7.14 (t, 1H), 3.87 (s, 3H).
According to the method described for intermediate 3-1, 2,3-dihydro-1H-inden-4-amine (CAS 32202-61-2, 1.00 g, 7.51 mmol) as the starting material, 1.10 g of the title compound were prepared (84% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=7.12-7.26 (m, 3H), 2.93 (dt, 4H), 2.06 (quin, 2H).
According to the method described for intermediate 3-1; 3-chloro-5-fluoro-2-methylaniline (CAS 886761-87-1, 1.00 g, 6.27 mmol) as the starting material, 1.10 g of the title compound were prepared (78% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=7.52 (dd, 1H), 7.48 (dd, 1H), 2.33 (d, 3H).
According to the method described for intermediate 3-1; 2-chloro-3-methylaniline (CAS 29027-17-6, 4.97 g, 35.1 mmol) as the starting material and stirring overnight, 6.4 g of the title compound were prepared (94% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=7.29-7.43 (m, 3H), 2.31-2.38 (m, 3H).
Using an analogous method as described for intermediate 3-1, 3-chloro-5-fluoro-2-methoxyaniline (1.00 g, 5.70 mmol) as the starting material, 1.17 g of the title compound were prepared (95% purity, 90% yield).
1H NMR (400 MHz, DMSO-d6) δ [ppm]=3.86 (s, 3H) 7.38 (dd, 1H) 7.58 (dd, 1H)
Using an analogous method as described for intermediate 3-1, 3-chloro-2-ethylaniline (5.00 g, 85% purity, 27.3 mmol) as the starting material, 6.29 g of the title compound were prepared (85% purity, 99% yield).
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.114 (6.85), 1.133 (16.00), 1.152 (7.16), 1.162 (0.49), 1.181 (0.89), 1.200 (0.45), 2.073 (1.13), 2.769 (1.71), 2.788 (5.41), 2.806 (5.30), 2.825 (1.61), 5.755 (0.54), 7.279 (2.39), 7.299 (6.16), 7.319 (4.11), 7.408 (2.91), 7.411 (3.70), 7.428 (2.42), 7.431 (2.41), 7.449 (3.40), 7.452 (3.01), 7.469 (2.72), 7.473 (2.47).
To an ice-cooled solution of 1-chloro-3-isothiocyanato-2-methoxybenzene (intermediate 3-1, 4.00 g, 20.0 mmol) and tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 4.27 g, 20.0 mmol) in acetonitrile (92 ml) was added dropwise DBU (4.5 ml, 30 mmol). The reaction was stirred at RT overnight. To the reaction mixture was added ice-water (200 mL) and conc. HCl (2 mL). The mixture was stirred for 20 min. and extracted with DCM. The organic phase was filtered over a water-repellent filter, concentrated under reduced pressure and purified by flash chromatography (silica, hexane/EtOAc gradient 0-50%) to give 6.54 g of the title compound (71% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=13.36 (br s, 1H), 7.73 (d, 1H), 7.47 (dd, 1H), 7.22 (t, 1H), 3.76-3.82 (m, 5H), 2.88 (t, 2H), 1.48 (s, 9H).
LC-MS (method 2): Rt=0.67 min; MS (ESIpos): m/z=413.2 [M+H]+
Using an analogous method as described for Intermediate 4-1; tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 7.26 g, 34.1 mmol) and 1-fluoro-3-isothiocyanato-2-methoxybenzene (intermediate 3-4, 6.24 g, 34.1 mmol) as the starting materials, 9.49 g of the title compound were prepared (67% yield) after stirring the product in MeOH, filtration and drying of the precipitate in vacuo.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=13.37 (br s, 1H), 7.58 (br d, 1H), 7.23-7.30 (m, 1H), 7.09-7.21 (m, 1H), 4.10 (br s, 1H), 3.78 (t, 2H), 3.17 (s, 3H), 2.88 (br t, 2H), 1.48 (s, 9H).
LC-MS (method 2): Rt=0.66 min; MS (ESIpos): m/z=397.3 [M+H]+
According to the method described for intermediate 4-1; 1-chloro-3-isothiocyanato-2-methylbenzene (CAS 19241-35-1; 2.50 g, 13.6 mmol) and tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 2.9 g, 13.6 mmol) as the starting materials, 4.68 g of the title compound were prepared (78% yield), after addition of HCl, filtration, and drying of the precipitate in vacuo.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=15.73 (s, 1H), 12.77 (br s, 1H), 7.45 (d, 1H), 7.30 (t, 1H), 7.19 (d, 1H), 3.78 (t, 2H), 2.85 (t, 2H), 2.20 (s, 3H), 1.48 (s, 9H).
LC-MS (method 2): Rt=0.72 min; MS (ESIpos): m/z=397.3 [M+H]+
1,2-Dichloro-3-isothiocyanatobenzene (CAS 6590-97-2, 5.00 g, 24.5 mmol) and tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 5.22 g, 24.5 mmol) were solubilised in acetonitrile (55 ml), DBU (5.5 ml, 37 mmol) was added carefully at 0° C. under argon atmosphere and the mixture was stirred overnight at RT. The reaction mixture was diluted with HCl (200 ml, 1 N in water) and stirred for 30 min. at RT. The resulting solid was filtered off, the filter cake was washed with water and dried at 50° C. in a vacuo oven overnight to give 9.40 g of the title compound (92% yield).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.467 (16.00), 1.484 (0.56), 1.622 (0.34), 1.644 (0.25), 1.661 (0.20), 1.674 (0.17), 1.898 (0.19), 1.913 (0.31), 1.927 (0.19), 2.075 (0.20), 2.327 (0.18), 2.518 (0.60), 2.523 (0.39), 2.621 (0.30), 2.647 (0.32), 2.665 (0.24), 2.669 (0.29), 2.673 (0.25), 3.249 (0.25), 3.459 (0.23), 3.473 (0.39), 3.487 (0.22), 3.538 (0.28), 3.561 (0.29), 3.727 (0.50), 7.357 (0.17), 7.377 (0.36), 7.383 (0.28), 7.397 (0.28), 7.544 (0.27), 7.547 (0.33), 7.560 (0.29), 7.564 (0.31), 7.568 (0.25), 7.580 (0.18).
LC-MS (method 2): Rt=0.70 min; MS (ESIpos): m/z=416 [M−H]−
According to the method described for Intermediate 4-1, 2-chloro-1-fluoro-4-isothiocyanato-3-methoxybenzene (intermediate 3-13, 5.42 g, 24.9 mmol) as the starting material, 5.96 g of the title compound were prepared (53% yield) after purification by flash chromatography (silica, hexane/EtOAc gradient 0-50%).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=1.45-1.52 (s, 9H), 2.88 (t, 2H), 3.76-3.81 (t, 2H), 3.81 (s, 3H), 7.30 (t, 1H), 7.64 (dd, 1H), 13.15 (br s, 1H), 16.08 (br s, 1H).
LC-MS (method 1): Rt=1.50 min; MS (ESIpos): m/z=431 [M+H]+
According to the method described for intermediate 4-1, 1-chloro-2-(difluoromethoxy)-3-isothiocyanatobenzene (intermediate 3-17, 2.44 g, 9.73 mmol) as the starting material, 2.11 g of the title compound were prepared (38% yield) after flash chromatography (silica, hexane/EtOAc gradient 0-30%).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.475 (16.00), 1.486 (2.52), 2.083 (3.92), 2.518 (3.00), 2.522 (1.89), 3.749 (0.52), 3.764 (0.85), 3.780 (0.52), 7.036 (0.76), 7.411 (0.52), 7.599 (0.44).
LC-MS (method 2): Rt=0.70 min; MS (ESIpos): m/z=449 [M+H]+
According to the method described for Intermediate 4-10; 2-chloro-1-isothiocyanato-3-methylbenzene (intermediate 3-37, 6.45 g, 35.1 mmol) and tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 7.49 g, 35.1 mmol) as the starting materials, 6.79 g of the title compound were prepared (46% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=13.32 (br s, 1H), 7.30-7.39 (m, 3H), 3.79 (t, 2H), 2.90 (t, 2H), 2.39 (s, 3H), 1.48 (s, 9H).
LC-MS (method 2): Rt=0.69 min; MS (ESIneg): m/z=397.3 [M−H]−
According to the method described for Intermediate 4-1, 1-chloro-5-fluoro-3-isothiocyanato-2-methylbenzene (intermediate 3-40, 1.10 g, 5.45 mmol) and tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 1.16 g, 5.45 mmol) as the starting materials, 2.01 g of the title compound were prepared (75% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=15.56 (br s, 1H), 12.80 (br s, 1H), 7.48 (dd, 1H), 7.19 (br d, 1H), 3.78 (t, 2H), 2.84 (br t, 2H), 2.16 (s, 3H), 1.48 (s, 9H).
LC-MS (method 2): Rt=0.73 min; MS (ESIneg): m/z=413 [M−H]−
Using an analogous method as described for Intermediate 4-1; tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 1.15 g, 5.38 mmol) and 1-chloro-5-fluoro-3-isothiocyanato-2-methoxybenzene (intermediate 3-65, 1.17 g, 5.38 mmol) as the starting materials, the title compound was prepared 1.42 g (75% purity, 46% yield).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.154 (0.88), 1.172 (1.70), 1.189 (0.80), 1.484 (16.00), 1.987 (3.28), 2.518 (0.89), 2.522 (0.61), 2.883 (0.73), 2.899 (0.40), 3.359 (0.69), 3.644 (4.46), 3.760 (7.94), 3.774 (0.44), 3.782 (1.03), 3.798 (0.53), 4.017 (0.69), 4.035 (0.69), 6.400 (0.60), 6.405 (0.53), 6.427 (1.00), 7.498 (0.41).
Using an analogous method as described for Intermediate 4-1; tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 5.77 g, 27.0 mmol) and 2-chloro-1-ethyl-3-isothiocyanatobenzene (intermediate 3-77, 6.29 g, 85% purity, 27.0 mmol) as the starting materials, 6.35 g of the title compound were prepared (85% purity, 49% yield).
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.063 (0.89), 1.082 (2.14), 1.100 (0.95), 1.362 (0.65), 1.478 (16.00), 1.486 (1.29), 2.518 (0.64), 2.523 (0.44), 2.631 (0.70), 2.650 (0.69), 2.850 (0.41), 2.866 (0.78), 2.883 (0.43), 3.775 (0.51), 3.791 (0.90), 3.807 (0.46), 7.212 (0.46), 7.214 (0.44), 7.232 (0.62), 7.234 (0.61), 7.293 (0.62), 7.313 (1.08), 7.332 (0.53), 7.440 (0.61), 7.443 (0.62), 7.460 (0.49), 7.463 (0.45).
Using an analogous method as described for Intermediate 4-1 with tert-butyl 2,4-dioxopiperidine-1-carboxylate (CAS 845267-78-9, 8.19 g, 38.4 mmol) and 1-fluoro-3-isothiocyanato-2-methylbenzene (CAS 363179-58-2, 6.42 g, 38.4 mmol) as the starting materials; 11.1 g of the title compound were prepared (95% purity, 72% yield) after stirring the product in MeOH, filtration and drying of the precipitate in vacuo.
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.479 (16.00), 2.084 (2.80), 2.088 (2.74), 2.834 (0.58), 2.850 (1.12), 2.866 (0.61), 3.768 (0.64), 3.784 (1.16), 3.800 (0.59), 7.073 (0.61), 7.093 (0.71), 7.186 (0.59), 7.299 (0.45), 7.316 (0.42).
To a solution of tert-butyl 5-[(3-chloro-2-methoxyphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-1, 6.54 g, 15.8 mmol) in dichloromethane (94 ml) was added TFA (12 ml, 160 mmol) and the mixture was stirred 1.5 h at RT. The reaction mixture was concentrated under reduced pressure and the residue was solved in EtOAc and washed with sat. sodium bicarbonate solution and brine. The organic layer was filtered through a water resistant filter and the filtrate was dried to dryness. The residue was purified by flash chromatography (silica, hexane/EtOAc gradient 20-100%) to give 4.06 g of the title compound (78% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=16.45 (d, 1H), 14.69 (s, 1H), 14.33 (s, 1H), 9.37 (br s, 1H), 8.18 (br s, 1H), 7.76-7.87 (m, 1H), 7.37-7.45 (m, 1H), 7.15-7.23 (m, 1H), 3.73-3.76 (m, 3H), 3.43 (td, 1H), 3.27-3.32 (m, 1H), 2.79 (t, 1H), 2.59-2.69 (m, 1H).
LC-MS (method 1): Rt=1.19 min; MS (ESIpos): m/z=313 [M+H]+
Using an analogous method as described for intermediate 5-1; tert-butyl 5-[(3-fluoro-2-methoxyphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-4, 9.49 g, 23.9 mmol) as the starting material, 6.98 g of the title compound were prepared (89% yield) after stirring for 15 min. without further purification.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=16.48 (d, 1H), 14.63 (s, 0.5H), 14.28 (s, 0.5H), 9.34 (br s, 0.5H), 8.16 (br s, 0.5H), 7.65 (t, 1H), 6.97-7.37 (m, 2H), 3.79-3.85 (m, 3H), 3.35-3.46 (m, 1H), 3.26-3.32 (m, 1H), 2.78 (t, 1H), 2.63 (t, 1H).
LC-MS (method 2): Rt=0.46 min; MS (ESIpos): m/z=297.1 [M+H]+
Using an analogous method as described for intermediate 5-1, tert-butyl 5-[(3-chloro-2-methylphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-7, 4.67 g, 11.8 mmol) as the starting material, 3.54 g of the title compound were prepared (91% yield) after 3 h without further purification.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=16.42 (d, 1H), 14.01-14.37 (m, 1H), 8.14-9.40 (m, 1H), 7.43 (br t, 1H), 7.16-7.32 (m, 2H), 3.42-3.48 (m, 1H), 3.26-3.34 (m, 1H), 2.78 (t, 1H), 2.60-2.68 (m, 1H), 2.12-2.21 (m, 3H).
LC-MS (method 2): Rt=0.60 min; MS (ESIpos): m/z=297.4 [M+H]+
Using an analogous method as described for intermediate 5-1, tert-butyl 5-[(2,3-dichlorophenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-10, 9.40 g, 22.5 mmol) as the starting material, 5.71 g of the title compound were prepared (62% yield) after stirring overnight.
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.018 (1.18), 1.050 (1.33), 1.072 (0.81), 1.102 (0.59), 1.132 (0.81), 1.154 (1.84), 1.172 (3.17), 1.189 (1.84), 1.199 (0.88), 1.231 (1.92), 1.259 (1.40), 1.486 (0.66), 1.593 (1.33), 1.626 (1.18), 1.695 (1.25), 1.727 (1.18), 1.907 (2.14), 1.987 (5.97), 2.322 (3.17), 2.326 (4.28), 2.331 (3.17), 2.518 (15.85), 2.522 (9.44), 2.638 (11.06), 2.664 (7.82), 2.669 (7.52), 2.673 (5.53), 2.798 (8.11), 3.436 (9.81), 4.017 (1.18), 4.035 (1.11), 5.560 (1.33), 5.579 (1.25), 7.392 (4.42), 7.410 (10.03), 7.430 (9.22), 7.565 (12.24), 7.585 (16.00), 7.605 (9.51), 8.134 (0.74), 8.197 (4.35), 9.418 (3.91), 14.273 (6.93), 14.665 (6.64), 16.114 (1.03), 16.295 (9.95), 16.352 (5.82), 16.503 (1.11).
LC-MS (method 2): Rt=0.55 min; MS (ESIpos): m/z=316 [M−H]−
Using an analogous method as described for intermediate 5-1, tert-butyl 5-[(3-chloro-4-fluoro-2-methoxyphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-13, 790 mg, 1.83 mmol) as the starting material, 507 mg of the title compound were prepared (75% yield) after 1 h without further purification.
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.172 (0.76), 1.987 (1.52), 2.518 (1.13), 2.522 (0.77), 2.635 (0.46), 2.664 (0.49), 2.668 (0.54), 2.673 (0.42), 3.422 (0.40), 3.778 (16.00), 7.265 (0.52), 16.377 (0.95).
LC-MS (method 2): Rt=1.23 min; MS (ESIpos): m/z=331.2 [M+H]+
To an ice-cooled suspension of 2-chloro-1-fluoro-3-isothiocyanatobenzene (intermediate 3-16, 10.2 g, 54.6 mmol) and piperidine-2,4-dione (CAS 50607-30-2, 6.17 g, 54.6 mmol) in acetonitrile (250 ml) was added dropwise DBU (12 ml, 82 mmol). The solution was stirred at 0° C. for 1 h and 2 h at RT. To the reaction mixture was added ice-cooled HCl (2 mL, 2 N). The mixture was stirred for 30 min. and the solid that precipitated from this procedure was collected by filtration and dried in vacuum. 7.12 g of the title compound were obtained (39% yield).
1H NMR (400 MHz, DMSO-d6): δ [ppm]=2.65 (t, 1H), 2.81 (t, 1H), 3.46 (dt, 1H), 7.36-7.50 (m, 3H) 1×CH obscured by NMR solvent.
LC-MS (method 4): Rt=0.30 min., 90%. MS (ESIpos): m/z=(M+H)+ 301
Using an analogous method as described for intermediate 5-1; tert-butyl 5-{[3-chloro-2-(difluoromethoxy)phenyl]carbamothioyl}-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-17, 1.10 g, 2.16 mmol) as the starting material, 989 mg of the title compound were prepared (100% yield) after 3 h without further purification.
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.390 (0.59), 2.084 (0.44), 2.331 (3.08), 2.336 (1.39), 2.518 (16.00), 2.523 (10.94), 2.541 (0.95), 2.612 (2.35), 2.629 (4.40), 2.647 (2.64), 2.673 (3.30), 2.678 (1.54), 2.694 (0.44), 2.713 (0.81), 2.730 (0.51), 2.775 (2.06), 2.793 (3.74), 2.810 (2.13), 3.300 (3.30), 3.426 (6.09), 3.433 (6.02), 6.825 (1.47), 6.839 (1.25), 7.006 (3.08), 7.020 (2.50), 7.115 (0.51), 7.134 (0.73), 7.188 (1.54), 7.201 (1.25), 7.296 (0.44), 7.318 (0.81), 7.327 (0.81), 7.335 (1.76), 7.357 (1.39), 7.377 (3.01), 7.397 (2.42), 7.409 (2.57), 7.429 (1.39), 7.549 (2.50), 7.569 (2.42), 7.585 (2.42), 7.605 (4.26), 7.626 (4.33), 7.646 (1.91), 8.132 (0.37), 8.166 (2.06), 8.183 (0.66), 8.192 (0.59), 8.202 (0.44), 9.392 (2.13), 12.389 (0.66), 12.678 (0.44), 14.169 (3.60), 14.610 (2.72), 16.074 (0.44), 16.326 (4.40), 16.361 (3.30).
LC-MS (method 2): Rt=0.57 min; MS (ESIpos): m/z=349 [M+H]+
To a suspension of 1-bromo-3-isothiocyanato-2-methoxybenzene (intermediate 3-33, 4.18 g, 17.1 mmol) and piperidine-2,4-dione (CAS 50607-30-2, 2.13 g, 18.8 mmol) in acetonitrile (50 ml) was added DBU (3.8 ml, 26 mmol) slowly at 5° C. The resulting solution was stirred at RT overnight. The reaction was quenched with aq. HCl (1 M), stirred for 1 h and then extracted with DCM. The organic layer was separated, filtered through hydrophobic filter paper and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, hexane/EtOAc gradient 0-50%) to give 950 mg of the title compound (15% yield).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.153 (0.58), 1.171 (1.11), 1.189 (0.58), 1.986 (2.03), 2.518 (0.60), 2.625 (1.39), 2.642 (2.58), 2.661 (1.54), 2.772 (1.28), 2.790 (2.74), 2.808 (1.46), 3.278 (0.90), 3.286 (0.99), 3.296 (1.73), 3.303 (1.74), 3.314 (1.04), 3.321 (1.19), 3.338 (10.17), 3.411 (0.92), 3.419 (0.98), 3.429 (1.58), 3.437 (1.54), 3.448 (0.88), 3.455 (0.81), 3.714 (16.00), 3.719 (15.82), 4.016 (0.46), 4.034 (0.45), 7.093 (1.11), 7.113 (2.34), 7.125 (1.20), 7.134 (1.34), 7.146 (2.34), 7.166 (1.25), 7.517 (1.39), 7.521 (1.44), 7.538 (1.32), 7.542 (1.25), 7.559 (1.37), 7.563 (1.44), 7.580 (1.29), 7.583 (1.25), 7.789 (1.24), 7.792 (1.23), 7.809 (1.20), 7.812 (1.13), 7.830 (1.25), 7.833 (1.23), 7.850 (1.19), 7.853 (1.11), 8.184 (1.01), 9.369 (0.87), 14.321 (2.01), 14.689 (1.63), 16.445 (5.20), 16.456 (5.02).
LC-MS (method 1): Rt=1.26 min; MS (ESIpos): m/z=357 [M+H]+
Using an analogous method as described for intermediate 5-33, 4-isothiocyanato-2,3-dihydro-1H-indene (intermediate 3-36, 1.10 g, 6.28 mmol) and piperidine-2,4-dione (CAS 50607-30-2, 781 mg, 6.90 mmol) as the starting materials, 340 mg of the title compound were prepared (19% yield).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: −4.193 (3.48), −4.123 (1.35), 1.154 (0.58), 1.172 (0.91), 1.190 (0.55), 1.231 (0.70), 1.907 (0.60), 1.979 (2.05), 1.987 (3.14), 1.997 (6.76), 2.015 (9.97), 2.033 (6.98), 2.052 (2.07), 2.322 (0.73), 2.326 (0.99), 2.331 (0.72), 2.522 (3.01), 2.597 (3.24), 2.615 (6.16), 2.634 (3.53), 2.664 (0.85), 2.669 (1.14), 2.673 (0.82), 2.744 (4.44), 2.761 (14.38), 2.779 (16.00), 2.795 (6.45), 2.835 (0.55), 2.854 (0.79), 2.899 (8.47), 2.918 (15.01), 2.936 (7.56), 3.266 (2.99), 3.273 (3.40), 3.284 (5.67), 3.291 (5.74), 3.302 (3.28), 3.308 (3.02), 3.395 (2.29), 3.401 (2.60), 3.412 (4.08), 3.419 (4.00), 3.430 (2.32), 7.087 (0.70), 7.106 (0.48), 7.151 (8.40), 7.167 (5.11), 7.181 (11.73), 7.189 (6.92), 7.194 (6.56), 7.213 (0.85), 7.329 (2.20), 7.348 (2.49), 7.359 (3.42), 7.372 (3.30), 7.381 (2.32), 8.111 (3.33), 9.251 (2.24), 9.290 (0.44), 14.130 (4.35), 14.424 (4.90).
LC-MS (method 1): Rt=1.28 min; MS (ESIpos): m/z=289 [M+H]+
Using an analogous method as described for intermediate 5-1, tert-butyl 5-[(2-chloro-3-methylphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-37, 6.80 g, 17.1 mmol) as the starting material, 5.1 g of the title compound were prepared (95% yield) after stirring over two days and purification by flash chromatography (silica, hexane/EtOAc gradient 0-80%).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=2.38 (s, 3H), 2.63 (t, 1H), 2.79 (t, 1H), 3.24-3.31 (m, 1H), 3.43 (m, 1H), 7.24-7.35 (m, 2H), 7.38-7.46 (m, 1H), 8.15 (br s, 0.5H), 9.34 (br s, 0.5H), 14.20 (s, 0.5H), 14.55 (s, 0.5H).
LC-MS (method 2): Rt=0.56 min; MS (ESIneg): m/z=313 [M−H]
Using an analogous method as described for intermediate 5-1, tert-butyl 5-[(3-chloro-5-fluoro-2-methylphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-40, 2.01 g, 4.84 mmol) as the starting material, 1.03 g of the title compound were prepared (64% yield) after stirring for 30 min and purification by flash chromatography (silica, hexane/EtOAc gradient 0-100%).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=16.32 (d, 1H), 14.04-14.50 (m, 1H), 8.08-9.47 (m, 1H), 7.45 (ddd, 1H), 7.25 (ddd, 1H), 3.43 (td, 1H), 3.27-3.32 (m, 1H), 2.79 (t, 1H), 2.60-2.68 (m, 1H), 2.14 (s, 3H).
LC-MS (method 2): Rt=0.54 min; MS (ESIpos): m/z=397.2 [M+H]+
Using an analogous method as described for intermediate 5-1, tert-butyl 5-[(3-chloro-5-fluoro-2-methoxyphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-65, 1.42 g, 3.30 mmol) as the starting material, 690 mg of the title compound were prepared (60% yield).
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.172 (0.74), 1.988 (1.47), 2.518 (1.06), 2.523 (0.74), 2.632 (0.75), 2.651 (1.44), 2.669 (1.12), 2.786 (0.56), 2.803 (1.21), 2.821 (0.65), 3.286 (0.42), 3.296 (0.75), 3.304 (0.74), 3.314 (0.42), 3.330 (16.00), 3.415 (0.49), 3.422 (0.53), 3.433 (0.87), 3.440 (0.85), 3.451 (0.49), 3.459 (0.44), 7.393 (0.58), 7.401 (0.66), 7.414 (0.61), 7.421 (0.66), 7.454 (0.47), 7.461 (0.53), 7.474 (0.48), 7.482 (0.51), 7.912 (0.48), 7.920 (0.52), 7.927 (0.53), 7.936 (0.69), 7.945 (0.53), 7.953 (0.51), 7.960 (0.45), 8.235 (0.45), 9.460 (0.44), 14.534 (1.11), 14.929 (0.77), 16.292 (2.68), 16.362 (2.11).
LC-MS (method 2): Rt=0.57 min; MS (ESIneg): m/z=329 [M−H]−
Using an analogous method as described for intermediate 5-1; tert-butyl 5-[(3-chloro-2-ethylphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-77, 2.35 g, 75% purity, 4.29 mmol) as the starting material, 1.22 g of the title compound were prepared (95% purity, 87% yield).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.048 (7.06), 1.067 (16.00), 1.086 (7.43), 1.107 (1.58), 1.124 (1.32), 1.143 (0.50), 1.154 (1.34), 1.172 (2.43), 1.190 (1.24), 1.232 (0.50), 1.988 (4.56), 2.318 (0.47), 2.323 (1.05), 2.327 (1.56), 2.332 (1.11), 2.336 (0.47), 2.518 (5.61), 2.523 (4.03), 2.581 (2.03), 2.600 (6.41), 2.619 (6.62), 2.642 (4.59), 2.661 (2.69), 2.669 (1.90), 2.673 (1.27), 2.678 (0.58), 2.771 (2.35), 2.789 (4.82), 2.807 (2.58), 3.287 (1.61), 3.294 (1.79), 3.305 (3.11), 3.312 (3.14), 3.323 (2.06), 3.330 (2.35), 3.415 (1.53), 3.422 (1.66), 3.433 (2.56), 3.441 (2.45), 3.452 (1.40), 3.459 (1.24), 4.017 (1.03), 4.035 (1.00), 7.166 (0.95), 7.176 (0.58), 7.179 (0.58), 7.248 (0.71), 7.259 (6.30), 7.268 (3.58), 7.275 (4.43), 7.287 (7.20), 7.294 (3.90), 7.303 (3.56), 7.324 (0.87), 7.392 (1.85), 7.400 (1.37), 7.407 (1.85), 7.415 (1.74), 7.419 (2.40), 7.426 (1.77), 7.436 (1.77), 7.443 (1.45), 8.174 (1.77), 9.335 (1.42), 14.111 (2.98), 14.452 (2.74), 16.428 (7.88), 16.441 (7.38).
LC-MS (method 6): Rt=1.28 min; MS (ESIpos): m/z=311 [M+H]+
Using an analogous method as described for intermediate 5-1 with tert-butyl 5-[(3-fluoro-2-methylphenyl)carbamothioyl]-4-hydroxy-6-oxo-3,6-dihydropyridine-1(2H)-carboxylate (intermediate 4-38, 11.1 g, 29.1 mmol) as the starting material, 7.25 g of the title compound was prepared (84% yield) after 15 min of stirring and used in the next steps without further purification.
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.172 (0.55), 1.987 (1.05), 2.063 (16.00), 2.518 (1.49), 2.523 (1.01), 2.612 (1.94), 2.631 (3.67), 2.649 (2.11), 2.761 (1.99), 2.779 (4.22), 2.798 (2.26), 3.280 (1.36), 3.287 (1.47), 3.298 (2.58), 3.305 (2.57), 3.315 (1.39), 3.322 (1.33), 3.410 (1.40), 3.417 (1.48), 3.428 (2.27), 3.435 (2.22), 3.446 (1.27), 3.454 (1.15), 7.112 (1.56), 7.119 (0.96), 7.132 (2.25), 7.141 (3.30), 7.161 (2.66), 7.168 (2.15), 7.192 (1.12), 7.243 (0.73), 7.262 (1.15), 7.272 (0.99), 7.279 (1.25), 7.292 (1.27), 7.308 (1.21), 7.328 (0.45), 8.150 (1.43), 9.317 (1.16), 14.003 (2.32), 14.321 (2.05), 16.439 (5.35), 16.468 (4.62).
LC-MS (method 2): Rt=0.47 min; MS (ESIpos): m/z=281 [M+H]+.
A mixture of N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 1.15 g, 3.68 mmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 1.00 g, 5.15 mmol) was stirred for 2 h at 120° C. The reaction mixture was purified by flash chromatography (amino phase silica, DCM/EtOAc gradient 0-100%) to give 560 mg of the title compound (30% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.79 (s, 1H), 13.72 (br t, 1H), 8.43 (s, 1H), 8.25 (d, 1H), 7.80 (dd, 1H), 7.73 (br s, 1H), 7.27-7.34 (m, 2H), 7.11 (t, 1H), 5.04 (dddd, 1H), 4.72 (d, 2H), 4.48-4.57 (m, 2H), 4.26-4.37 (m, 2H), 3.71 (s, 3H), 3.16 (td, 2H), 2.58-2.80 (m, 4H).
LC-MS (method 1): Rt=1.08 min; MS (ESIpos): m/z=490.7 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 200 mg, 575 μmol) and 1-(3-{[(2R)-oxetan-2-yl]methoxy}pyridin-4-yl)methanamine (intermediate 2-2, 145 mg, 748 μmol) as the starting materials, 92.1 mg of the title compound were prepared (29% yield) after heating for 3 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.79 (s, 1H), 13.72 (br t, 1H), 8.43 (s, 1H), 8.25 (d, 1H), 7.80 (dd, 1H), 7.73 (br s, 1H), 7.28-7.33 (m, 2H), 7.11 (t, 1H), 5.01-5.07 (m, 1H), 4.72 (d, 2H), 4.49-4.57 (m, 2H), 4.27-4.36 (m, 2H), 3.71 (s, 3H), 3.16 (td, 2H), 2.59-2.80 (m, 4H).
LC-MS (method 2): Rt=1.09 min; MS (ESIpos): m/z=489.4 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-4, 500 mg, 1.69 mmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 524 mg, 2.70 mmol) as the starting materials, 340 mg of the title compound were prepared (34% yield) after heating for 3 h and flash chromatography (amino phase silica, DCM/EtOH gradient 0-10%).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.74 (s, 1H), 13.72 (br t, 1H), 8.43 (s, 1H), 8.25 (d, 1H), 7.64-7.73 (m, 2H), 7.31 (d, 1H), 7.03-7.14 (m, 2H), 5.01-5.08 (m, 1H), 4.72 (d, 2H), 4.48-4.57 (m, 2H), 4.26-4.37 (m, 2H), 4.10 (q, 2H), 3.78 (d, 3H), 2.59-2.79 (m, 4H).
LC-MS (method 2): Rt=1.00 min; MS (ESIpos): m/z=473.2 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-methylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-7, 250 mg, 842 μmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 180 mg, 927 μmol) as the starting materials, 126 mg of the title compound were prepared (30% yield) after purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.54 (s, 1H), 13.66 (br t, 1H), 8.42 (s, 1H), 8.25 (d, 1H), 7.69 (br s, 1H), 7.28-7.36 (m, 2H), 7.14-7.24 (m, 2H), 5.00-5.07 (m, 1H), 4.70 (d, 2H), 4.48-4.57 (m, 2H), 4.26-4.35 (m, 2H), 3.16 (td, 2H), 2.58-2.80 (m, 4H), 2.16 (s, 3H).
LC-MS (method 2): Rt=1.09 min; MS (ESIpos): m/z=473.5 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(2,3-dichlorophenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-10, 400 mg, 757 μmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 180 mg, 927 μmol) as the starting materials, 126 mg of the title compound were prepared (32% yield) after preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.89 (s, 1H), 13.69 (t, 1H), 8.43 (s, 1H), 8.25 (d, 1H), 7.75 (br s, 1H), 7.49-7.56 (m, 2H), 7.35 (t, 1H), 7.31 (d, 1H), 5.04 (dddd, 1H), 4.72 (d, 2H), 4.49-4.57 (m, 2H), 4.27-4.36 (m, 2H), 3.13-3.20 (m, 2H), 2.75-2.81 (m, 2H), 2.58-2.74 (m, 2H).
LC-MS (method 2): Rt=1.09 min; MS (ESIpos): m/z=493.4 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-4-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-13, 500 mg, 1.51 mmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 524 mg, 2.70 mmol) as the starting materials, 200 mg of the title compound were prepared (18% yield) after heating for 3 h and flash chromatography (amino phase silica, DCM/EtOH gradient 0-10%).
LC-MS (method 2): Rt=1.09 min; MS (ESIpos): m/z=507.2 [M+H]+
N-(2-chloro-3-fluorophenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-16, 594 mg, 1.98 mmol) and 1-{3-[(oxetan-3-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-3, 767 mg, 3.95 mmol) were stirred for 90 min. at 130° C. The mixture was cooled down to RT. The residue was purified by reverse phase chromatography (Biotage Isolera, eluent: 20-70% acetonitrile in ammonium hydroxide pH=10) to give 388 mg of the title compound (38% yield).
1H-NMR (400 MHz, CDCl3) δ [ppm]=2.73-2.76 (m, 2H), 3.55-3.38 (m, 2H), 3.47-3.56 (m, 1H), 4.39 (d, 2H), 4.59-4.63 (m, 4H), 4.91-4.95 (m, 2H), 5.84 (s, 1H), 7.08 (dd, 1H), 7.24-7.31 (m, 2H), 7.51 (d, 1H), 8.32-8.33 (m, 2H), 14.11-14.14 (m, 1H), 14.33 (s, 1H).
LC-MS (method 4) Rt=0.68 min, 92%, MS (ESIneg) m/z=(M−H)−=475
A mixture of N-[3-chloro-2-(difluoromethoxy)phenyl]-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-17, 359 mg, 1.03 mmol) and 1-{3-[(oxetan-3-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-3, 400 mg, 2.06 mmol) in minimum amount of DCM (1 ml) were heated at 130° C. for 90 min. The product was sonicated in MeOH for 45 min. The solid was filtered of and the filtrate was concentrated under reduced pressure to give 705 mg of the title compound (99% yield).
LC-MS (method 3): Rt=1.43 min, 76%. MS (ESIpos): m/z=(M+H)+=525 1
1H-NMR (400 MHz, CDCl3): δ [ppm]=2.67-2.80 (m, 2H), 3.30-3.43 (m, 2H), 3.44-3.59 (m, 2H), 3.87-3.99 (m, 1H), 4.30-4.44 (m, 2H), 4.56-4.69 (m, 4H), 4.87-4.98 (m, 2H), 5.55 (br s, 1H), 6.24-6.75 (t, 1H), 7.24 (t, 1H), 7.34-7.40 (m, 2H), 7.63 (d, 1H), 8.25-8.31 (m, 1H), 8.32-8.36 (m, 2H), 14.05 (br s, 1H).
A mixture of 4-(aminomethyl)pyridin-3-ol (CAS 20485-35-2, 75 g, 0.604 mol) and N-(3-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-4, 150 g, 0.506 mol) in DMA (1.2 L) was stirred at 120° C. for 2.5 h under nitrogen. The mixture was concentrated in vacuum to remove most of the solvent. The dark brown solution was slowly added to EtOAc (8 L) with stirring. The resulting mixture was washed with water (2.5 L) and brine (2.5, 2×). The organic phase was dried over sodium sulphate, filtered and concentrated in vacuum. The residue was slurried with EtOAc (300 mL) and filtered. The filter cake was dried in vacuum to afford the title compound (87 g, 47% yield) as a yellow solid.
1H NMR (400 MHz, DMSO-d6): δ [ppm]=14.73 (s, 1H), 13.69 (t, 1H), 10.28 (s, 1H), 8.21-8.13 (m, 2H), 7.67-7.66 (m, 2H), 7.10 (br.s, 1H), 7.09-7.04 (m, 2H), 4.61 (d, 2H), 3.79 (s, 3H), 3.16 (t, 2H), 2.77 (t, 2H).
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 400 mg, 1.15 mmol) and 1-{3-[(2-ethyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-4, 333 mg, 1.50 mmol) as the starting materials, 254 mg of the title compound were prepared (42% yield) after heating for 3 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.78 (s, 1H), 13.65 (br t, 1H), 8.47 (s, 1H), 8.26 (d, 1H), 7.80 (dd, 1H), 7.73 (br s, 1H), 7.27-7.34 (m, 2H), 7.10 (t, 1H), 4.67 (d, 2H), 4.49 (d, 2H), 4.34 (d, 2H), 4.28 (s, 2H), 3.71 (s, 3H), 3.16 (td, 2H), 2.76 (t, 2H), 1.81 (q, 2H), 0.89 (t, 3H).
LC-MS (method 2): Rt=1.17 min; MS (ESIpos): m/z=517.5 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-methylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-7, 400 mg, 1.35 mmol) and 1-{3-[(2-ethyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-4, 330 mg, 1.48 mmol) as the starting material, 250 mg of the title compound were prepared (36% yield) after heating for 3 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.53 (s, 1H), 13.60 (t, 1H), 8.46 (s, 1H), 8.25 (d, 1H), 7.68 (br s, 1H), 7.29-7.35 (m, 2H), 7.13-7.24 (m, 2H), 4.65 (d, 2H), 4.49 (d, 2H), 4.34 (d, 2H), 4.27 (s, 2H), 3.16 (td, 2H), 2.67-2.78 (m, 2H), 2.15 (s, 3H), 1.80 (q, 2H), 0.89 (t, 3H).
LC-MS (method 2): Rt=1.18 min; MS (ESIpos): m/z=501.3 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 250 mg, 759 μmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-8, 190 mg, 911 μmol) as the starting materials, 60 mg of the title compound were prepared (15% yield) after heating for 3 h and preparative HPLC (method 10, flow: 250 mL/min, gradient: 0.00-2.00 min 30% B, 2.00-14.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.79 (s, 1H), 13.68 (br t, 1H), 8.37 (s, 1H), 8.23 (d, 1H), 7.81 (dd, 1H), 7.73 (br s, 1H), 7.26-7.34 (m, 2H), 7.11 (t, 1H), 4.96 (quin, 1H), 4.65 (d, 2H), 4.37-4.57 (m, 2H), 4.12-4.28 (m, 2H), 3.71 (s, 3H), 3.10-3.21 (m, 2H), 2.63-2.82 (m, 3H), 2.40 (ddt, 1H), 2.07-2.26 (m, 2H).
LC-MS (method 2): Rt=1.12 min; MS (ESIpos): m/z=503.4 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 1.19 g, 3.82 mmol) and tert-butyl (2S)-2-({[4-(aminomethyl)pyridin-3-yl]oxy}methyl)azetidine-1-carboxylate (intermediate 2-5, 700 mg, 2.39 mmol) as the starting materials, 300 mg of the title compound were prepared (12% yield) after flash chromatography (amino phase silica, DCM/EtOAc gradient 0-100%, then DCM/EtOH gradient 0-10%).
1H-NMR (400 MHz, DMSO-d6): b [ppm]=14.76-14.81 (m, 1H), 13.68-13.76 (m, 1H), 8.39-8.44 (m, 1H), 8.23-8.27 (m, 1H), 7.78-7.82 (m, 1H), 7.72-7.76 (m, 1H), 7.27-7.34 (m, 2H), 7.08-7.14 (m, 1H), 4.64-4.76 (m, 2H), 4.43-4.51 (m, 2H), 4.21-4.29 (m, 1H), 3.68-3.87 (m, 5H), 3.12-3.21 (m, 2H), 2.73-2.81 (m, 2H), 2.19-2.33 (m, 2H), 1.24-1.35 (m, 9H).
LC-MS (method 2): Rt=1.28 min; MS (ESIpos): m/z=588.5 [M+H]+
Using an analogous method as described for intermediate 6-1; N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 250 mg, 719 μmol) and tert-butyl (2R)-2-({[4-(aminomethyl)pyridin-3-yl]oxy}methyl)azetidine-1-carboxylate (intermediate 2-6, 295 mg, 935 μmol) as the starting materials, 181 mg of the title compound were prepared (42% yield) after preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.79 (s, 1H), 13.73 (br t, 1H), 8.42 (s, 1H), 8.25 (d, 1H), 7.73-7.81 (m, 2H), 7.28-7.33 (m, 2H), 7.11 (t, 1H), 4.64-4.77 (m, 2H), 4.43-4.52 (m, 2H), 4.25 (br d, 1H), 3.74-3.86 (m, 2H), 3.71 (s, 3H), 3.17 (td, 2H), 2.77 (br d, 2H), 2.19-2.33 (m, 2H), 1.29 (br s, 9H).
LC-MS (method 2): Rt=1.26 min; MS (ESIpos): m/z=588.5 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 211 mg, 676 μmol) and tert-butyl 2-({[4-(aminomethyl)pyridin-3-yl]oxy}methyl)-2-methylazetidine-1-carboxylate (intermediate 2-7, 270 mg, 878 μmol) as the starting materials, 192 mg of the title compound were prepared (42% yield) after purification by flash chromatography (amino phase silica, DCM/EtOAc gradient then DCM/ethanol gradient).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.71-14.84 (m, 1H), 13.65-13.81 (m, 1H), 8.38-8.46 (m, 1H), 8.22-8.29 (m, 1H), 7.70-7.81 (m, 2H), 7.27-7.34 (m, 2H), 7.05-7.15 (m, 1H), 4.55-4.86 (m, 2H), 4.25-4.51 (m, 1H), 3.95-4.15 (m, 2H), 3.68-3.87 (m, 4H), 3.10-3.25 (m, 2H), 2.69-2.86 (m, 2H), 2.40-2.46 (m, 1H), 1.99-2.07 (m, 1H), 1.38-1.46 (m, 3H), 1.30 (s, 2H), 1.17-1.22 (m, 7H).
LC-MS (method 1): Rt=1.25 min; MS (ESIpos): m/z=602.3 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-bromo-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-33, 350 mg, 980 μmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 247 mg, 1.27 mmol) as the starting materials, 303 mg of the title compound were prepared (49% yield) after heating at 100° C. for 12 h and purification by preparative HPLC (method 9, gradient: 0.00-0.50 min 20% B, 0.50-6.00 min 20-60% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.76 (br d, 1H), 13.65-13.81 (m, 1H), 8.43 (s, 1H), 8.25 (d, 1H), 7.78-7.86 (m, 1H), 7.71-7.76 (m, 1H), 7.44 (dd, 1H), 7.32 (d, 1H), 7.02-7.10 (m, 1H), 4.72 (br d, 2H), 4.45-4.59 (m, 4H), 4.22-4.32 (m, 3H), 3.69 (s, 3H), 3.12-3.20 (m, 2H), 2.78 (br t, 2H).
LC-MS (method 1): Rt=1.02 min; MS (ESIpos): m/z=533 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(2,3-dihydro-1H-inden-4-yl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-36, 340 mg, 1.18 mmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 298 mg, 1.53 mmol) as the starting materials, 196 mg of the title compound were prepared (35% yield) after heating for 12 h at 100° C. and purification by preparative HPLC (method 9, gradient: 0.00-0.50 min 20% B, 0.50-6.00 min 20-60% B).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.949 (0.42), 1.968 (1.46), 1.986 (2.12), 2.005 (1.56), 2.023 (0.51), 2.073 (10.69), 2.083 (16.00), 2.522 (0.96), 2.539 (1.40), 2.607 (0.43), 2.617 (0.47), 2.629 (0.52), 2.635 (0.80), 2.652 (0.64), 2.656 (0.73), 2.673 (0.84), 2.691 (0.93), 2.698 (0.47), 2.708 (0.98), 2.712 (0.92), 2.725 (1.40), 2.729 (1.41), 2.740 (3.74), 2.757 (4.13), 2.775 (1.50), 2.874 (1.52), 2.893 (2.66), 2.912 (1.37), 3.123 (0.97), 3.131 (1.13), 3.140 (1.69), 3.146 (1.65), 3.359 (0.88), 4.274 (0.43), 4.283 (0.52), 4.302 (1.90), 4.310 (2.28), 4.313 (2.30), 4.326 (1.85), 4.342 (0.43), 4.354 (0.48), 4.506 (1.13), 4.514 (1.21), 4.523 (1.30), 4.528 (1.61), 4.533 (1.58), 4.545 (1.11), 4.553 (1.00), 4.690 (2.18), 4.705 (2.23), 5.030 (0.47), 5.038 (0.71), 5.046 (0.79), 5.050 (0.62), 5.054 (0.66), 5.062 (0.41), 7.064 (0.76), 7.081 (2.10), 7.093 (1.71), 7.112 (1.98), 7.131 (0.74), 7.307 (1.87), 7.318 (1.91), 7.351 (1.44), 7.369 (1.32), 7.640 (1.39), 8.177 (2.36), 8.246 (2.28), 8.258 (2.21), 8.426 (3.70), 13.725 (0.51), 13.739 (0.95), 13.753 (0.50), 14.566 (2.20).
LC-MS (method 1): Rt=0.99 min; MS (ESIpos): m/z=465 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(2-chloro-3-methylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-37, 387 mg, 1.30 mmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 304 mg, 1.57 mmol) as the starting materials, 199 mg of the title compound were prepared (31% yield) after heating for 3 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.72 (s, 1H), 13.74 (t, 1H), 8.43 (s, 1H), 8.25 (d, 1H), 7.69 (br s, 1H), 7.35-7.42 (m, 1H), 7.19-7.32 (m, 3H), 5.00-5.11 (m, 1H), 4.71 (d, 2H), 4.48-4.57 (m, 2H), 4.25-4.36 (m, 2H), 3.16 (td, 2H), 2.58-2.80 (m, 4H), 2.32-2.37 (m, 3H).
LC-MS (method 6): Rt=0.87 min; MS (ESIpos): m/z=473.2 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-5-fluoro-2-methylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-40, 200 mg, 635 μmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 197 mg, 1.02 mmol) as the starting materials, 196 mg of the title compound were prepared (50% yield) after heating for 5 h and purification by flash chromatography (amino phase silica, DCM/EtOH gradient 0-10%).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.154 (0.42), 1.172 (0.72), 1.190 (0.42), 1.232 (0.86), 1.988 (1.34), 2.023 (0.58), 2.124 (16.00), 2.198 (0.61), 2.322 (1.20), 2.326 (1.61), 2.332 (1.20), 2.336 (0.56), 2.518 (8.46), 2.522 (5.40), 2.554 (0.56), 2.571 (0.67), 2.576 (0.50), 2.581 (0.89), 2.587 (0.50), 2.593 (0.78), 2.599 (1.53), 2.604 (1.17), 2.609 (1.20), 2.615 (1.25), 2.620 (1.75), 2.627 (1.61), 2.630 (1.17), 2.637 (1.25), 2.644 (1.22), 2.647 (1.39), 2.665 (2.00), 2.668 (2.59), 2.673 (1.73), 2.689 (1.73), 2.697 (1.47), 2.705 (1.92), 2.710 (1.53), 2.713 (1.70), 2.717 (1.53), 2.725 (1.50), 2.734 (1.31), 2.741 (0.86), 2.760 (1.86), 2.776 (3.51), 2.793 (2.06), 3.142 (1.28), 3.149 (1.53), 3.159 (6.12), 3.166 (2.62), 3.172 (4.87), 3.744 (6.82), 3.767 (1.00), 4.082 (0.50), 4.095 (0.95), 4.108 (0.83), 4.197 (0.45), 4.206 (0.67), 4.209 (0.70), 4.217 (1.11), 4.225 (2.98), 4.232 (3.92), 4.243 (2.64), 4.264 (1.17), 4.273 (1.75), 4.281 (1.14), 4.301 (3.23), 4.309 (3.65), 4.312 (3.67), 4.325 (3.23), 4.341 (0.78), 4.353 (0.95), 4.475 (0.78), 4.489 (1.61), 4.501 (2.20), 4.505 (2.25), 4.511 (3.48), 4.517 (3.37), 4.523 (2.70), 4.533 (3.67), 4.539 (3.09), 4.551 (2.42), 4.557 (1.34), 4.565 (0.67), 4.571 (0.64), 4.705 (3.34), 4.720 (3.48), 4.991 (0.50), 5.003 (0.64), 5.012 (1.42), 5.020 (1.59), 5.024 (1.50), 5.028 (1.67), 5.032 (1.84), 5.036 (1.45), 5.040 (1.92), 5.044 (1.28), 5.048 (1.53), 5.057 (0.78), 5.061 (0.75), 5.068 (0.53), 7.174 (1.86), 7.180 (2.28), 7.198 (1.92), 7.204 (2.17), 7.299 (3.37), 7.311 (3.45), 7.328 (2.09), 7.335 (2.14), 7.350 (2.14), 7.356 (2.25), 7.399 (1.86), 7.411 (1.86), 7.430 (0.50), 7.442 (0.53), 7.739 (2.37), 7.752 (1.47), 7.757 (0.86), 7.764 (0.75), 7.768 (1.14), 8.177 (3.06), 8.181 (1.22), 8.189 (3.06), 8.193 (1.28), 8.206 (0.45), 8.243 (5.34), 8.255 (5.09), 8.272 (4.95), 8.304 (1.25), 8.319 (0.64), 8.336 (0.86), 8.428 (8.24), 8.706 (1.09), 8.710 (0.58), 8.716 (0.56), 8.721 (1.06), 13.620 (0.72), 13.634 (1.39), 14.688 (1.42).
LC-MS (method 2): Rt=1.15 min; MS (ESIpos): m/z=491 [M+H]+
Using an analogous method as described for intermediate 6-1; N-(3-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-4, 200 mg, 614 μmol) and 1-{3-[2-(oxetan-2-yl)ethoxy]pyridin-4-yl}methanamine (intermediate 2-8, 192 mg, 921 μmol) as the starting materials, 84.2 mg of the title compound were prepared (28% yield) after preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.74 (s, 1H), 13.69 (br t, 1H), 8.37 (s, 1H), 8.23 (d, 1H), 7.65-7.73 (m, 2H), 7.30 (d, 1H), 7.03-7.13 (m, 2H), 4.96 (quin, 1H), 4.64 (d, 2H), 4.38-4.56 (m, 2H), 4.14-4.26 (m, 3H), 3.78 (d, 3H), 2.63-2.80 (m, 3H), 2.32-2.47 (m, 2H), 2.07-2.24 (m, 2H).
LC-MS (method 2): Rt=1.05 min; MS (ESIpos): m/z=487 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 300 mg, 959 μmol) and 1-{3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-9, 374 mg, 1.79 mmol) as the starting materials, 208 mg of the title compound were prepared (41% yield) after flash chromatography (amino phase silica, DCM/EtOAc gradient 0-100%; DCM/EtOH gradient 0-10%).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.79 (s, 1H), 13.71 (br t, 1H), 8.43 (s, 1H), 8.25 (d, 1H), 7.80 (dd, 1H), 7.74 (br s, 1H), 7.28-7.33 (m, 2H), 7.11 (t, 1H), 4.70-4.80 (m, 2H), 4.34-4.47 (m, 2H), 4.16 (d, 2H), 3.71 (s, 3H), 3.16 (td, 2H), 2.72-2.80 (m, 3H), 2.36-2.47 (m, 1H), 1.45 (s, 3H).
LC-MS (method 2): Rt=1.10 min; MS (ESIpos): m/z=503.2 [M+H]+
Using an analogous method as described for intermediate 6-1; N-(3-chloro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-1, 250 mg, 799 μmol) and 1-{3-[(3,3-dimethyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-10, 243 mg, 1.04 mmol) as the starting materials, 156 mg of the title compound were prepared (37% yield) after preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.79 (s, 1H), 13.68 (br t, 1H), 8.46 (s, 1H), 8.25 (d, 1H), 7.81 (dd, 1H), 7.73 (br s, 1H), 7.28-7.33 (m, 2H), 7.11 (t, 1H), 4.60-4.69 (m, 3H), 4.32-4.47 (m, 2H), 4.17-4.25 (m, 2H), 3.71 (s, 3H), 3.15 (td, 2H), 2.75 (t, 2H), 1.29 (s, 3H), 1.20 (s, 3H).
LC-MS (method 2): Rt=1.19 min; MS (ESIpos): m/z=517.6 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-4, 250 mg, 844 μmol) and 1-{3-[(3,3-dimethyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-10, 257 mg, 1.10 mmol) as the starting materials, 145 mg of the title compound were prepared (34% yield) after preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.74 (s, 1H), 13.69 (br t, 1H), 8.46 (s, 1H), 8.25 (d, 1H), 7.63-7.74 (m, 2H), 7.30 (d, 1H), 7.03-7.13 (m, 2H), 4.58-4.69 (m, 3H), 4.33-4.46 (m, 2H), 4.17-4.26 (m, 2H), 3.78 (d, 3H), 3.11-3.32 (m, 2H), 2.74 (t, 2H), 1.27-1.36 (m, 3H), 1.13-1.24 (m, 3H).
LC-MS (method 2): Rt=1.13 min; MS (ESIpos): m/z=501.6 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(2-chloro-3-methylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-37, 390 mg, 1.31 mmol) and 1-{3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-9, 328 mg, 1.57 mmol) as the starting materials, 223 mg of the title compound were prepared (33% yield) after heating for 3 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.71 (s, 1H), 13.73 (t, 1H), 8.43 (s, 1H), 8.25 (d, 1H), 7.69 (br s, 1H), 7.39 (t, 1H), 7.30 (d, 1H), 7.22 (s, 1H), 7.21 (s, 1H), 4.66-4.81 (m, 2H), 4.33-4.49 (m, 2H), 4.12-4.20 (m, 2H), 3.16 (td, 2H), 2.70-2.80 (m, 3H), 2.32-2.44 (m, 4H), 1.45 (s, 3H).
LC-MS (method 6): Rt=0.92 min; MS (ESIpos): m/z=487.2 [M+H]+
Using an analogous method as described for intermediate 6-1; N-(2-chloro-3-methylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-37, 305 mg, 1.03 mmol) and 1-{3-[(3,3-dimethyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-10, 274 mg, 1.23 mmol) as the starting materials, 189 mg of the title compound were prepared (35% yield) after heating for 3 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=14.72 (s, 1H), 13.70 (t, 1H), 8.45 (s, 1H), 8.24 (d, 1H), 7.68 (br s, 1H), 7.39 (t, 1H), 7.30 (d, 1H), 7.21 (d, 2H), 4.59-4.68 (m, 3H), 4.31-4.46 (m, 2H), 4.17-4.25 (m, 2H), 3.13-3.21 (m, 2H), 2.74 (t, 2H), 2.33-2.36 (m, 3H), 1.29 (s, 3H), 1.20 (s, 3H).
LC-MS (method 6): Rt=1.01 min; MS (ESIpos): m/z=501.2 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(2-chloro-3-methylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-37, 314 mg, 1.06 mmol) and 1-{3-[2-(oxetan-2-yl)ethoxy]pyridin-4-yl}methanamine (intermediate 2-8, 264 mg, 1.27 mmol) as the starting materials, 59.8 mg of the title compound were prepared (10% yield) after heating for 2.5 h preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6) δ [ppm]=1.230 (0.63), 2.074 (2.34), 2.131 (0.66), 2.146 (1.25), 2.155 (0.69), 2.164 (1.22), 2.169 (1.40), 2.186 (1.24), 2.200 (0.61), 2.221 (0.43), 2.229 (1.99), 2.318 (0.58), 2.322 (0.68), 2.326 (0.76), 2.332 (0.66), 2.336 (0.46), 2.354 (16.00), 2.373 (0.69), 2.377 (0.59), 2.382 (0.61), 2.390 (0.54), 2.396 (0.73), 2.401 (0.79), 2.404 (0.59), 2.413 (0.49), 2.418 (0.64), 2.422 (0.69), 2.441 (0.46), 2.518 (2.60), 2.522 (1.70), 2.629 (0.46), 2.644 (0.54), 2.650 (0.73), 2.656 (0.56), 2.665 (1.12), 2.669 (1.42), 2.673 (0.84), 2.677 (0.82), 2.684 (0.64), 2.690 (0.63), 2.692 (0.59), 2.696 (0.49), 2.711 (0.44), 2.741 (1.25), 2.758 (2.47), 2.774 (1.47), 3.141 (0.94), 3.148 (1.07), 3.157 (1.73), 3.164 (1.63), 3.174 (0.92), 3.181 (0.79), 4.175 (1.17), 4.181 (1.30), 4.189 (1.66), 4.196 (2.41), 4.207 (1.43), 4.211 (1.30), 4.221 (0.43), 4.393 (0.87), 4.407 (2.03), 4.416 (1.09), 4.422 (1.37), 4.430 (1.96), 4.445 (1.02), 4.496 (0.97), 4.510 (0.92), 4.516 (1.48), 4.529 (1.14), 4.535 (1.07), 4.550 (0.76), 4.628 (2.90), 4.643 (2.90), 4.938 (1.05), 4.955 (1.29), 4.971 (0.97), 7.199 (0.41), 7.207 (7.09), 7.219 (3.41), 7.221 (4.88), 7.240 (0.41), 7.295 (2.46), 7.307 (2.49), 7.383 (1.43), 7.397 (2.26), 7.407 (1.09), 7.681 (1.68), 8.217 (4.09), 8.229 (3.64), 8.367 (5.73), 13.691 (0.63), 13.705 (1.22), 13.720 (0.56), 14.717 (3.41).
LC-MS (method 6): Rt=0.89 min; MS (ESIpos): m/z=487 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(2,3-dichlorophenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-10, 434 mg, 1.37 mmol) and 1-{3-[2-(oxetan-2-yl)ethoxy]pyridin-4-yl}methanamine (intermediate 2-8, 342 mg, 1.64 mmol) as the starting materials, 243 mg of the title compound were prepared (23% yield) after preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.751 (0.64), 1.793 (0.41), 1.810 (0.41), 1.825 (0.46), 1.844 (0.43), 1.907 (0.41), 2.134 (0.84), 2.154 (0.58), 2.170 (0.64), 2.181 (0.61), 2.200 (0.55), 2.322 (1.22), 2.326 (1.65), 2.332 (1.24), 2.401 (0.41), 2.518 (7.90), 2.522 (4.95), 2.539 (0.75), 2.653 (0.46), 2.660 (0.84), 2.665 (1.56), 2.669 (2.03), 2.673 (1.65), 2.678 (0.95), 2.698 (0.49), 2.713 (0.61), 2.750 (1.16), 2.767 (1.48), 2.782 (0.78), 3.164 (1.68), 3.700 (0.46), 3.718 (0.64), 3.734 (0.58), 3.821 (0.58), 4.208 (1.30), 4.223 (1.91), 4.237 (1.56), 4.260 (1.50), 4.275 (1.50), 4.397 (0.90), 4.411 (1.27), 4.419 (0.95), 4.426 (1.07), 4.433 (1.30), 4.449 (1.01), 4.499 (1.07), 4.513 (0.95), 4.519 (1.13), 4.532 (0.98), 4.538 (0.84), 4.553 (0.69), 4.691 (1.94), 4.707 (1.94), 4.940 (0.61), 4.957 (0.69), 4.972 (0.55), 6.982 (14.12), 7.002 (0.43), 7.109 (16.00), 7.237 (13.97), 7.336 (1.01), 7.347 (0.67), 7.351 (0.87), 7.357 (1.97), 7.377 (1.30), 7.403 (0.43), 7.414 (1.19), 7.426 (1.13), 7.489 (0.46), 7.493 (0.49), 7.503 (1.53), 7.506 (1.74), 7.523 (1.50), 7.527 (1.45), 7.531 (1.74), 7.535 (1.45), 7.551 (1.36), 7.554 (1.22), 7.761 (1.13), 8.293 (1.42), 8.305 (1.36), 8.416 (0.41), 8.440 (1.97), 8.455 (1.07), 13.661 (0.67), 14.898 (1.97).
Using an analogous method as described for intermediate 6-1, N-(3-chloro-5-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-58, 243 mg, 735 μmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 200 mg, 1.03 mmol) as the starting materials, 260 mg (70% yield) of the title compound were prepared after heating for 4 h and purification by flash chromatography (silica, DCM/EtOH gradient, 0-20%)).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.035 (8.59), 1.053 (16.00), 1.070 (8.20), 1.232 (0.53), 2.337 (0.47), 2.518 (5.88), 2.523 (4.24), 2.540 (12.32), 2.633 (0.42), 2.678 (0.53), 2.694 (0.47), 2.711 (0.45), 2.772 (0.56), 2.789 (1.11), 2.805 (0.64), 3.152 (0.78), 3.159 (0.72), 3.404 (1.31), 3.417 (1.31), 3.422 (4.04), 3.435 (4.18), 3.439 (3.37), 3.452 (3.34), 3.457 (1.34), 3.469 (1.37), 3.701 (13.71), 4.312 (1.11), 4.321 (1.34), 4.323 (1.34), 4.335 (1.11), 4.343 (2.68), 4.355 (5.10), 4.368 (2.43), 4.505 (0.64), 4.515 (0.61), 4.522 (0.72), 4.527 (0.75), 4.533 (0.75), 4.535 (0.78), 4.543 (0.59), 4.554 (0.56), 4.730 (1.09), 4.745 (1.09), 5.048 (0.42), 5.759 (2.09), 7.274 (0.92), 7.282 (1.11), 7.294 (0.95), 7.302 (1.06), 7.315 (1.06), 7.326 (1.09), 7.800 (0.75), 8.022 (0.72), 8.029 (0.78), 8.048 (0.72), 8.056 (0.72), 8.252 (1.64), 8.264 (1.62), 8.439 (2.56), 13.680 (0.56), 15.074 (1.48).
LC-MS (method 2): Rt=1.13 min; MS (ESIpos): m/z=507 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-5-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-68, 300 mg, 907 μmol) and 1-{3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-9, 353 mg, 1.70 mmol) as the starting materials, 190 mg (73% purity, 29% yield) of the title compound were prepared after purification by flash chromatography (amino phase silica, DCM/EtOAc gradient, 0-100%, then DCM/EtOH gradient, 0-10%).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=15.07 (s, 1H), 13.67 (t, 1H), 13.22-13.27 (m, 1H), 8.44 (s, 1H), 8.23-8.32 (m, 1H), 7.98-8.07 (m, 1H), 7.80 (br s, 1H), 7.31 (br d, 2H), 4.72-4.82 (m, 2H), 4.67-4.82 (m, 2H), 4.34-4.49 (m, 3H), 4.15-4.18 (m, 2H), 4.06-4.07 (m, 1H), 3.77-3.82 (m, 1H), 3.66-3.72 (m, 3H), 3.09-3.25 (m, 2H), 2.68-2.86 (m, 3H), 2.37-2.45 (m, 1H), 1.42-1.49 (m, 4H).
LC-MS (method 2): Rt=1.18 min; MS (ESIpos): m/z=522 [M+H]+
Using an analogous method as described for intermediate 6-1, N-[3-chloro-2-(difluoromethoxy)phenyl]-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-17, 150 mg, 344 μmol) and 1-{3-[(3,3-dimethyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-10, 105 mg, 447 μmol) as the starting materials, 44.3 mg (99% purity, 23% yield) of the title compound were prepared after heating for 3 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-3.69 min 30-53% B, 3.69-4.68 min 53% B, 4.68-7.00 min 53-70% B).
LC-MS (method 2): Rt=1.21 min; MS (ESIpos): m/z=553 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.203 (15.39), 1.289 (16.00), 2.326 (1.71), 2.669 (1.71), 2.742 (1.62), 2.758 (3.03), 2.774 (1.85), 3.147 (2.36), 3.153 (2.34), 4.186 (2.17), 4.199 (3.96), 4.224 (3.98), 4.238 (2.21), 4.336 (0.97), 4.347 (1.12), 4.362 (1.91), 4.374 (2.03), 4.403 (1.81), 4.420 (2.01), 4.430 (1.02), 4.446 (1.10), 4.631 (3.21), 4.636 (3.43), 4.648 (4.24), 4.665 (1.66), 6.722 (1.38), 6.905 (2.82), 7.087 (1.36), 7.288 (2.64), 7.294 (2.25), 7.300 (2.82), 7.315 (3.25), 7.335 (2.01), 7.452 (2.11), 7.455 (2.25), 7.472 (1.77), 7.476 (1.69), 7.587 (2.03), 7.607 (1.79), 7.721 (2.01), 8.237 (3.27), 8.249 (3.19), 8.458 (5.38), 13.609 (1.30), 14.783 (2.56).
Using an analogous method as described for intermediate 6-1; N-[3-chloro-2-(difluoromethoxy)phenyl]-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-17, 150 mg, 80% purity, 344 μmol) and 1-{3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-9, 97.0 mg, 96% purity, 447 μmol) as the starting materials, 37.8 mg (70% purity, 14% yield) of the title compound were prepared after heating for 3 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-3.69 min 30-53% B, 3.69-4.68 min 53% B, 4.68-7.00 min 53-70% B).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.446 (16.00), 2.373 (0.46), 2.390 (0.59), 2.396 (0.71), 2.400 (0.66), 2.413 (0.62), 2.418 (0.82), 2.423 (0.64), 2.441 (0.56), 2.518 (3.18), 2.523 (2.17), 2.715 (0.52), 2.730 (0.63), 2.737 (0.82), 2.742 (0.66), 2.753 (0.90), 2.758 (1.21), 2.764 (1.25), 2.780 (1.80), 2.794 (0.89), 3.135 (0.74), 3.141 (0.86), 3.150 (1.37), 3.157 (1.34), 3.167 (0.75), 4.128 (0.62), 4.154 (3.92), 4.161 (3.96), 4.187 (0.60), 4.351 (0.48), 4.366 (1.01), 4.373 (0.66), 4.381 (0.90), 4.388 (1.02), 4.403 (0.68), 4.411 (0.71), 4.425 (0.66), 4.428 (0.89), 4.433 (0.95), 4.442 (0.64), 4.447 (0.68), 4.451 (0.79), 4.464 (0.44), 4.740 (1.33), 4.753 (1.39), 5.383 (1.03), 6.642 (0.70), 6.646 (0.80), 6.662 (0.79), 6.666 (0.83), 6.717 (1.77), 6.721 (1.21), 6.737 (0.97), 6.741 (0.84), 6.903 (2.24), 6.951 (0.91), 6.972 (1.34), 6.992 (0.67), 7.088 (1.26), 7.293 (1.81), 7.304 (1.60), 7.315 (1.66), 7.335 (1.01), 7.454 (1.01), 7.473 (0.78), 7.580 (0.87), 7.600 (0.78), 7.728 (0.90), 8.243 (3.22), 8.255 (2.99), 8.430 (4.31), 13.642 (0.58), 14.782 (0.97).
LC-MS (method 2): Rt=1.14 min; MS (ESIpos): m/z=539 [M+H]+
Using an analogous method as described for intermediate 6-1; N-(3-chloro-2-ethylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-77, 408 mg, 85% purity, 1.12 mmol) and 1-{3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-1, 325 mg, 1.67 mmol) as the starting materials, 283 mg (85% purity, 44% yield) of the title compound were prepared after purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.005 (1.47), 1.024 (3.78), 1.038 (5.72), 1.042 (3.26), 1.057 (13.98), 1.076 (5.68), 2.075 (10.69), 2.323 (0.87), 2.327 (1.24), 2.332 (0.87), 2.518 (4.44), 2.523 (3.16), 2.573 (1.38), 2.592 (4.60), 2.599 (1.94), 2.604 (2.97), 2.610 (5.24), 2.623 (2.54), 2.627 (2.85), 2.638 (0.95), 2.644 (1.47), 2.649 (1.53), 2.665 (2.29), 2.669 (1.61), 2.673 (1.03), 2.685 (2.00), 2.692 (0.74), 2.702 (1.90), 2.706 (1.65), 2.713 (1.16), 2.723 (1.18), 2.730 (1.09), 2.734 (0.74), 2.755 (2.42), 2.772 (4.75), 2.789 (2.75), 3.146 (1.75), 3.153 (2.02), 3.162 (3.28), 3.169 (3.16), 3.179 (1.78), 3.186 (1.53), 4.270 (0.93), 4.278 (1.20), 4.297 (4.50), 4.306 (5.24), 4.309 (5.45), 4.321 (4.65), 4.336 (0.95), 4.349 (1.18), 4.487 (0.81), 4.501 (2.62), 4.510 (2.50), 4.517 (3.12), 4.523 (3.10), 4.528 (3.10), 4.532 (3.22), 4.539 (2.42), 4.546 (1.40), 4.549 (2.33), 4.563 (0.66), 4.695 (4.44), 4.710 (4.62), 5.010 (0.72), 5.019 (0.81), 5.022 (1.05), 5.031 (1.57), 5.035 (1.11), 5.039 (1.84), 5.043 (1.36), 5.047 (1.45), 5.050 (1.09), 5.055 (0.83), 5.059 (0.89), 5.067 (0.66), 5.200 (0.91), 6.528 (0.52), 6.531 (0.87), 6.535 (0.85), 6.538 (0.50), 6.547 (0.47), 6.550 (1.07), 6.555 (1.01), 6.558 (0.45), 6.821 (0.72), 6.841 (1.09), 6.860 (0.58), 7.196 (0.93), 7.207 (16.00), 7.216 (6.94), 7.221 (8.86), 7.241 (1.42), 7.303 (4.95), 7.309 (4.98), 7.316 (6.28), 7.323 (3.92), 7.332 (2.77), 7.343 (0.41), 7.708 (3.22), 8.243 (7.80), 8.255 (7.68), 8.425 (11.83), 13.670 (1.09), 13.685 (2.13), 13.699 (1.03), 14.636 (5.86).
LC-MS (method 6): Rt=0.98 min; MS (ESIpos): m/z=487 [M+H]+
Using an analogous method as described for intermediate 6-1, N-(3-chloro-2-ethylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-77, 270 mg, 85% purity, 739 μmol) and 1-{3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-9, 200 mg, 960 μmol) as the starting materials, 148 mg (90% purity, 36% yield) of the title compound were prepared after purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
LC-MS (method 6): Rt=1.03 min; MS (ESIpos): m/z=501 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.024 (0.65), 1.036 (2.48), 1.055 (6.17), 1.074 (2.53), 1.445 (16.00), 2.075 (2.07), 2.372 (0.48), 2.390 (0.62), 2.395 (0.69), 2.399 (0.68), 2.412 (0.63), 2.417 (0.86), 2.422 (0.66), 2.439 (0.60), 2.518 (3.24), 2.523 (2.28), 2.572 (0.62), 2.590 (1.96), 2.609 (1.93), 2.627 (0.57), 2.715 (0.51), 2.731 (0.65), 2.737 (0.85), 2.743 (0.68), 2.753 (1.63), 2.758 (1.33), 2.771 (2.11), 2.781 (1.02), 2.788 (1.25), 3.147 (0.79), 3.154 (0.89), 3.163 (1.48), 3.170 (1.42), 3.180 (0.77), 3.187 (0.65), 4.125 (0.59), 4.152 (4.22), 4.157 (4.15), 4.184 (0.54), 4.352 (0.45), 4.366 (1.00), 4.374 (0.62), 4.381 (0.88), 4.389 (0.99), 4.404 (0.66), 4.411 (0.71), 4.425 (0.65), 4.428 (0.88), 4.433 (0.92), 4.442 (0.63), 4.447 (0.68), 4.450 (0.79), 4.464 (0.42), 4.718 (1.53), 4.723 (1.51), 4.734 (1.56), 4.737 (1.57), 7.192 (0.46), 7.204 (6.80), 7.212 (2.91), 7.219 (3.64), 7.239 (0.71), 7.297 (2.25), 7.307 (3.81), 7.315 (1.22), 7.322 (1.71), 7.331 (1.22), 7.712 (1.45), 8.243 (3.33), 8.255 (3.05), 8.425 (4.98), 13.666 (0.51), 13.680 (0.96), 13.695 (0.46), 14.634 (2.64).
Using an analogous method as described for intermediate 6-1, N-(3-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 5-4, 670 mg, 2.26 mmol) and tert-butyl (2R)-2-({[4-(aminomethyl)pyridin-3-yl]oxy}methyl)azetidine-1-carboxylate (intermediate 2-6, 785 mg, 93% purity, 2.49 mmol) as the starting materials, 550 mg (90% purity, 38% yield) of the title compound were prepared after purification by flash chromatography (amino phase silica, hexane/EtOAc gradient, 0-100%).
LC-MS (method 6): Rt=1.08 min; MS (ESIpos): m/z=572 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.154 (4.80), 1.172 (10.04), 1.190 (4.93), 1.295 (0.72), 1.988 (16.00), 2.518 (0.42), 3.780 (1.68), 3.782 (1.70), 3.999 (1.29), 4.017 (3.81), 4.035 (3.71), 4.053 (1.18).
Using an analogous method as described for intermediate 6-1, N-(2-chloro-3-methylphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (461 mg, 1.55 mmol) and 1-{3-[(3-ethyloxetan-3-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-4, 414 mg, 1.86 mmol) as the starting materials, 200.3 mg (24% yield) of the title compound were prepared after purification by reversed phase HPLC (basic conditions).
LC-MS (method 1): Rt=0.98 min; MS (ESIpos): m/z=501 [M+H]+
1H-NMR (400 MHz, DMSO-d6) delta [ppm]: 0.868 (0.58), 0.877 (3.86), 0.895 (9.75), 0.914 (4.13), 1.779 (1.02), 1.798 (3.26), 1.816 (3.16), 1.835 (0.91), 2.074 (0.64), 2.229 (0.58), 2.313 (0.50), 2.322 (0.44), 2.327 (0.62), 2.332 (0.52), 2.349 (16.00), 2.518 (1.79), 2.523 (1.20), 2.669 (0.48), 2.734 (1.25), 2.751 (2.56), 2.768 (1.48), 3.140 (0.94), 3.147 (1.08), 3.157 (1.73), 3.163 (1.64), 3.173 (0.94), 3.181 (0.79), 4.271 (8.64), 4.336 (5.51), 4.351 (6.53), 4.485 (6.69), 4.500 (5.55), 4.656 (2.91), 4.670 (2.91), 7.202 (6.67), 7.215 (5.22), 7.303 (2.49), 7.316 (2.54), 7.368 (1.47), 7.381 (2.26), 7.392 (1.12), 7.679 (1.75), 8.246 (3.87), 8.258 (3.84), 8.460 (5.76), 13.660 (0.64), 13.675 (1.27), 13.689 (0.60), 14.706 (3.55).
Using an analogous method as described for intermediate 6-1, N-(2,3-dichlorophenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (419 mg, 1.32 mmol) and 1-{3-[(3-ethyloxetan-3-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-4, 352 mg, 1.58 mmol) as the starting materials, 176 mg (24% yield) of the title compound were prepared after purification by reversed phase HPLC (basic conditions).
LC-MS (method 1): Rt=1.02 min; MS (ESIpos): m/z=521 [M+H]+
1H-NMR (400 MHz, DMSO-d6) delta [ppm]: 0.875 (6.52), 0.894 (16.00), 0.913 (6.89), 1.231 (0.44), 1.777 (1.76), 1.795 (5.47), 1.814 (5.28), 1.833 (1.51), 2.074 (2.86), 2.518 (2.70), 2.523 (1.80), 2.753 (2.24), 2.769 (4.44), 2.786 (2.51), 3.148 (1.69), 3.155 (1.95), 3.164 (3.08), 3.171 (2.96), 3.180 (1.69), 3.188 (1.38), 4.271 (14.37), 4.335 (9.35), 4.350 (10.55), 4.483 (10.98), 4.498 (9.09), 4.669 (4.98), 4.683 (4.89), 7.303 (4.15), 7.315 (4.20), 7.326 (3.01), 7.346 (6.79), 7.367 (4.41), 7.493 (3.43), 7.497 (4.99), 7.514 (3.79), 7.517 (7.35), 7.519 (3.56), 7.536 (3.12), 7.540 (2.51), 7.742 (3.03), 8.247 (6.36), 8.259 (5.67), 8.463 (9.22), 13.611 (1.14), 13.624 (2.22), 13.638 (1.03), 14.883 (6.03).
Using an analogous method as described for intermediate 6-1, N-(3-fluoro-2-methoxyphenyl)-4-hydroxy-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (150 mg, 91% purity, 461 μmol) and 1-{3-[(3-ethyloxetan-3-yl)methoxy]pyridin-4-yl}methanamine (intermediate 2-4, 154 mg, 691 μmol) as the starting materials, 109 mg (47% yield) of the title compound were prepared after purification by reversed phase HPLC (basic conditions).
LC-MS (method 1): Rt=0.91 min; MS (ESIpos): m/z=501 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.877 (2.18), 0.896 (5.36), 0.915 (2.33), 1.782 (0.59), 1.801 (1.86), 1.819 (1.77), 1.838 (0.51), 2.074 (16.00), 2.518 (0.85), 2.522 (0.57), 2.732 (0.73), 2.749 (1.47), 2.766 (0.84), 3.133 (0.54), 3.140 (0.61), 3.150 (0.99), 3.156 (0.94), 3.166 (0.54), 3.173 (0.45), 3.776 (8.24), 3.779 (8.45), 4.277 (4.88), 4.338 (3.14), 4.354 (3.52), 4.487 (3.68), 4.501 (3.05), 4.663 (1.68), 4.678 (1.68), 5.758 (6.11), 7.046 (0.84), 7.062 (0.98), 7.065 (1.81), 7.072 (1.03), 7.081 (0.87), 7.093 (0.90), 7.098 (0.91), 7.309 (1.42), 7.321 (1.45), 7.649 (0.88), 7.654 (0.54), 7.666 (0.72), 7.672 (0.54), 7.703 (0.99), 8.250 (2.21), 8.262 (2.12), 8.465 (3.31), 13.662 (0.69), 14.734 (1.80).
To a suspension of N-(3-fluoro-2-methoxyphenyl)-4-(((3-hydroxypyridin-4-yl)methyl)amino)-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-18, 41 g, 101.88 mmol) in MeOH (410 mL) was added TFA (0.75 mL, 10.13 mmol), followed by hydrogen peroxide (18 mL, 30% in water). The mixture was heated to 60° C. and stirred for 16 h. Additional TFA (6.8 mL, 91.84 mmol) and hydrogen peroxide (1.5 mL, 30% in water) were added. The suspension was stirred at 60° C. for further 3 h. The mixture was cooled to room temperature and stand overnight. The suspension was combined with a second batch that was generated identically. The combined suspension was filtered and the cake was washed with water (250 mL) and MeOH (150 mL), and then slurried in MeOH (150 mL). The suspension was filtered. The cake was washed with MeOH (75 mL) and dried in vacuum to afford the title compound (25.4 g, 33.8% yield) as a yellow solid.
1H NMR (400 MHz, DMSO-d6): 5=11.45 (s, 1H), 8.18 (s, 1H), 7.98 (d, 1H), 7.39 (d, 1H), 7.18 (s, 1H), 6.67 (t, 1H), 6.54 (t, 1H), 6.04 (d, 1H), 3.92 (s, 3H), 3.40 (t, 2H), 2.90 (t, 2H).
LC-MS (method 5): Rt=1.851 min; m/z=369.0 (M+H)+
To a suspension of tert-butyl (2S)-2-[({4-[({5-[(3-chloro-2-methoxyphenyl)carbamothioyl]-6-oxo-1,2,3,6-tetrahydropyridin-4-yl}amino)methyl]pyridin-3-yl}oxy)methyl]azetidine-1-carboxylate (intermediate 6-25, 530 mg, 541 μmol) in MeOH (7.1 ml) was added TFA (42 μl, 540 μmol) followed by aqueous hydrogen peroxide (95 μl, 35% purity, 1.1 mmol) and heated at 50° C. for 17 h. The reaction mixture was allowed to cool down to RT. Sat. sodium thiosulfate solution and sat. sodium bicarbonate solution were added and the reaction mixture was stirred for 15 min. The mixture was diluted with water and extracted with DCM /EtOH (9/1; 3×), the combined organic layers were filtered through a water-repellent filter and concentrated under reduced pressure. The residue was purified by preparative HPLC ((method 10, gradient: 0.00-0.50 min 30% B, 0.50-7.00 min 30-70% B).) to give 160 mg of the title compound (51% yield).
LC-MS (method 2): Rt=1.22 min; MS (ESIpos): m/z=554 [M+H]+
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.45 (br s, 1H), 8.40 (s, 1H), 8.00 (br d, 1H), 7.55 (br s, 1H), 7.28-7.41 (m, 1H), 7.09-7.20 (m, 1H), 6.65-6.72 (m, 2H), 6.13 (t, 1H), 4.66 (br s, 1H), 4.47 (br s, 1H), 4.31 (br d, 1H), 3.89 (s, 5H), 3.37-3.47 (m, 2H), 2.91 (br s, 2H), 2.33 (dt, 1H), 2.07 (br s, 1H), 1.39 (br s, 9H).
To a solution of tert-butyl (2S)-2-[({4-[3-(3-chloro-2-methoxyanilino)-4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-yl]pyridin-3-yl}oxy)methyl]azetidine-1-carboxylate (intermediate 25-1, 70.0 mg, 126 μmol) in DCM (890 μl) TFA (190 μl, 2.5 mmol) was added and the mixture was stirred for 2 h at RT. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative HPLC ((method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-65% B).) to give 20 mg of the title compound (33% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=12.70-13.84 (m, 1H), 8.43 (s, 1H), 7.98 (d, 1H), 7.61 (s, 1H), 7.31 (d, 1H), 7.09-7.17 (m, 1H), 6.68-6.76 (m, 3H), 6.22 (dd, 1H), 5.76 (s, 1H), 4.44 (dd, 1H), 4.31 (br t, 1H), 3.91 (s, 4H), 3.61-3.70 (m, 1H), 3.38-3.43 (m, 2H), 2.75-2.85 (m, 2H), 2.38-2.47 (m, 1H), 2.18-2.27 (m, 1H).
LC-MS (method 2): Rt=0.99 min; MS (ESIpos): m/z=454 [M+H]+
Using an analogous method as described for intermediate 25-1; tert-butyl (2R)-2-[({4-[({5-[(3-chloro-2-methoxyphenyl)carbamothioyl]-6-oxo-1,2,3,6-tetrahydropyridin-4-yl}amino)methyl]pyridin-3-yl}oxy)methyl]azetidine-1-carboxylate (intermediate 6-30, 178 mg, 303 μmol) as the starting material, 164 mg of the title compound were prepared (78% yield, 80% purity) after heating overnight and purification by selective solubilization with ethanol.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.45 (br s, 1H), 8.40 (s, 1H), 8.00 (br d, 1H), 7.55 (br s, 1H), 7.35 (br d, 1H), 7.16 (br s, 1H), 6.69 (d, 2H), 6.13 (t, 1H), 4.66 (br s, 1H), 4.47 (br s, 1H), 4.31 (br d, 1H), 3.67-3.94 (m, 5H), 3.36-3.46 (m, 2H), 2.91 (br s, 2H), 2.25-2.45 (m, 1H), 2.08 (s, 1H), 1.39 (br s, 9H).
LC-MS (method 2): Rt=1.23 min; MS (ESIpos): m/z=554.6 [M+H]+
Using an analogous method as described for intermediate 25-2; tert-butyl (2R)-2-[({4-[3-(3-chloro-2-methoxyanilino)-4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-yl]pyridin-3-yl}oxy)methyl]azetidine-1-carboxylate (intermediate 30-1, 134 mg, 230 μmol) as the starting material, 58 mg of the title compound were prepared (54% yield) after stirring overnight and purification by preparative HPLC (method 9, gradient: 0-0.50 min 10% B, 0.50-6.00 min 10-50% B).
Optical rotation:[α]D=−93.9+/−1.29° (c=5.7 mg/ml, methanol)
LC-MS (method 2): Rt=0.99 min; MS (ESIpos): m/z=454.5 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.154 (0.87), 1.172 (1.76), 1.190 (0.89), 1.987 (3.18), 2.216 (0.56), 2.236 (0.63), 2.322 (0.63), 2.326 (0.86), 2.331 (0.63), 2.399 (0.67), 2.421 (0.82), 2.447 (0.73), 2.522 (3.27), 2.664 (0.62), 2.668 (0.83), 2.673 (0.62), 2.771 (0.82), 2.778 (0.92), 2.788 (1.61), 2.793 (1.56), 2.805 (0.99), 2.812 (0.90), 3.190 (0.42), 3.198 (0.50), 3.217 (0.79), 3.229 (0.52), 3.237 (0.44), 3.380 (0.79), 3.397 (1.41), 3.417 (1.33), 3.429 (0.63), 3.649 (0.80), 3.669 (0.79), 3.911 (16.00), 3.934 (1.10), 3.942 (1.00), 4.017 (0.75), 4.035 (0.75), 4.312 (0.80), 4.331 (0.43), 4.426 (1.16), 4.432 (1.16), 4.452 (1.10), 4.458 (1.00), 6.205 (1.39), 6.212 (1.36), 6.222 (1.43), 6.229 (1.46), 6.707 (0.72), 6.721 (4.96), 6.727 (2.81), 6.739 (2.24), 6.759 (0.53), 7.143 (1.84), 7.301 (2.64), 7.313 (2.72), 7.615 (3.54), 7.974 (3.24), 7.987 (2.95), 8.435 (4.99).
Using an analogous method as described for intermediate 25-1; tert-butyl 2-[({4-[({5-[(3-chloro-2-methoxyphenyl)carbamothioyl]-6-oxo-1,2,3,6-tetrahydropyridin-4-yl}amino)methyl]pyridin-3-yl}oxy)methyl]-2-methylazetidine-1-carboxylate (intermediate 6-31, 220 mg, 365 μmol) as the starting material, 99.5 mg of the title compound were prepared (43% yield) after purification by preparative HPLC (method 10, gradient: 0-0.75 min 10% B, 0.75-10.00 min 10-50% B, 10.00-12.40 min 50% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=8.35-8.50 (m, 1H), 7.96-8.09 (m, 1H), 7.24-7.58 (m, 2H), 7.08-7.19 (m, 1H), 6.58-6.74 (m, 2H), 6.05-6.24 (m, 1H), 4.30-4.52 (m, 1H), 4.06-4.24 (m, 1H), 3.81-3.91 (m, 3H), 3.52-3.82 (m, 2H), 3.39-3.48 (m, 2H), 2.73-3.02 (m, 2H), 2.17-2.31 (m, 1H), 1.82-2.10 (m, 1H), 1.51-1.65 (m, 2H), 1.32-1.43 (m, 6H), 1.15-1.29 (m, 5H).
LC-MS (method 1): Rt=1.05 min; MS (ESIpos): m/z=568.3 [M+H]+
Using an analogous method as described for intermediate 25-2; tert-butyl 2-[({4-[3-(3-chloro-2-methoxyanilino)-4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-yl]pyridin-3-yl}oxy)methyl]-2-methylazetidine-1-carboxylate (intermediate 31-1, 98.1 mg, 173 μmol) as the starting material, 65 mg of the title compound were prepared (72% yield) after purification by preparative HPLC (method 10, gradient: 0-0.50 min 1% B, 0.50-5.00 min 1-25% B, 5.00-5.35 min 25% B, 5.35-10.40 min 25-40% B, 10.40-11.80 min 40% B, 11.80-17.0 min 40-60% B).
LC-MS (method 2): Rt=1.05 min; MS (ESIpos): m/z=468 [M+H]+
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=13.70-14.01 (m, 1H), 8.44 (s, 1H), 7.96-7.99 (m, 1H), 7.60-7.65 (m, 1H), 7.29-7.34 (m, 1H), 7.12-7.18 (m, 1H), 6.73 (d, 2H), 6.22 (dd, 1H), 4.32-4.39 (m, 1H), 3.86-3.95 (m, 3H), 3.60-3.71 (m, 2H), 3.36-3.47 (m, 2H), 3.13-3.25 (m, 1H), 2.90-3.14 (m, 1H), 2.70-2.84 (m, 2H), 2.54-2.60 (m, 1H), 1.84-2.00 (m, 1H), 1.39-1.47 (m, 3H).
Using an analogous method as described for intermediate 25-1, tert-butyl (2R)-2-[({4-[({5-[(3-fluoro-2-methoxyphenyl)carbamothioyl]-6-oxo-1,2,3,6-tetrahydropyridin-4-yl}amino)methyl]pyridin-3-yl}oxy)methyl]azetidine-1-carboxylate (intermediate 6-79, 240 mg, 420 μmol) as the starting material, 135 mg (95% purity, 57% yield) of the title compound were prepared after heating overnight at 60° C. and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
LC-MS (method 2): Rt=1.22 min; MS (ESIpos): m/z=538 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.234 (0.63), 1.395 (7.72), 2.074 (0.42), 2.318 (1.61), 2.322 (3.37), 2.326 (4.63), 2.331 (3.30), 2.336 (1.75), 2.518 (15.09), 2.522 (10.32), 2.659 (1.47), 2.664 (3.23), 2.668 (4.28), 2.673 (3.02), 2.678 (1.33), 2.911 (1.68), 3.400 (1.82), 3.417 (2.95), 3.918 (16.00), 4.317 (0.91), 4.474 (0.56), 4.666 (0.56), 5.977 (2.53), 5.998 (2.53), 6.480 (0.70), 6.503 (1.26), 6.529 (0.98), 6.614 (0.77), 6.634 (1.33), 6.649 (1.33), 6.670 (0.49), 7.162 (1.96), 7.352 (1.47), 7.364 (1.54), 7.546 (0.98), 7.992 (1.75), 8.004 (1.68), 8.399 (3.23).
Using an analogous method as described for intermediate 25-2, tert-butyl (2R)-2-[({4-[3-(3-fluoro-2-methoxyanilino)-4-oxo-4,5,6,7-tetrahydro-1H-pyrrolo[3,2-c]pyridin-2-yl]pyridin-3-yl}oxy)methyl]azetidine-1-carboxylate (intermediate 79-1, 210 mg, 391 μmol) as the starting material, 77 mg (92% purity, 42% yield) of the title compound were prepared after stirring overnight and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B).
LC-MS (method 2): Rt=0.95 min; MS (ESIpos): m/z=438 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 2.232 (0.54), 2.253 (0.62), 2.261 (0.51), 2.397 (0.71), 2.419 (0.83), 2.446 (0.63), 2.468 (0.52), 2.518 (1.80), 2.522 (1.16), 2.770 (0.85), 2.779 (0.98), 2.787 (1.73), 2.798 (1.51), 2.804 (1.04), 2.813 (0.93), 3.245 (0.71), 3.252 (0.71), 3.264 (0.49), 3.364 (0.50), 3.371 (0.48), 3.379 (0.76), 3.397 (1.48), 3.402 (1.26), 3.411 (1.33), 3.416 (1.35), 3.428 (0.63), 3.433 (0.57), 3.645 (0.40), 3.666 (1.04), 3.686 (1.01), 3.934 (16.00), 3.961 (0.90), 3.969 (0.84), 4.321 (0.43), 4.342 (0.83), 4.362 (0.42), 4.431 (1.28), 4.437 (1.21), 4.457 (1.21), 4.463 (1.06), 5.759 (2.90), 6.061 (1.73), 6.081 (1.82), 6.502 (0.85), 6.506 (0.86), 6.523 (1.18), 6.527 (1.19), 6.530 (1.04), 6.533 (0.89), 6.551 (1.09), 6.554 (1.00), 6.658 (0.83), 6.673 (0.96), 6.679 (1.44), 6.694 (1.41), 6.699 (0.73), 6.714 (0.63), 7.143 (2.06), 7.310 (3.45), 7.323 (3.46), 7.593 (4.18), 7.976 (4.25), 7.989 (4.14), 8.430 (6.38).
To a solution of 2-chloro-1,3-difluoro-4-nitrobenzene (CAS 3847-58-3, 500 mg, 2.58 mmol) in DMSO (500 μl) was added potassium hydroxide solution (1.6 ml, 10% in water, 3.1 mmol) and the mixture was stirred for 4 h at RT. Water was added and the mixture was extracted with DCM (2×). The combined organic layers were washed with water, filtered through a water-repellent filter and concentrated under reduced pressure. The residue was purified by flash chromatography (silica, hexane/EtOAc gradient 0-50%) to give 135 mg of the title compound (26% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=7.12 (dd, 1H), 8.06 (dd, 1H).
To a solution of 2-chloro-3-fluoro-6-nitrophenol (intermediate A-13, 120 mg, 626 μmol) in acetone (12.0 ml) was added iodomethane (780 μl, 25.2 mmol) and potassium carbonate (260 mg, 1.88 mmol) and the mixture was stirred for 3 days at 50° C. The mixture was concentrated under reduced pressure and purified by flash chromatography (silica, hexane/EtOAc gradient 0-50%) to give 92 mg of the title compound (68% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=3.98 (s, 3H), 7.50 (dd, 1H), 8.09 (dd, 1H).
2-chloro-1-fluoro-3-methoxy-4-nitrobenzene (intermediate B-13, 1.00 g, 4.86 mmol) was solved in THF (60 ml) and methanol (60 ml). Platinum 1% and vanadium 2% (on activated carbon, 50-70% wetted powder, 285 mg, 14.6 μmol) was added and the mixture was stirred under hydrogen atmosphere for 5 h at RT. The reaction mixture was filtered through celite, the filter cake was washed with methanol. The filtrate was concentrated under reduced pressure and purified by flash chromatography (basic silica, hexane/EtOAc gradient 20-100%) to give 718 mg of the title compound (80% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=3.70 (s, 3H), 5.08 (s, 2H), 6.63 (dd, 1H), 6.89 (t, 1H).
LC-MS (method 1): Rt=0.94 min; MS (ESIpos): m/z=176 [M+H]+
Using an analogous method as described for intermediate C-13; 1-chloro-2-(difluoromethoxy)-3-nitrobenzene (intermediate D-17, 4.95 g, 22.1 mmol) as the starting material, 4.28 g of the title compound were prepared (89% yield) after stirring in EtOH for 16 h.
1H NMR (400 MHz, CDCl3): d [ppm]=4.05 (br s, 2H), 6.50 (t, 1H), 6.65-6.75 (m, 1H), 6.80 (d, 1H), 6.99 (t, 1H).
LC-MS (method 3): Rt=0.86 min., 89%. MS (ESIpos): m/z=174 no ionisation
To a solution of 2-chloro-6-nitrophenol (CAS 603-86-1, 20.0 g, 115 mmol) in DMF (400 ml) and water (40 ml) was added cesium carbonate (150 g, 461 mmol). The mixture was degassed with nitrogen for 30 min. then added sodium chloro(difluoro)acetate (43.9 g, 288 mmol) and the mixture was stirred overnight at 80° C. The reaction mixture was diluted with EtOAc and the organic layer was washed with water, dried over sodium sulphate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (1/9 EtOAc/heptane and 15/85 acetone/heptane) to give 3.5 g of the title compound (13% yield).
1H NMR (400 MHz, CDCl3): d [ppm]=6.48-6.92 (t, 1H), 7.36-7.47 (m, 1H), 7.73-7.80 (dd, 1H), 7.83-7.90 (dd, 1H).
LC-MS (method 3): Rt=0.91 min., 96%. MS (ESIpos): m/z=no ionisation
To a suspension of N-(3-chloro-2-methoxyphenyl)-4-({[3-(oxetan-2-yl)methoxy)pyridin-4-yl]methyl}amino)-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-1; 540 mg, 1.10 mmol) in MeOH (11 ml) was added TFA (85 μl, 1.1 mmol) followed by aqueous hydrogen peroxide (190 μl, 35% purity, 2.2 mmol) and the mixture was heated at 50° C. for 16 h. The reaction mixture was allowed to cool down to RT. Sat. sodium thiosulfate solution and sat. sodium bicarbonate solution were added and the reaction mixture was stirred for 15 min. The mixture was diluted with water and extracted with DCM (3×), the combined organic layers were filtered through a water-repellent filter and concentrated under reduced pressure. The residue was purified by preparative HPLC (method 9, gradient: 0.50 min 5% B, 0.50-8.00 min 5-40% B, 8.00-11.20 min 40% B) to give 326 mg of the title compound (62% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.09-11.34 (m, 1H), 8.42-8.51 (m, 1H), 8.03-8.09 (m, 1H), 7.39-7.50 (m, 1H), 7.23-7.34 (m, 1H), 7.03-7.16 (m, 1H), 6.60-6.73 (m, 2H), 6.10-6.21 (m, 1H), 5.06-5.18 (m, 1H), 4.20-4.64 (m, 4H), 3.80-3.90 (m, 3H), 3.38-3.43 (m, 2H), 2.56-2.92 (m, 4H).
LC-MS (method 1): Rt=0.78 min; MS (ESIpos): m/z=456.6 [M+H]+
The racemic title compound from example 1 (326 mg) was separated into enantiomers by preparative chiral HPLC to give the title compound (enantiomer 1, 175 mg, at Rt=15.6-16.8 min, 33% yield) and enantiomer 2 (202 mg at Rt=13.9-15.2 min, used for direct comparison to example 3 and assignment of absolute stereochemistry)
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Amylose SA 5μ 250×30 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-50% B in 17 min; flow 40.0 ml/min; UV 254 nm
Instrument: Agilent HPLC 1260; column: Amylose SA 3μ 100×4.6 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 7 min; flow 1.4 ml/min; temperature: 25° C.; DAD 254 nm
Analytical chiral HPLC: Rt=3.99 min.
Optical rotation:[α]D=57.7°+/−1.74° (c=4.67 mg/ml, methanol).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.21 (s, 1H), 8.46 (s, 1H), 8.06 (d, 1H), 7.44 (s, 1H), 7.28 (d, 1H), 7.11 (s, 1H), 6.64-6.69 (m, 2H), 6.14-6.19 (m, 1H), 5.10-5.16 (m, 1H), 4.56-4.62 (m, 1H), 4.39-4.50 (m, 2H), 4.29 (dd, 1H), 3.86 (s, 3H), 3.35-3.43 (m, 2H), 2.77-2.85 (m, 2H), 2.66-2.74 (m, 1H), 2.55-2.64 (m, 1H).
Absolute stereochemistry of example 2 was assigned by comparison of enantiomer 2 obtained from example 1 by preparative chiral HPLC (Analytical chiral HPLC: Rt=3.64 min, optical rotation:[α]D=−60.8°+/−1.77° at c=2.9 mg/ml, methanol) to example 3 synthesized from enantiopure intermediate 6-3.
In analogy to example 1 N-(3-chloro-2-methoxyphenyl)-4-{[(3-{[(2R)-oxetan-2-yl]methoxy}pyridin-4-yl)methyl]amino}-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-3, 92 mg, 188 μmol) was used to prepare 30.1 mg of the title compound (33% yield) after heating overnight and purification by preparative HPLC (method 9, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B)
Optical rotation:[α]D=−61.5°+/−0.67° (c=4.3 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 2.322 (0.43), 2.327 (0.61), 2.332 (0.45), 2.518 (2.74), 2.523 (1.76), 2.597 (0.46), 2.602 (0.64), 2.606 (0.40), 2.619 (0.43), 2.624 (0.56), 2.665 (0.70), 2.669 (0.71), 2.673 (0.52), 2.680 (0.41), 2.685 (0.58), 2.701 (0.59), 2.706 (0.54), 2.713 (0.41), 2.786 (1.05), 2.804 (2.23), 2.820 (1.23), 3.382 (0.79), 3.388 (0.87), 3.399 (1.55), 3.404 (1.57), 3.416 (0.75), 3.422 (0.68), 3.857 (16.00), 4.273 (0.81), 4.280 (0.87), 4.301 (1.23), 4.307 (1.22), 4.392 (1.18), 4.406 (1.24), 4.420 (0.81), 4.434 (0.85), 4.442 (0.51), 4.458 (0.95), 4.465 (0.61), 4.472 (0.78), 4.480 (0.93), 4.495 (0.54), 4.568 (0.54), 4.582 (0.59), 4.586 (0.75), 4.589 (0.84), 4.600 (0.65), 4.603 (0.68), 4.607 (0.64), 4.621 (0.43), 5.122 (0.59), 5.128 (0.47), 5.132 (0.46), 5.135 (0.52), 5.139 (0.56), 6.152 (1.38), 6.160 (1.23), 6.168 (1.37), 6.176 (1.44), 6.638 (0.63), 6.649 (5.92), 6.658 (2.67), 6.666 (2.27), 6.686 (0.40), 7.110 (1.52), 7.277 (2.35), 7.290 (2.37), 7.444 (3.40), 8.051 (2.92), 8.064 (2.63), 8.461 (4.18), 11.214 (1.58).
Using an analogous method as described for example 1, N-(3-fluoro-2-methoxyphenyl)-4-[({3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-4, 340 mg, 720 μmol) as the starting material, 160 mg of the title compound (48% yield) were prepared after heating for 17 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=2.60 (s, 2H), 2.80 (t, 2H), 3.40 (td, 2H), 3.89 (s, 3H), 4.29 (dd, 1H), 4.37-4.51 (m, 2H), 4.56-4.63 (m, 1H), 5.08-5.18 (m, 1H), 6.02 (d, 1H), 6.43-6.50 (m, 1H), 6.58-6.65 (m, 1H), 7.11 (s, 1H), 7.29 (d, 1H), 7.45 (s, 1H), 8.05 (d, 1H), 8.46 (s, 1H), 11.19 (s, 1H).
LC-MS (method 2): Rt=0.92 min; MS (ESIpos): m/z=439 [M+H]+
The title compound from example 4 (430 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 140 mg at Rt=19.0-21.8 min) and enantiomer 2 (150 mg at Rt=14.5-21.8 min, see example 6).
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Amylose SA 5μ 250×30 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; isocratic: 90% A+10% B; flow 60.0 ml/min; UV 254 nm
Instrument: Agilent HPLC 1260; column: Amylose SA 3μ 100×4.6 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; isocratic 90% A+10% B; flow 1.4 ml/min; temperature: 25° C.; DAD 254 nm
Analytical chiral HPLC: Rt=2.98 min.
Optical rotation:[α]D=58.4°+/−2.81° (c=4.7 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=2.56-2.75 (m, 2H), 2.80 (t, 2H), 3.40 (td, 2H), 3.89 (s, 3H), 4.29 (dd, 1H), 4.38-4.50 (m, 2H), 4.56-4.63 (m, 1H), 5.10-5.17 (m, 1H), 6.02 (d, 1H), 6.46 (ddd, 1H), 6.62 (td, 1H), 7.11 (s, 1H), 7.29 (d, 1H), 7.45 (s, 1H), 8.06 (d, 1H), 8.46 (s, 1H), 11.19 (s, 1H).
Absolute stereochemistry was assigned by comparison of optical rotation and retention behaviour in analytical chiral HPLC runs to examples 2 and 3. In addition a X-ray structure was solved for crystals obtained from example 6 by precipitation out of dioxane. An image of the solved X-ray structure depicting the absolute configuration of example 6 is given as
For the preparation of the racemic title compound see example 4. Separation of enantiomers by preparative chiral HPLC (method see example 5) to give 150 mg of the title compound.
Analytical chiral HPLC (method see example 5): Rt=2.40 min.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.19 (s, 1H), 8.46 (s, 1H), 8.05 (d, 1H), 7.45 (s, 1H), 7.29 (d, 1H), 7.01-7.15 (m, 1H), 6.62 (td, 1H), 6.42-6.50 (m, 1H), 6.00-6.04 (m, 1H), 5.10-5.17 (m, 1H), 4.55-4.63 (m, 1H), 4.27-4.50 (m, 3H), 3.89 (s, 3H), 3.40 (td, 2H), 2.80 (t, 2H), 2.57-2.75 (m, 2H).
Optical rotation:[α]D=−60.3°+/−4.02° (c=3 mg/ml, methanol)
Using an analogous method as described for example 1 with N-(3-chloro-2-methylphenyl)-4-[({3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl]amino}-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-7, 120 mg, 254 μmol) as the starting material, 26 mg of the title compound were prepared (22% yield) after heating overnight and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.18 (s, 1H), 8.43 (s, 1H), 8.02 (d, 1H), 7.26 (s, 1H), 7.24 (d, 1H), 7.13 (s, 1H), 6.69-6.77 (m, 2H), 6.22 (dd, 1H), 5.09-5.15 (m, 1H), 4.56-4.64 (m, 1H), 4.36-4.51 (m, 2H), 4.27 (dd, 1H), 3.35-3.45 (m, 2H), 2.81 (t, 2H), 2.56-2.75 (m, 2H), 2.29-2.38 (m, 3H).
LC-MS (method 2): Rt=1.01 min; MS (ESIpos): m/z=439.3 [M+H]+
The title compound from example 7 (26 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 8 mg, at Rt=15.8-17.6 min) and enantiomer 2 (9 mg at Rt=13.6-15.2 min, see example 9).
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Amylose SA 5μ 250×30 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 20 min; flow 40.0 ml/min; UV 280 nm
Instrument: Agilent HPLC 1260; column: Amylose SA 3μ 100×4.6 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 7 min.; flow 1.4 ml/min; temperature: 25° C.; DAD 280 nm
Analytical chiral HPLC: Rt=4.15 min.
Optical rotation:[α]D=98.4°+/−2.02° (c=1.1 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.18 (s, 1H), 8.44 (s, 1H), 8.02 (d, 1H), 7.26 (s, 1H), 7.22-7.25 (m, 1H), 7.13 (s, 1H), 6.69-6.77 (m, 2H), 6.22 (dd, 1H), 5.09-5.15 (m, 1H), 4.60 (ddd, 1H), 4.37-4.51 (m, 2H), 4.27 (dd, 1H), 3.35-3.43 (m, 2H), 2.81 (t, 2H), 2.66-2.73 (m, 1H), 2.56-2.65 (m, 1H), 2.33 (s, 3H).
Absolute stereochemistry was assigned by comparison of optical rotation and retention behaviour in analytical chiral HPLC runs to examples 2, 3, 5 and 6.
For the preparation of the racemic title compound see example 7. Separation of enantiomers by preparative chiral HPLC (method see example 8) gave 9 mg of the title compound.
Analytical chiral HPLC (method see example 8): Rt=3.61 min.
Optical rotation:[α]D=−35.9°+/−0.35° (c=3.7 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.18 (s, 1H), 8.44 (s, 1H), 8.02 (d, 1H), 7.26 (s, 1H), 7.24 (d, 1H), 7.14 (s, 1H), 6.69-6.77 (m, 2H), 6.22 (dd, 1H), 5.09-5.15 (m, 1H), 4.60 (ddd, 1H), 4.37-4.51 (m, 2H), 4.27 (dd, 1H), 3.36-3.44 (m, 2H), 2.81 (t, 2H), 2.65-2.75 (m, 1H), 2.55-2.65 (m, 1H), 2.33 (s, 3H).
Using an analogous method as described for example 1 with N-(2,3-dichlorophenyl)-4-[({3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-10, 120 mg, 243 μmol) as the starting material, 55 mg of the title compound were prepared (47% yield) after heating overnight and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.35 (s, 1H), 8.45 (s, 1H), 8.09 (d, 1H), 7.56 (s, 1H), 7.26 (d, 1H), 7.13 (s, 1H), 6.86 (d, 1H), 6.81-6.84 (m, 1H), 6.29 (dd, 1H), 5.05-5.11 (m, 1H), 4.57 (ddd, 1H), 4.34-4.46 (m, 2H), 4.22 (dd, 1H), 3.35-3.47 (m, 2H), 2.82 (t, 2H), 2.65-2.72 (m, 1H), 2.53-2.59 (m, 1H).
LC-MS (method 2): Rt=1.00 min; MS (ESIpos): m/z=459.2 [M+H]+
The title compound from example 10 (55 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 16 mg, at Rt=14.4-16.2 min) and enantiomer 2 (21 mg at Rt=16.6-20.0 min, see example 12).
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Chiralcel OH-H 5μ 250×20 mm; eluent A: hexane+0.1 vol. % diethylamine (99%); eluent B: ethanol; isocratic: 75% A+25% B; flow 20.0 ml/min; UV 254 nm
Analytical Chiral HPLC Method: Instrument: Agilent HPLC 1260; column: Chiralcel OD-H 3μ 100×4.6 mm; eluent A: hexane+0.1 vol. % diethylamine (99%); eluent B: ethanol; isocratic: 70% A+30% B; flow 1.4 ml/min; temperature: 25° C.; DAD 254 nm
Analytical chiral HPLC: Rt=5.69 min.
Optical rotation:[α]D=44.6°+/−2.90° (c=2.05 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.35 (s, 1H), 8.45 (s, 1H), 8.09 (d, 1H), 7.56 (s, 1H), 7.26 (d, 1H), 7.13 (s, 1H), 6.81-6.89 (m, 2H), 6.29 (dd, 1H), 5.05-5.11 (m, 1H), 4.57 (ddd, 1H), 4.34-4.46 (m, 2H), 4.22 (dd, 1H), 3.41 (td, 2H), 2.82 (t, 2H), 2.66-2.72 (m, 1H), 2.54-2.61 (m, 1H).
For the preparation of the racemic title compound see example 10. Separation of enantiomers by preparative chiral HPLC (method see example 11) gave 21 mg of the title compound.
Analytical chiral HPLC (method see example 11): Rt=6.63 min.
Optical rotation:[α]D=−40.3°+/−0.84° (c=3.7 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.35 (s, 1H), 8.45 (s, 1H), 8.09 (d, 1H), 7.56 (s, 1H), 7.26 (d, 1H), 7.13 (s, 1H), 6.81-6.89 (m, 2H), 6.29 (dd, 1H), 5.05-5.11 (m, 1H), 4.53-4.60 (m, 1H), 4.34-4.46 (m, 2H), 4.22 (dd, 1H), 3.36-3.45 (m, 2H), 2.86-2.95 (m, 1H), 2.82 (t, 2H), 2.64-2.73 (m, 1H).
Using an analogous method as described for example 1, N-(3-chloro-4-fluoro-2-methoxyphenyl)-4-[({3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-13, 200 mg, 394 μmol) as the starting material, 50 mg of the title compound were prepared (24% yield) after heating for 17 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=2.56-2.77 (m, 2H), 2.80 (t, 2H), 3.40 (td, 2H), 3.89 (s, 3H), 4.24-4.38 (m, 1H), 4.38-4.53 (m, 2H), 4.53-4.63 (m, 1H), 5.10-5.17 (m, 1H), 6.15 (dd, 1H), 6.77 (t, 1H), 7.11 (s, 1H), 7.28 (d, 1H), 7.32 (s, 1H), 8.07 (d, 1H), 8.47 (s, 1H), 11.22 (s, 1H).
LC-MS (method 2): Rt=0.99 min; MS (ESIpos): m/z=473.2 [M+H]+
The title compound from example 13 (50 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 20 mg at Rt=11.5-13.4 min) and enantiomer 2 (20 mg at Rt=13.7-15.0 min, see example 15).
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Amylose SA 5μ 250×30 mm; eluent A: MTBE 0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 20 min; flow 50.0 ml/min; UV 254 nm
Instrument: Agilent HPLC 1260; column: Amylose SA 3μ 100×4.6 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 7 min; flow 1.4 ml/min; temperature: 25° C.; DAD 254 nm
Analytical chiral HPLC: Rt=3.65 min.
Optical rotation:[α]D=−47.9°+/−0.31° (c=7.3 mg/ml, methanol)
For the preparation of the racemic title compound see example 13. Separation of enantiomers by preparative chiral HPLC (method see example 14) gave 20 mg of the title compound.
Analytical chiral HPLC (method see example 14): Rt=4.22 min.
Optical rotation:[α]D=59.5°+/−0.71° (c=7.8 mg/ml, methanol)
To a solution of N-(2-chloro-3-fluorophenyl)-4-[({3-[(oxetan-3-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-16, 388 mg, 813 μmol) in MeOH (5 ml) was added aqueous hydrogen peroxide (140 μl, 35%, 1.6 mmol) and the mixture heated at 70° C. overnight. The mixture was cooled down to RT and concentrated under reduced pressure. The crude residue was purified by reverse phase chromatography (Biotage Isolera, 60 g; SNAP C18 Ultra Biotage cartridge; acetonitrile+0.1% ammonium hydroxide/water+0.1% ammonium hydroxide; 5/95; 100/0) to give 100 mg of the title compound (36% yield).
1H NMR (400 MHz, CDCl3): δ [ppm]=2.91 (t, 2H), 3.31-3.36 (m, 1H), 3.50 (t, 2H), 4.49 (s, 2H), 4.76-4.79 (m, 2H), 5.25 (t, 2H), 5.31 (s, 1H), 6.23 (d, 1H), 6.62 (dd, 1H), 6.86 (dd, 1H), 7.50 (d, 1H), 7.72 (s, 1H), 8.05 (d, 1H), 8.37 (s, 1H), 11.37 (s, 1H).
LC-MS (method 4): Rt=0.69 min; MS (ESIpos): m/z=443 [M+H]+
To a solution of N-[3-chloro-2-(difluoromethoxy)phenyl]-4-[({3-[(oxetan-3-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-17, 705 mg, 1.34 mmol) in MeOH (12 ml) was added TFA (210 μl, 2.7 mmol) and stirred for 10 min. then added meta-chloroperoxybenzoic acid (463 mg, 2.69 mmol) and the mixture heated at 50° C. for 4 h. The reaction mixture was cooled down to RT and concentrated under reduced pressure. The residue was purified by reverse phase chromatography (Biotage Isolera, 60 g; SNAP C18 Ultra Biotage cartridge; acetontirle+0.1% formic acid/water+0.1% formic acid; 5/95, 100/0) then treated with sat. sodium bicarbonate solution, extracted with DCM and concentrated under reduced pressure, to give 40 mg of the title compound (6% yield).
1H NMR (400 MHz, CD3OD+CDCl3 drops): δ [ppm]=2.88 (t, 2H), 3.33-3.41 (m, 1H), 3.52 (t, 2H), 4.45 (d, 2H), 4.66 (t, 2H), 5.06 (t, 2H), 6.32-6.39 (m, 1H), 6.59-7.06 (m, 3H), 7.14 (s, 1H), 7.58 (d, 1H), 7.78 (s, 1H), 7.96 (d, 1H), 8.30 (s, 1H).
LC-MS (method 3): Rt=1.25 min; MS (ESIpos): m/z=491 [M+H]+
Triphenylphosphine (318 mg, 1.21 mmol) was solved in THF and diisopropyl azodicarboxylate (240 μl, 1.2 mmol) was added. Then (oxetan-3-yl)methanol (49 μl, 610 μmol, CAS 6246-06-6) and 3-(3-fluoro-2-methoxyanilino)-2-(3-hydroxypyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 18, 223 mg, 605 μmol) were added and the mixture was stirred at RT for 2 days. The mixture was purified by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B). The impure product was purified by preparative HPLC again (instrument: Waters Acquity UPLC-MS SingleQuad; column: Acquity UPLC BEH C18 1.7 μm, 50×2.1 mm; eluent A: water (0.2 vol. % aqueous ammonia 32%), eluent B: acetonitrile; gradient: 0-1.6 min. 1-99% B, 1.6-2.0 min. 99% B; flow 0.8 ml/min; temperature: 60° C.; DAD scan: 210-400 nm) to give 5.2 mg of the title compound (2% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.23 (s, 1H), 8.42 (s, 1H), 8.05 (d, 1H), 7.47 (s, 1H), 7.34 (d, 1H), 7.11 (s, 1H), 6.63 (td, 1H), 6.48 (ddd, 1H), 5.99 (d, 1H), 4.80 (dd, 2H), 4.48 (t, 2H), 4.39 (d, 2H), 3.90 (s, 3H), 3.35-3.42 (m, 3H), 2.79 (t, 2H).
LC-MS (method 2): Rt=0.90 min; MS (ESIpos): m/z=439 [M+H]+
Using an analogous method as described for example 18; 3-(3-fluoro-2-methoxyanilino)-2-(3-hydroxypyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 18, 208 mg, 565 μmol) and (2-methyloxetan-2-yl)methanol (CAS 61266-71-5, 57.7 mg, 565 μmol) as the starting materials, 18.7 mg of the title compound were prepared (7% yield) after preparative HPLC (instrument: Waters Acquity UPLC-MS SingleQuad; column: Acquity UPLC BEH C18 1.7 μm, 50×2.1 mm; eluent A: water (0.1 vol. % formic acid 99%), eluent B: acetonitrile; gradient: 0-1.6 min. 1-99% B, 1.6-2.0 min. 99% B; flow 0.8 ml/min; temperature: 60° C.; DAD scan: 210-400 nm).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.32 (s, 1H), 8.48 (s, 1H), 8.06 (d, 1H), 7.42 (s, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.63 (td, 1H), 6.47 (ddd, 1H), 6.05 (d, 1H), 4.46 (dt, 1H), 4.29-4.38 (m, 2H), 4.12 (d, 1H), 3.86-3.90 (m, 3H), 3.35-3.42 (m, 2H), 2.74-2.82 (m, 3H), 2.33-2.46 (m, 1H), 1.47 (s, 3H).
LC-MS (method 2): Rt=0.99 min; MS (ESIpos): m/z=453 [M+H]+
The title compound from example 19 (30 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 7 mg, at Rt=10.1-10.8 min) and enantiomer 2 (11 mg at Rt=9.0-9.7 min, see example 21).
Instrument: Sepiatec: Prep SFC100; column: Chiralpak IB 5μ 250×30 mm; eluent A: CO2; eluent B: methanol+0.2 vol. % aq. ammonia (32%); isocratic: 20% B; flow 100 ml/min; temperature: 40° C.; BPR: 150 bar; UV: 254 nm
Instrument: Agilent: 1260, Aurora SFC-Modul; column: Chiralpak IB 5μ 100×4.6 mm; eluent A: CO2; eluent B: methanol+0.1 vol. % aq. ammonia (32%); isokratic: 20% B; flow 4 ml/min; temperature: 37.5° C.; BPR: 100 bar; UV: 254 nm
Analytical chiral HPLC: Rt=3.2 min.
Optical rotation:[α]D=−8.2°+/−0.01° (c=1.7 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.32 (s, 1H), 8.48 (s, 1H), 8.06 (d, 1H), 7.42 (s, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.63 (td, 1H), 6.47 (ddd, 1H), 6.05 (d, 1H), 4.46 (dt, 1H), 4.30-4.39 (m, 2H), 4.12 (d, 1H), 3.89 (s, 3H), 3.37-3.43 (m, 2H), 2.73-2.82 (m, 3H), 2.34-2.44 (m, 1H), 1.47 (s, 3H).
For the preparation of the racemic title compound see example 19. Separation of enantiomers by preparative chiral HPLC (method see example 20) gave 11 mg of the title compound.
Analytical chiral HPLC (method see example 20): Rt=2.4 min.
Optical rotation:[α]D=21.0°+/−0.3° (c=2 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.32 (s, 1H), 8.48 (s, 1H), 8.06 (d, 1H), 7.42 (s, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.63 (td, 1H), 6.47 (t, 1H), 6.05 (d, 1H), 4.46 (dt, 1H), 4.29-4.39 (m, 2H), 4.12 (d, 1H), 3.89 (s, 3H), 3.37-3.43 (m, 2H), 2.73-2.82 (m, 3H), 2.35-2.42 (m, 1H), 1.47 (s, 3H).
Using an analogous method as described for example 1, N-(3-chloro-2-methoxyphenyl)-4-[({3-[(3-ethyloxetan-3-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-22, 250 mg, 479 μmol) as the starting material, 133 mg of the title compound were prepared (55% yield) after heating overnight and purification by preparative HPLC (method 9, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.855 (2.78), 0.874 (6.75), 0.892 (3.04), 1.691 (0.76), 1.710 (2.35), 1.729 (2.26), 1.748 (0.66), 2.074 (0.42), 2.331 (0.68), 2.518 (3.97), 2.522 (2.43), 2.739 (1.23), 2.756 (2.63), 2.774 (1.35), 3.355 (1.20), 3.362 (1.16), 3.372 (1.87), 3.378 (1.79), 3.390 (0.91), 3.395 (0.83), 3.883 (16.00), 4.294 (5.91), 4.523 (2.82), 4.538 (4.61), 4.569 (4.71), 4.585 (2.75), 6.128 (1.43), 6.140 (2.23), 6.152 (1.47), 6.676 (4.88), 6.687 (3.63), 6.689 (3.38), 7.117 (1.64), 7.373 (2.75), 7.386 (2.75), 7.438 (3.56), 8.046 (3.44), 8.059 (3.14), 8.132 (0.44), 8.496 (4.52), 11.334 (1.72).
LC-MS (method 2): Rt=1.11 min; MS (ESIpos): m/z=483.5 [M+H]+
Using an analogous method as described for example 1, N-(3-chloro-2-methylphenyl)-4-[({3-[(3-ethyloxetan-3-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-23, 250 mg, 499 μmol) as the starting material, 47.4 mg of the title compound were prepared (19% yield) after heating overnight and purification by preparative HPLC (method 9, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.32 (s, 1H), 8.47 (s, 1H), 8.01 (d, 1H), 7.34 (d, 1H), 7.28 (s, 1H), 7.14 (s, 1H), 6.72-6.80 (m, 2H), 6.20 (dd, 1H), 4.52-4.61 (m, 4H), 4.28 (s, 2H), 3.35-3.41 (m, 2H), 2.76 (t, 2H), 2.35 (s, 3H), 1.72 (q, 2H), 0.88 (t, 3H).
LC-MS (method 1): Rt=0.90 min; MS (ESIpos): m/z=467 [M+H]+Example 24
To a suspension of N-(3-chloro-2-methoxyphenyl)-4-[({3-[2-(oxetan-2-yl)ethoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-24, 60.0 mg, 119 μmol) in MeOH (2 ml) was added TFA (9.2 μl, 120 μmol) followed by aqueous hydrogen peroxide (21 μl, 35% purity, 240 μmol) and heated at 50° C. overnight. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC (method 9, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B). To the impure produce was added water and the mixture was neutralised with 1 N NaOH to pH=7 and extracted with DCM. The organic phase was washed with brine, dried (hydrophobic filter paper) and concentrated under reduced pressure to give 26.8 mg of the title compound (47% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.28 (s, 1H), 8.36 (s, 1H), 7.99 (d, 1H), 7.54 (s, 1H), 7.33 (d, 1H), 7.14 (s, 1H), 6.69 (d, 2H), 6.12 (t, 1H), 5.04-5.11 (m, 1H), 4.56-4.69 (m, 2H), 4.37 (dt, 1H), 4.25 (td, 1H), 3.89 (s, 3H), 3.35-3.43 (m, 2H), 2.68-2.79 (m, 3H), 2.37-2.47 (m, 2H), 1.98-2.18 (m, 1H).
LC-MS (method 1): Rt=0.83 min; MS (ESIpos): m/z=469 [M+H]+
The title compound from example 24 (22 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 8 mg at Rt=12.3-13.5 min) and enantiomer 2 (9 mg at Rt=16.2-17.7 min, see example 26).
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Amylose SA 5μ 250×30 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 20 min; flow 50.0 ml/min; UV 254 nm
Instrument: Agilent HPLC 1260; column: Amylose SA 3μ 100×4.6 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 7 min; flow 1.4 ml/min; temperature: 25° C.; DAD 254 nm
Optical rotation:[α]D=154°+/−8.21° (c=3.9 mg/2 ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.28 (s, 1H), 8.36 (s, 1H), 7.99 (br d, 1H), 7.54 (s, 1H), 7.33 (d, 1H), 7.14 (s, 1H), 6.69 (d, 2H), 6.12 (t, 1H), 5.04-5.11 (m, 1H), 4.56-4.69 (m, 2H), 4.37 (dt, 1H), 4.25 (td, 1H), 3.89 (s, 3H), 3.37-3.44 (m, 2H), 2.66-2.79 (m, 3H), 2.32-2.48 (m, 2H), 2.04-2.14 (m, 1H).
For the preparation of the racemic title compound see example 24. Separation of enantiomers by preparative chiral HPLC (method see example 25) gave 9 mg of the title compound.
Analytical chiral HPLC (method see example 25): Rt=4.9 min.
Optical rotation:[α]D=−53.6°+/−10.97° (c=4.1 mg/2 ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.28 (s, 1H), 8.36 (br s, 1H), 7.99 (br d, 1H), 7.54 (s, 1H), 7.33 (d, 1H), 7.14 (s, 1H), 6.69 (d, 2H), 6.10-6.14 (m, 1H), 5.04-5.11 (m, 1H), 4.56-4.69 (m, 2H), 4.37 (dt, 1H), 4.25 (td, 1H), 3.89 (s, 3H), 3.37-3.44 (m, 2H), 2.66-2.81 (m, 3H), 2.38-2.48 (m, 2H), 2.04-2.14 (m, 1H).
To a solution of 2-(3-{[(2S)-azetidin-2-yl]methoxy}pyridin-4-yl)-3-(3-chloro-2-methoxyanilino)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 25-2, 25.0 mg, 55.1 μmol) in methanol (750 μl) were added formaldehyde (8.3 μl, 37% purity, 110 μmol) and acetic acid (3.2 μl, 55 μmol) and the mixture was stirred for 15 min. at RT. After that sodium trisacetoxyborohydride (17.5 mg, 82.6 μmol) was added and the mixture was stirred for 1.5 h at RT. The reaction mixture was passed through a SCX-2 cartridge, eluted with 7N ammonia in methanol and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (method 10, gradient: 0.00-0.50 min 5% B, 0.50-8.00 min 5-50% B, 8.00-11.20 min 50% B) to give 1.57 mg of the title compound (5% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=12.85 (br s, 1H), 8.43 (s, 1H), 7.99 (br d, 1H), 7.56-7.66 (m, 1H), 7.30 (d, 1H), 7.16 (s, 1H), 6.69-6.75 (m, 2H), 6.20 (dd, 1H), 4.44-4.50 (m, 1H), 4.08 (br s, 1H), 3.91 (s, 3H), 3.37-3.59 (m, 4H), 2.87-3.02 (m, 1H), 2.76-2.84 (m, 2H), 2.34-2.41 (m, 2H), 2.21-2.30 (m, 1H), 2.07 (s, 1H).
LC-MS (method 2): Rt=1.08 min; MS (ESIpos): m/z=468 [M+H]+
Using an analogous method as described for example 25; 2-(3-{[(2S)-azetidin-2-yl]methoxy}pyridin-4-yl)-3-(3-chloro-2-methoxyanilino)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 25-2, 25.0 mg, 55.1 μmol) and acetaldehyde (6.2 μl, 110 μmol) as the starting materials, 15.1 mg of the title compound were prepared (54% yield) after stirring for 2.5 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 5% B, 0.50-8.00 min 5-50% B, 8.00-11.20 min 50% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=12.53-12.64 (m, 1H), 8.40 (s, 1H), 7.98 (d, 1H), 7.63 (s, 1H), 7.30 (d, 1H), 7.12-7.24 (m, 1H), 6.67-6.77 (m, 2H), 6.15-6.26 (m, 1H), 4.36-4.52 (m, 1H), 4.04-4.14 (m, 1H), 3.84-3.97 (m, 3H), 3.52-3.64 (m, 1H), 3.38-3.48 (m, 3H), 2.85-2.97 (m, 1H), 2.81 (s, 1H), 2.76-2.86 (m, 1H), 2.67 (br d, 2H), 2.13-2.29 (m, 1H), 1.93-2.10 (m, 1H), 0.86-0.96 (m, 3H).
LC-MS (method 2): Rt=1.11 min; MS (ESIpos): m/z=482.3 [M+H]+
To a suspension of 2-(3-{[(2S)-azetidin-2-yl]methoxy}pyridin-4-yl)-3-(3-chloro-2-methoxyanilino)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 25-2, 25.0 mg, 55.1 μmol) in DMF (830 μl) and triethylamine (46 μl, 330 μmol) was added 2,2,2-trifluoroethyl trifluoromethanesulfonate (12 μl, 83 μmol) and the mixture was stirred for 5 h at RT. The reaction mixture was purified by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B) to give 11 mg of the title compound (33% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.56 (s, 1H), 8.38 (s, 1H), 8.01 (d, 1H), 7.56 (s, 1H), 7.31 (d, 1H), 7.16 (s, 1H), 6.66-6.70 (m, 2H), 6.13-6.18 (m, 1H), 4.32 (dd, 1H), 4.16 (dd, 1H), 3.86-3.92 (m, 4H), 3.49-3.53 (m, 1H), 3.44-3.49 (m, 1H), 3.38-3.44 (m, 2H), 3.22-3.30 (m, 2H), 2.76-2.84 (m, 2H), 2.22-2.31 (m, 1H), 2.08-2.14 (m, 1H).
LC-MS (method 2): Rt=1.13 min; MS (ESIpos): m/z=536.2 [M+H]+
2-(3-{[(2R)-azetidin-2-yl]methoxy}pyridin-4-yl)-3-(3-chloro-2-methoxyanilino)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 30-2, 58.0 mg, 128 μmol) was solved in DMF (2.0 ml), triethylamine (110 μl, 770 μmol) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (28 μl, 190 μmol) were added and the mixture was stirred at RT for 4 h. The solvent was evaporated. The mixture was purified by preparative HPLC (method X) to give 40.1 mg of the title compound (56% yield).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.56 (s, 1H), 8.38 (s, 1H), 8.01 (d, 1H), 7.57 (s, 1H), 7.31 (d, 1H), 7.17 (s, 1H), 6.69 (s, 1H), 6.68 (s, 1H), 6.16 (t, 1H), 4.32 (dd, 1H), 4.16 (dd, 1H), 3.85-3.93 (m, 4H), 3.35-3.54 (m, 4H), 3.21-3.31 (m, 2H), 2.74-2.87 (m, 2H), 2.22-2.31 (m, 1H), 2.05-2.15 (m, 1H).
LC-MS (method 2): Rt=1.15 min; MS (ESIpos): m/z=536.4 [M+H]+
Using an analogous method as described for example 25; 3-(3-chloro-2-methoxyanilino)-2-{3-[(2-methylazetidin-2-yl)methoxy]pyridin-4-yl}-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 29-2, 30.0 mg, 64.1 μmol) as the starting material, 8.2 mg of the title compound were prepared (25% yield) after purification by preparative HPLC (method 10, gradient: 0-0.50 min 1% B, 0.50-5.00 min 1-25% B, 5.00-5.35 min 25% B, 5.35-10.35 min 25-41% B, 10.35-11.80 min 41% B, 11.80-17.60 min 60% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=13.09-13.23 (m, 1H), 8.42 (s, 1H), 7.93-8.00 (m, 1H), 7.63-7.70 (m, 1H), 7.27-7.33 (m, 1H), 7.15-7.22 (m, 1H), 6.69-6.77 (m, 2H), 6.18-6.25 (m, 1H), 4.28-4.36 (m, 1H), 3.87-3.95 (m, 3H), 3.73-3.81 (m, 1H), 3.37-3.47 (m, 3H), 3.05-3.18 (m, 1H), 2.76-2.86 (m, 2H), 2.20-2.26 (m, 3H), 1.64-1.73 (m, 1H), 1.62-1.78 (m, 1H), 1.21-1.30 (m, 4H).
Using an analogous method as described for example 28, 3-(3-chloro-2-methoxyanilino)-2-{3-[(2-methylazetidin-2-yl)methoxy]pyridin-4-yl}-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 29-2, 30.0 mg, 64.1 μmol) as the starting material, 4.18 mg of the title compound were prepared (11% yield) after purification by preparative HPLC (method 10, gradient: 0-0.50 min 1% B, 0.50-5.00 min 1-25% B, 5.00-5.35 min 25% B, 5.35-10.35 min 25-41% B, 10.35-11.80 min 41% B, 11.80-17.60 min 60% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.51 (s, 1H), 8.38 (s, 1H), 8.01 (d, 1H), 7.56 (s, 1H), 7.32 (d, 1H), 7.12-7.23 (m, 1H), 6.69 (d, 2H), 6.14-6.20 (m, 1H), 4.18 (d, 1H), 3.82-3.97 (m, 4H), 3.34-3.54 (m, 6H), 3.15-3.30 (m, 1H), 2.72-2.89 (m, 2H), 1.74-1.82 (m, 1H), 1.34 (s, 3H).
LC-MS (method 1): Rt=0.97 min; MS (ESIpos): m/z=550.2 [M+H]+
Using an analogous method as described for example 1, N-(3-bromo-2-methoxyphenyl)-4-[({3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-33, 300 mg, 562 μmol) as the starting material, 41 mg of the title compound were prepared (13% yield) after heating overnight and purification by preparative HPLC (method 9, gradient: 0-0.50 min 20% B, 0.50-6.00 min 20-60% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.22 (s, 1H), 8.40-8.52 (m, 1H), 8.06 (d, 1H), 7.44 (s, 1H), 7.28 (d, 1H), 7.11 (s, 1H), 6.79 (dd, 1H), 6.60 (t, 1H), 6.20 (dd, 1H), 5.05-5.17 (m, 1H), 4.55-4.62 (m, 1H), 4.38-4.51 (m, 2H), 4.29 (dd, 1H), 3.84 (s, 3H), 3.36-3.44 (m, 2H), 2.74-2.83 (m, 2H), 2.56-2.71 (m, 2H).
LC-MS (method 1): Rt=0.84 min; MS (ESIpos): m/z=499.3 [M+H]+
The title compound from example 33 (35 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 10.0 mg at Rt=13.2-14.9 min, 90% purity) and enantiomer 2 (11 mg at 11.6-12.9 min, see example 35).
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Amylose SA 5μ 250×30 mm; eluent A: MtBE+0.1 Vol-% diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 20 min; flow 50.0 ml/min; UV 280 nm
Instrument: Agilent HPLC 1260; column: Amylose SA 3μ 100×4.6 mm; eluent A: MtBE+0.1 Vol-% diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 7 min; flow 1.4 ml/min; temperature: 25° C.; DAD 280 nm
Analytical chiral HPLC: Rt=4.19 min.
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.104 (0.99), 1.114 (0.55), 1.133 (0.42), 1.137 (0.50), 1.233 (0.92), 1.905 (0.92), 2.323 (0.82), 2.327 (1.16), 2.332 (0.82), 2.518 (4.54), 2.523 (3.11), 2.596 (0.51), 2.601 (0.67), 2.618 (0.46), 2.623 (0.57), 2.665 (1.14), 2.669 (1.28), 2.673 (0.90), 2.679 (0.59), 2.685 (0.63), 2.701 (0.61), 2.707 (0.55), 2.713 (0.42), 2.786 (1.05), 2.803 (2.23), 2.820 (1.26), 3.381 (0.84), 3.387 (0.92), 3.398 (1.58), 3.404 (1.60), 3.415 (0.76), 3.655 (0.57), 3.844 (16.00), 4.270 (0.78), 4.278 (0.84), 4.298 (1.20), 4.305 (1.18), 4.393 (1.20), 4.406 (1.26), 4.421 (0.80), 4.434 (0.86), 4.441 (0.55), 4.456 (1.03), 4.464 (0.63), 4.471 (0.82), 4.478 (0.97), 4.494 (0.57), 4.567 (0.57), 4.582 (0.63), 4.586 (0.78), 4.588 (0.86), 4.599 (0.67), 4.603 (0.71), 4.606 (0.65), 4.620 (0.42), 5.120 (0.61), 5.133 (0.51), 5.137 (0.55), 6.187 (1.47), 6.190 (1.47), 6.207 (1.60), 6.211 (1.49), 6.583 (1.32), 6.604 (2.61), 6.624 (1.37), 6.777 (1.98), 6.781 (2.08), 6.798 (1.64), 6.801 (1.41), 7.109 (1.51), 7.276 (1.33), 7.288 (1.35), 7.435 (3.32), 8.052 (0.76), 8.064 (0.74), 8.461 (1.16), 11.217 (1.60).
For the preparation of the racemic title compound see example 33. Separation of enantiomers by preparative chiral HPLC (method see example 34) gave 11.0 mg (90% purity, 4% yield) of the title compound.
Analytical chiral HPLC (method see example 34): Rt=3.75 min.
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.116 (0.48), 1.233 (0.85), 1.905 (0.71), 2.323 (0.68), 2.327 (0.93), 2.332 (0.67), 2.518 (3.82), 2.523 (2.55), 2.596 (0.53), 2.601 (0.70), 2.618 (0.48), 2.623 (0.59), 2.665 (0.98), 2.669 (1.06), 2.673 (0.76), 2.679 (0.53), 2.685 (0.65), 2.701 (0.64), 2.706 (0.59), 2.713 (0.45), 2.729 (0.42), 2.786 (1.10), 2.803 (2.34), 2.820 (1.32), 3.381 (0.87), 3.387 (0.96), 3.398 (1.66), 3.404 (1.68), 3.415 (0.84), 3.422 (0.76), 3.844 (16.00), 4.270 (0.82), 4.278 (0.87), 4.298 (1.24), 4.305 (1.21), 4.393 (1.21), 4.406 (1.27), 4.421 (0.82), 4.434 (0.87), 4.441 (0.57), 4.456 (1.02), 4.464 (0.65), 4.471 (0.82), 4.478 (0.98), 4.494 (0.56), 4.567 (0.59), 4.582 (0.65), 4.586 (0.81), 4.588 (0.88), 4.599 (0.70), 4.603 (0.73), 4.606 (0.67), 4.620 (0.43), 5.120 (0.65), 5.133 (0.54), 5.137 (0.59), 6.187 (1.54), 6.190 (1.51), 6.207 (1.65), 6.211 (1.54), 6.583 (1.33), 6.604 (2.62), 6.624 (1.38), 6.777 (2.02), 6.781 (2.08), 6.798 (1.66), 6.801 (1.46), 7.109 (1.58), 7.276 (1.37), 7.288 (1.33), 7.436 (3.35), 8.052 (0.74), 8.064 (0.71), 8.462 (1.10), 11.217 (1.66).
Using an analogous method as described for example 1, N-(2,3-dihydro-1H-inden-4-yl)-4-({(3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-36, 190 mg, 409 μmol) as the starting material, 21.3 mg of the title compound were prepared (11% yield) after heating overnight and purification by preparative HPLC (method 9, gradient: 0.50 min 15% B, 0.50-6.00 min 15-55% B).
LC-MS (method 1): Rt=0.83 min; MS (ESIpos): m/z=431 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.137 (2.63), 1.232 (0.69), 1.905 (0.85), 2.024 (0.92), 2.043 (2.43), 2.062 (3.81), 2.073 (13.04), 2.084 (16.00), 2.115 (1.45), 2.327 (2.07), 2.539 (6.44), 2.584 (1.28), 2.612 (1.71), 2.634 (1.48), 2.652 (1.02), 2.669 (2.69), 2.687 (1.68), 2.703 (1.64), 2.723 (1.18), 2.730 (1.18), 2.751 (0.85), 2.779 (2.79), 2.796 (5.32), 2.813 (5.55), 2.829 (6.51), 2.846 (6.51), 2.864 (3.09), 3.251 (4.93), 3.843 (1.54), 4.249 (1.64), 4.256 (1.87), 4.276 (2.50), 4.283 (2.40), 4.387 (2.10), 4.401 (2.20), 4.415 (1.58), 4.428 (1.54), 4.476 (1.08), 4.490 (1.97), 4.498 (1.38), 4.505 (1.58), 4.513 (1.91), 4.528 (1.15), 4.590 (1.15), 4.611 (1.84), 4.626 (1.54), 4.644 (0.79), 5.139 (1.35), 5.989 (2.83), 6.009 (2.89), 6.541 (2.37), 6.559 (3.06), 6.656 (1.91), 6.675 (2.92), 6.694 (1.41), 7.142 (3.22), 7.232 (3.91), 7.245 (4.21), 7.257 (5.22), 7.286 (0.43), 7.434 (0.43), 7.992 (4.07), 8.004 (3.88), 8.189 (4.14), 8.429 (6.70), 8.460 (0.56), 11.085 (3.15).
Using an analogous method as described for example 1, N-(2-chloro-3-methylphenyl)-4-[({3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-37, 197 mg, 395 μmol) as the starting material, 43 mg of the title compound were prepared (22% yield) after heating overnight at 60° C. and purification by preparative HPLC (method 10, gradient: 0.50 min 15% B, 0.50-6.00 min 15-55% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.23 (s, 1H), 8.43 (s, 1H), 8.03 (d, 1H), 7.54 (s, 1H), 7.20 (d, 1H), 7.14-7.17 (m, 1H), 6.74 (t, 1H), 6.61 (d, 1H), 6.20 (dd, 1H), 5.08-5.16 (m, 1H), 4.59 (ddd, 1H), 4.35-4.49 (m, 2H), 4.21 (dd, 1H), 3.37-3.49 (m, 2H), 2.53-2.84 (m, 4H), 2.28-2.31 (m, 3H).
LC-MS (method 2): Rt=0.99 min; MS (ESIpos): m/z=439.5 [M+H]+
The title compound from example 37 (43 mg) was separated into enantiomers by preparative chiral HPLC to give the title compound (enantiomer 1, 16.0 mg at Rt=8.1-9.5 min, 95% purity) and enantiomer 2 (18 mg at Rt=9.7-11.4 min, see example 39).
Instrument: PrepCon Labomatic HPLC; Column: Chiralcel OD-H 5μ, 250×20; eluent A: acetonitrile+0.1 vol % diethylamine; eluent B: ethanol; isocratic: 93% A+7% B; flow: 20 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; Column: Chiralcel OD-H 5μ, 100×4.6; eluent A: acetonitrile+0.1 vol % diethylamine; eluent B: ethanol; isocratic: 90% A+10% B; flow: 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=3.75 min.
Optical rotation:[α]D=53.5°+/−0.75° (c=4 mg/ml in DMSO)
For the preparation of the racemic title compound see example 37. Separation of enantiomers by preparative chiral HPLC (method see example 38) gave 18.0 mg (95% purity) of the title compound.
Analytical chiral HPLC (method see example 38): Rt=4.55 min.
Optical rotation:[α]D=−59.1°+/−0.48° (c=3.6 mg/ml in DMSO)
Using an analogous method as described for example 1, N-(3-chloro-5-fluoro-2-methylphenyl)-4-[({3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-40, 196 mg, 399 μmol) as the starting material, 35.5 mg of the title compound were prepared (17% yield) after preparative HPLC (method 10, gradient: 0.50 min 30% B, 0.50-7.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=2.25 (s, 3H), 2.58-2.73 (m, 2H), 2.78-2.85 (t, 2H), 3.38-3.45 (m, 2H), 4.20-4.30 (dd, 1H), 4.30-4.40 (dd, 1H), 4.40-4.48 (m, 1H), 4.48-4.65 (m, 1H), 5.01-5.21 (m, 1H), 5.88-5.96 (dd, 1H), 6.53-6.60 (dd, 1H), 7.25-7.32 (d, 1H), 7.32-7.39 (s, 1H), 8.06-8.14 (d, 1H), 8.46 (s, 1H), 11.31 (s, 1H).
LC-MS (method 2): Rt=1.03 min; MS (ESIpos): m/z=457 [M+H]+
The title compound from example 40 (32 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 14 mg at Rt=17.4-19.6 min) and enantiomer 2 (12 mg at Rt=21.2-23.3 min, see example 42).
Instrument: PrepCon Labomatic HPLC; column: YMC Amylose SA 5μ, 250×30; eluent A: MTBE+0.1 vol. % diethylamine; eluent B: ethanol; gradient: 0-20 min 2-60% B; flow 40 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; column: YMC Amylose SA 3μ, 100×4.6; eluent A: MTBE+0.1 vol. % diethylamine; eluent B: ethanol; gradient: 2-60% B 0-7 min; flow 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=4.75 min.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.32 (s, 1H), 8.46 (s, 1H), 8.10 (d, 1H), 7.36 (d, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.53-6.61 (m, 1H), 5.91 (dd, 1H), 5.03-5.11 (m, 1H), 4.50-4.60 (m, 1H), 4.21-4.47 (m, 4H), 3.39-3.44 (m, 2H), 2.82 (t, 2H), 2.64-2.72 (m, 1H), 2.25 (s, 3H).
For the preparation of the racemic title compound see example 40. Separation of enantiomers by preparative chiral HPLC (method see example 41) gave 12 mg of the title compound.
Analytical chiral HPLC (method see example 41): Rt=5.55 min.
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.31 (s, 1H), 8.46 (s, 1H), 8.10 (d, 1H), 7.36 (s, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.57 (dd, 1H), 5.91 (dd, 1H), 5.07 (dtd, 1H), 4.57 (ddd, 1H), 4.33-4.47 (m, 2H), 4.23-4.30 (m, 1H), 3.37-3.44 (m, 2H), 2.82 (t, 2H), 2.64-2.72 (m, 1H), 2.52-2.60 (m, 1H), 2.25 (s, 3H).
Using an analogous method as described for example 1, N-(3-fluoro-2-methoxyphenyl)-4-[({3-[2-(oxetan-2-yl)ethoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-43, 80.0 mg, 164 μmol) as the starting material, 32.6 mg of the title compound were prepared (43% yield) after heating overnight and purification by preparative HPLC (method 10, gradient: 0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.26 (s, 1H), 8.35 (s, 1H), 7.98 (d, 1H), 7.45-7.58 (m, 1H), 7.34 (d, 1H), 7.14 (s, 1H), 6.65 (td, 1H), 6.44-6.56 (m, 1H), 5.98 (d, 1H), 5.04-5.14 (m, 1H), 4.53-4.74 (m, 2H), 4.31-4.42 (m, 1H), 4.19-4.31 (m, 1H), 3.92 (s, 3H), 3.38-3.44 (m, 2H), 2.68-2.79 (m, 3H), 2.37-2.47 (m, 2H), 1.97-2.18 (m, 1H).
LC-MS (method 6): Rt=0.66 min; MS (ESIpos): m/z=453.2 [M+H]+
The title compound from example 43 (28 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 12 mg at Rt=9.8-12.3 min) and enantiomer 2 (11 mg at Rt=13.7-15.7 min, see example 45).
Instrument: Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000, column: Amylose SA 5μ 250×30 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 20 min; flow 50.0 ml/min; UV 280 nm
Instrument: Agilent HPLC 1260; column: Amylose SA 3μ 100×4.6 mm; eluent A: MTBE+0.1 vol. % diethylamine (99%); eluent B: methanol; gradient: 2-60% B in 7 min; flow 1.4 ml/min; temperature: 25° C.; DAD 280 nm
Analytical chiral HPLC: Rt=3.66 min.
Optical rotation:[α]D=79.0°+/−2.59° (c=2.2 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.27 (s, 1H), 8.35 (br s, 1H), 7.98 (br d, 1H), 7.53 (s, 1H), 7.34 (d, 1H), 7.14 (br s, 1H), 6.65 (td, 1H), 6.50 (t, 1H), 5.98 (d, 1H), 5.04-5.11 (m, 1H), 4.56-4.69 (m, 2H), 4.37 (dt, 1H), 4.25 (td, 1H), 3.88-3.94 (m, 3H), 3.38-3.43 (m, 2H), 2.66-2.79 (m, 3H), 2.38-2.48 (m, 2H), 1.95-2.19 (m, 1H).
For the preparation of the racemic title compound see example 43. Separation of enantiomers by preparative chiral HPLC (method see example 44) gave 11 mg of the title compound.
Analytical chiral HPLC (method see example 44): Rt=4.65 min.
Optical rotation:[α]D=−69.4°+/−0.88° (c=1.85 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.27 (s, 1H), 8.35 (br s, 1H), 7.99 (br d, 1H), 7.53 (s, 1H), 7.34 (d, 1H), 7.14 (s, 1H), 6.65 (td, 1H), 6.50 (ddd, 1H), 5.98 (d, 1H), 5.04-5.11 (m, 1H), 4.56-4.69 (m, 2H), 4.37 (dt, 1H), 4.20-4.29 (m, 1H), 3.92 (s, 3H), 3.37-3.44 (m, 2H), 2.66-2.79 (m, 3H), 2.38-2.47 (m, 2H), 2.05-2.13 (m, 1H).
Using an analogous method as described for example 1, N-(3-chloro-2-methoxyphenyl)-4-[({3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-46, 208 mg, 413 μmol) as the starting material, 65 mg of the title compound were prepared (32% yield) after heating for 17 h and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.19-11.40 (m, 1H), 8.49 (s, 1H), 8.06 (d, 1H), 7.37-7.47 (m, 1H), 7.20-7.32 (m, 1H), 7.04-7.13 (m, 1H), 6.66 (s, 1H), 6.58-6.76 (m, 1H), 6.12-6.24 (m, 1H), 4.41-4.49 (m, 1H), 4.28-4.38 (m, 2H), 4.04-4.16 (m, 1H), 3.86 (s, 3H), 3.37-3.44 (m, 2H), 2.71-2.85 (m, 3H), 2.35-2.44 (m, 1H), 1.47 (s, 3H).
LC-MS (method 2): Rt=1.03 min; MS (ESIpos): m/z=469.2 [M+H]+
The title compound from example 46 (75 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 25 mg at Rt=12.9-14.4 min.) and enantiomer 2 (27 mg at Rt=14.7-16.6 min, see example 48).
Instrument: PrepCon Labomatic HPLC; column: YMC Amylose SA 5μ, 250×30; eluent A: MTBE+0.1 vol. % diethylamine; eluent B: methanol+0.1 vol. % diethylamine; gradient: 0-20 min. 2-60% B; flow 40 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; column: YMC Amylose SA 3μ, 100×4.6; eluent A: MTBE+0.1 vol. % diethylamine; eluent B: methanol; gradient: 0-7 min. 2-60% B; flow 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=3.51 min.
Optical rotation:[α]D=−58.8°+/−1.29° (c=3.82 mg/ml, methanol).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.34 (s, 1H), 8.49 (s, 1H), 8.06 (d, 1H), 7.42 (s, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.63-6.69 (m, 2H), 6.19 (d, 1H), 4.29-4.49 (m, 3H), 4.12 (d, 1H), 3.86 (s, 3H), 3.39-3.45 (m, 2H), 2.74-2.84 (m, 3H), 2.33-2.44 (m, 1H), 1.47 (s, 3H).
For the preparation of the racemic title compound see example 46. Separation of enantiomers by preparative chiral HPLC (method see example 47) gave 27 mg of the title compound.
Analytical chiral HPLC (method see example 47): Rt=3.87 min.
Optical rotation:[α]D=58.9°+/−1.29° (c=2.75 mg/ml, methanol).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.43 (s, 1H), 8.58 (s, 1H), 8.15 (d, 1H), 7.47-7.55 (m, 1H), 7.38 (d, 1H), 7.20 (br s, 1H), 6.70-6.80 (m, 2H), 6.28 (d, 1H), 4.55 (dt, 1H), 4.35-4.49 (m, 2H), 4.21 (d, 1H), 3.95 (s, 3H), 3.46-3.53 (m, 2H), 2.82-2.92 (m, 3H), 2.44-2.54 (m, 1H), 1.56 (s, 3H).
Using an analogous method as described for example 1, N-(3-chloro-2-methoxyphenyl)-4-[({3-[(,3-dimethyloxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-49, 150 mg, 290 μmol) as the starting material, 82 mg of the title compound were prepared (58% yield) after heating overnight at 60° C. and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.16 (s, 1H), 8.46 (s, 1H), 8.06 (d, 1H), 7.45 (s, 1H), 7.29 (d, 1H), 7.06-7.15 (m, 1H), 6.65 (s, 1H), 6.63 (s, 1H), 6.12 (t, 1H), 4.73 (dd, 1H), 4.47 (dd, 1H), 4.21-4.31 (m, 3H), 3.86 (s, 3H), 3.36-3.44 (m, 2H), 2.81 (t, 2H), 1.23-1.33 (m, 3H), 1.15 (s, 3H).
LC-MS (method 2): Rt=1.09 min; MS (ESIpos): m/z=483.6 [M+H]+
The title compound from example 49 (80 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 42 mg at Rt=12.4-15.6 min.) and enantiomer 2 (45 mg at Rt=16.1-19.9 min., see example 51).
Instrument: PrepCon Labomatic HPLC; column: YMC Amylose SA 5μ, 250×30; eluent A: hexane+0.1 vol. % diethylamine; eluent B: 2-propanol; gradient: 0-7 min. 20-50% B; flow 50 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; column: YMC Amylose SA 3μ, 100×4.6; eluent A: hexane+0.1 vol. % diethylamine; eluent B: 2-propanol; gradient: 0-7 min. 20-50% B; flow 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=4.04 min.
Optical rotation:[α]D=34.5°+/−1.24° (c=3 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.17 (s, 1H), 8.46 (s, 1H), 8.07 (d, 1H), 7.47 (s, 1H), 7.29 (d, 1H), 7.09-7.13 (m, 1H), 6.65 (s, 1H), 6.63 (s, 1H), 6.12 (t, 1H), 4.73 (dd, 1H), 4.47 (dd, 1H), 4.21-4.31 (m, 3H), 3.86 (s, 3H), 3.36-3.44 (m, 2H), 2.81 (t, 2H), 1.25-1.28 (m, 3H), 1.15 (s, 3H).
For the preparation of the racemic title compound see example 49. Separation of enantiomers by preparative chiral HPLC (method see example 50) gave 45 mg of the title compound.
Analytical chiral HPLC (method see example 50): Rt=5.01 min.
Optical rotation:[α]D=−34.2°+/−0.78° (c=3.6 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.16 (s, 1H), 8.46 (s, 1H), 8.07 (d, 1H), 7.46 (s, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.65 (s, 1H), 6.63 (s, 1H), 6.12 (dd, 1H), 4.73 (dd, 1H), 4.47 (dd, 1H), 4.21-4.32 (m, 3H), 3.86 (s, 3H), 3.36-3.44 (m, 2H), 2.81 (t, 2H), 1.28 (s, 3H), 1.15 (s, 3H).
Using an analogous method as described for example 1, 4-[({3-[(3,3-dimethyloxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-N-(3-fluoro-2-methoxyphenyl)-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-52, 140 mg, 280 μmol) as the starting material, 69 mg of the title compound were prepared (52% yield) after heating overnight at 60° C. and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.14 (s, 1H), 8.45 (s, 1H), 8.06 (d, 1H), 7.46 (s, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.59 (td, 1H), 6.45 (ddd, 1H), 5.97 (d, 1H), 4.74 (dd, 1H), 4.46 (dd, 1H), 4.21-4.31 (m, 3H), 3.89 (s, 3H), 3.35-3.43 (m, 2H), 2.81 (t, 2H), 1.23-1.32 (m, 3H), 1.12-1.17 (m, 3H).
LC-MS (method 2): Rt=1.04 min; MS (ESIpos): m/z=467.6 [M+H]+
The title compound from example 52 (65 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 30 mg at Rt=8.1-11.0) and enantiomer 2 (34 mg at Rt=12.0-14.6 min, see example 54).
Instrument: PrepCon Labomatic HPLC; column: YMC Amylose SA 5μ, 250×30; eluent A: MTBE+0.1 vol. % diethylamine; eluent B: methanol; gradient: 0-20 min. 2-60% B; flow 50 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; column: YMC Amylose SA 3μ, 100×4.6; eluent A: MTBE+0.1 vol. % diethylamine; eluent B: methanol; gradient: 0-7 min. 2-60% B; flow 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=3.25 min.
Optical rotation:[α]D=−29.5°+/−0.36° (c=4.3 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.14 (s, 1H), 8.45 (s, 1H), 8.06 (d, 1H), 7.45 (s, 1H), 7.29 (d, 1H), 7.11 (s, 1H), 6.55-6.63 (m, 1H), 6.45 (ddd, 1H), 5.95-5.99 (m, 1H), 4.74 (dd, 1H), 4.46 (dd, 1H), 4.21-4.31 (m, 3H), 3.89 (s, 3H), 3.36-3.43 (m, 2H), 2.81 (t, 2H), 1.43 (s, 1H), 1.28 (s, 3H), 1.15 (s, 3H).
For the preparation of the racemic title compound see example 52. Separation of enantiomers by preparative chiral HPLC (method see example 53) gave 34 mg of the title compound.
Analytical chiral HPLC (method see example 53): Rt=4.15 min.
Optical rotation:[α]D=21.1°+/−0.68° (c=4.7 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.14 (s, 1H), 8.45 (s, 1H), 8.06 (d, 1H), 7.45 (s, 1H), 7.29 (d, 1H), 7.08-7.14 (m, 1H), 6.54-6.63 (m, 1H), 6.45 (ddd, 1H), 5.95-5.99 (m, 1H), 4.74 (dd, 1H), 4.46 (dd, 1H), 4.21-4.31 (m, 3H), 3.89 (s, 3H), 3.36-3.43 (m, 2H), 2.81 (t, 2H), 1.28 (s, 3H), 1.15 (s, 3H).
Using an analogous method as described for example 1, N-(2-chloro-3-methylphenyl)-4-[({3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-55, 223 mg, 435 μmol) as the starting material, 70.3 mg of the title compound were prepared (34% yield) after heating overnight at 60° C. and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.34 (s, 1H), 8.47 (s, 1H), 8.03 (d, 1H), 7.49 (s, 1H), 7.19 (d, 1H), 7.14-7.17 (m, 1H), 6.76 (t, 1H), 6.62 (d, 1H), 6.24 (d, 1H), 4.45 (dt, 1H), 4.25-4.39 (m, 2H), 4.11 (d, 1H), 3.36-3.44 (m, 2H), 2.73-2.84 (m, 3H), 2.28-2.40 (m, 4H), 1.42-1.48 (m, 3H).
LC-MS (method 6): Rt=0.71 min; MS (ESIpos): m/z=453.2 [M+H]+
The title compound from example 55 (70 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 27 mg at Rt=12.7-13.9 min) and enantiomer 2 (33 mg t Rt=14.1-15.4 min, see example 57).
Instrument: PrepCon Labomatic HPLC; column: YMC Cellulose SC SA 5μ, 250×30; eluent A: hexane+0.1 vol. % diethylamine; eluent B: ethanol; gradient: 0-20 min. 20-50% B; flow 50 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; column: YMC Amylose SA 3μ, 100×4.6; eluent A: hexane+0.1 vol. % diethylamine; eluent B: ethanol; gradient: 0-7 min. 20-50% B; flow 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=5.71 min.
Optical rotation:[α]D=34.3°+/−0.69° (c=4.9 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.34 (s, 1H), 8.47 (s, 1H), 8.03 (d, 1H), 7.49 (s, 1H), 7.12-7.22 (m, 2H), 6.76 (t, 1H), 6.62 (d, 1H), 6.24 (d, 1H), 4.45 (dt, 1H), 4.26-4.38 (m, 2H), 4.11 (d, 1H), 3.36-3.44 (m, 2H), 2.74-2.82 (m, 3H), 2.29-2.44 (m, 4H), 1.46 (s, 3H).
For the preparation of the racemic title compound see example 55. Separation of enantiomers by preparative chiral HPLC (method see example 56) gave 33 mg of the title compound.
Analytical chiral HPLC (method see example 56): Rt=4.46 min.
Optical rotation:[α]D=−36.1°+/−0.5° (c=4.4 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.34 (s, 1H), 8.47 (s, 1H), 8.03 (d, 1H), 7.49 (s, 1H), 7.19 (d, 1H), 7.14-7.17 (m, 1H), 6.75 (t, 1H), 6.62 (d, 1H), 6.24 (d, 1H), 4.45 (dt, 1H), 4.21-4.38 (m, 2H), 4.11 (d, 1H), 3.35-3.44 (m, 2H), 2.74-2.82 (m, 3H), 2.28-2.46 (m, 4H), 1.46 (s, 3H).
Using an analogous method as described for example 1, N-(2-chloro-3-methylphenyl)-4-[({3-[(3,3-dimethyloxetan-2-yl]methoxy}pyridin-4-yl)methyl]amino}-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-58, 189 mg, 359 μmol) as the starting material, 62.4 mg of the title compound were prepared (34% yield) after heating overnight at 60° C. and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.20 (s, 1H), 8.42 (s, 1H), 8.05 (d, 1H), 7.56 (s, 1H), 7.21 (d, 1H), 7.14 (s, 1H), 6.71 (t, 1H), 6.59 (d, 1H), 6.15 (dd, 1H), 4.71 (dd, 1H), 4.42 (dd, 1H), 4.13-4.29 (m, 3H), 3.37-3.46 (m, 2H), 2.82 (t, 2H), 2.29 (s, 3H), 1.27 (s, 3H), 1.14 (s, 3H).
LC-MS (method 6): Rt=0.77 min; MS (ESIpos): m/z=467.2 [M+H]+
The title compound from example 58 (62 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 33 mg at Rt=9.0-11.6 min) and enantiomer 2 (27 mg at Rt=12.9-14.9 min, see example 60).
Instrument: PrepCon Labomatic HPLC; column: YMC Cellulose SC SA 5μ, 250×30; eluent A: MTBE+0.1 vol. % diethylamine; eluent B: acetonitrile+0.1 vol. % diethylamine; isocratic: 50% A+50% B; flow 50 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; column: YMC Cellulose SC 3μ, 100×4.6; eluent A: MTBE+0.1 vol. % diethylamine; eluent B: acetonitrile; isocratic: 50% A+50% B, flow 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=4.73 min.
Optical rotation:[α]D=29.2°+/−0.90° (c=5 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.19 (s, 1H), 8.42 (s, 1H), 8.05 (d, 1H), 7.55 (s, 1H), 7.21 (d, 1H), 7.14 (s, 1H), 6.71 (t, 1H), 6.59 (d, 1H), 6.15 (d, 1H), 4.71 (dd, 1H), 4.42 (dd, 1H), 4.14-4.27 (m, 3H), 3.36-3.44 (m, 2H), 2.81 (t, 2H), 2.29 (s, 3H), 1.27 (s, 3H), 1.14 (s, 3H).
For the preparation of the racemic title compound see example 58. Separation of enantiomers by preparative chiral HPLC (method see example 59) gave 27 mg of the title compound.
Analytical chiral HPLC (method see example 59): Rt=6.35 min.
Optical rotation:[α]D=−28.0°+/−0.76° (c=5 mg/ml, methanol)
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.19 (s, 1H), 8.42 (s, 1H), 8.05 (d, 1H), 7.55 (s, 1H), 7.21 (d, 1H), 7.14 (s, 1H), 6.71 (t, 1H), 6.59 (d, 1H), 6.15 (dd, 1H), 4.71 (dd, 1H), 4.42 (dd, 1H), 4.14-4.27 (m, 3H), 3.36-3.44 (m, 2H), 2.81 (t, 2H), 2.29 (s, 3H), 1.27 (s, 3H), 1.14 (s, 3H).
3-(3-Fluoro-2-methoxyanilino)-2-(3-hydroxypyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 18, 163 mg, 442 μmol) and (tributylphosphoranylidene) acetonitrile (CAS 157141-27-0, 350 μl, 1.3 mmol) were dissolved in 1,4-dioxane (110 μl) under an argon atmosphere. (3-Fluorooxetan-3-yl)methanol (CAS 865451-85-0, 93.8 mg, 884 μmol) was added and the mixture was stirred at RT overnight. After stirring additional 20 h at 50° C. the solvent was evaporated and the residue was purified by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B) to give 12.2 mg of the title compound (5% yield).
LC-MS (method 6): Rt=0.67 min; MS (ESIpos): m/z=457 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.852 (0.41), 0.879 (0.46), 1.233 (1.80), 2.075 (1.89), 2.332 (2.03), 2.336 (0.88), 2.518 (12.96), 2.523 (8.76), 2.673 (2.03), 2.679 (0.92), 2.794 (1.52), 2.812 (3.32), 2.829 (1.71), 3.381 (1.24), 3.387 (1.34), 3.398 (2.21), 3.404 (2.21), 3.415 (1.11), 3.421 (0.97), 3.841 (1.57), 3.860 (0.69), 3.877 (15.22), 3.879 (16.00), 4.533 (0.41), 4.579 (4.33), 4.628 (4.29), 4.689 (0.88), 4.710 (3.41), 4.723 (3.69), 4.740 (1.24), 4.744 (1.38), 4.760 (3.37), 4.772 (3.73), 4.792 (0.97), 5.599 (0.41), 5.759 (1.38), 5.979 (1.80), 5.999 (1.89), 6.426 (0.97), 6.430 (0.97), 6.447 (1.34), 6.451 (1.29), 6.453 (1.11), 6.457 (0.97), 6.474 (1.20), 6.478 (1.11), 6.570 (0.92), 6.585 (1.01), 6.590 (1.61), 6.606 (1.57), 6.611 (0.83), 6.627 (0.65), 6.961 (0.41), 7.002 (0.41), 7.104 (2.07), 7.294 (3.60), 7.306 (3.55), 7.405 (4.52), 8.091 (4.56), 8.104 (4.33), 8.463 (6.04), 11.053 (2.12).
Using an analogous method as described for example 1 with N-(2-chloro-3-methylphenyl)-4-[({3-[2-(oxetan-2-yl)ethoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-62, 57.0 mg, 85% purity, 99.5 μmol) as the starting material, 4.00 mg of the title compound were prepared (8% yield) after heating overnight and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
LC-MS (method 6): Rt=0.70 min; MS (ESIpos): m/z=453 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.232 (1.03), 1.255 (1.43), 2.057 (0.69), 2.064 (0.74), 2.076 (0.44), 2.084 (0.79), 2.093 (0.89), 2.102 (0.84), 2.112 (0.44), 2.146 (0.84), 2.317 (16.00), 2.331 (0.98), 2.371 (0.44), 2.380 (0.69), 2.392 (0.89), 2.406 (0.79), 2.417 (0.89), 2.429 (1.18), 2.451 (1.38), 2.518 (4.09), 2.523 (2.66), 2.540 (0.49), 2.665 (1.03), 2.669 (1.03), 2.674 (0.84), 2.678 (0.84), 2.684 (0.84), 2.691 (0.54), 2.699 (0.84), 2.704 (0.89), 2.711 (0.74), 2.718 (0.64), 2.726 (0.69), 2.752 (1.92), 2.769 (3.84), 2.786 (2.07), 3.386 (1.58), 3.392 (1.72), 3.403 (2.51), 3.409 (2.66), 3.423 (1.23), 4.177 (0.44), 4.188 (0.59), 4.201 (1.08), 4.211 (1.08), 4.224 (0.79), 4.234 (0.64), 4.294 (0.64), 4.306 (1.43), 4.318 (1.18), 4.330 (0.98), 4.343 (0.44), 4.552 (0.64), 4.566 (1.58), 4.575 (0.89), 4.581 (1.18), 4.589 (1.58), 4.604 (0.84), 4.625 (0.84), 4.639 (0.89), 4.645 (1.67), 4.659 (1.23), 4.665 (1.03), 4.679 (0.59), 5.026 (0.39), 5.044 (0.98), 5.059 (0.98), 6.155 (1.97), 6.173 (2.02), 6.637 (1.72), 6.654 (2.26), 6.749 (2.22), 6.768 (3.00), 6.787 (1.43), 7.165 (2.56), 7.212 (3.20), 7.224 (3.20), 7.611 (5.27), 7.953 (2.17), 7.966 (2.07), 8.327 (3.30), 11.292 (2.66).
3-(2,3-dichloroanilino)-2-(3-{2-[oxetan-2-yl]ethoxy}pyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (Stereoisomer 1)
Using an analogous method as described for example 1, N-(2,3-dichlorophenyl)-4-[({3-[2-(oxetan-2-yl)ethoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-63, 243 mg, 65% purity, 311 μmol) as the starting material, 19.3 mg (85% purity, 11% yield) of the racemic title compound were prepared after heating for 48 h and purification by preparative HPLC (method 9, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-55% B). LC-MS (method 6): Rt=0.73 min; MS (ESIpos): m/z=473 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.232 (0.60), 1.850 (0.90), 1.865 (0.84), 1.940 (0.42), 2.044 (1.20), 2.054 (1.26), 2.069 (0.96), 2.081 (1.50), 2.090 (1.50), 2.159 (2.23), 2.336 (1.74), 2.346 (1.14), 2.359 (1.44), 2.382 (1.26), 2.396 (1.14), 2.409 (1.38), 2.418 (1.44), 2.436 (1.92), 2.459 (2.05), 2.518 (16.00), 2.522 (10.11), 2.539 (14.32), 2.653 (0.72), 2.678 (1.80), 2.687 (1.44), 2.693 (1.50), 2.700 (1.20), 2.707 (0.96), 2.714 (1.08), 2.734 (0.60), 2.767 (3.19), 2.784 (6.74), 2.801 (3.61), 2.824 (0.60), 2.843 (1.02), 2.858 (0.78), 2.932 (0.48), 3.166 (0.78), 3.188 (1.26), 3.204 (1.08), 3.214 (0.84), 3.219 (0.48), 3.275 (0.84), 3.389 (3.19), 3.395 (3.55), 3.405 (5.53), 3.411 (5.71), 3.422 (2.89), 3.697 (0.54), 3.714 (1.08), 3.731 (0.60), 4.171 (1.02), 4.182 (1.32), 4.194 (2.05), 4.205 (2.05), 4.216 (1.80), 4.227 (1.74), 4.270 (1.56), 4.283 (2.89), 4.295 (2.23), 4.307 (1.92), 4.320 (0.96), 4.421 (0.60), 4.537 (1.32), 4.551 (3.25), 4.560 (1.92), 4.565 (2.29), 4.573 (2.95), 4.588 (1.56), 4.612 (1.62), 4.632 (3.07), 4.646 (2.35), 4.651 (1.92), 4.666 (1.14), 4.991 (0.72), 4.999 (0.78), 5.010 (1.50), 5.018 (1.74), 5.031 (1.74), 5.049 (0.78), 5.057 (0.66), 5.186 (0.42), 5.200 (0.42), 6.046 (0.42), 6.051 (0.42), 6.231 (0.42), 6.242 (4.21), 6.247 (4.21), 6.261 (4.75), 6.266 (4.57), 6.280 (0.84), 6.285 (0.84), 6.290 (0.78), 6.310 (0.48), 6.834 (0.60), 6.838 (0.84), 6.843 (0.72), 6.856 (2.35), 6.861 (3.73), 6.875 (8.60), 6.880 (8.12), 6.883 (8.36), 6.903 (6.62), 6.922 (2.29), 6.945 (0.48), 7.038 (0.42), 7.072 (0.48), 7.159 (4.33), 7.187 (0.42), 7.200 (0.42), 7.270 (1.14), 7.282 (1.26), 7.292 (4.45), 7.305 (4.45), 7.474 (0.54), 7.486 (0.48), 7.566 (0.78), 7.621 (1.26), 7.634 (0.78), 7.658 (0.66), 7.663 (0.72), 7.676 (9.02), 8.022 (3.07), 8.034 (2.95), 8.082 (0.42), 8.132 (5.95), 8.297 (0.42), 8.352 (4.75), 8.430 (0.54), 11.368 (4.45), 11.512 (0.90).
Racemic 3-(2,3-dichloroanilino)-2-(3-{2-[oxetan-2-yl]ethoxy}pyridin-4-yl)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (17.5 mg) was separated into enantiomers by preparative chiral HPLC to give the title compound (enantiomer 1, 7.00 mg at Rt=17.3-18.4 min) and enantiomer 2 (6 mg at Rt=14.0-15.1 min, see example 65).
Instrument: PrepCon Labomatic HPLC; Column: YMC Amylose SA 5μ, 250×30; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: methanol; gradient: 0-20 min 2-60% B; flow: 50 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; Column: YMC Amylose SA 5μ, 100×4.6; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: methanol; gradient: 0-7 min 2-60% B; flow: 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=5.32 min.
Optical rotation:[α]D=−37.1°+/−1.80° (c=3.6 mg/ml in DMSO)
1H-NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 0.102 (0.85), 0.181 (1.32), 0.844 (2.26), 0.860 (2.81), 0.888 (2.87), 0.904 (1.53), 0.946 (0.49), 1.060 (0.79), 1.078 (0.78), 1.263 (16.00), 1.350 (1.31), 1.729 (1.57), 2.102 (1.45), 2.141 (1.68), 2.518 (0.49), 2.583 (0.54), 2.602 (1.15), 2.611 (1.05), 2.625 (2.02), 2.652 (2.36), 2.665 (1.16), 2.672 (1.30), 2.704 (0.54), 2.755 (0.42), 2.772 (0.83), 2.795 (1.44), 2.812 (2.84), 2.829 (2.19), 2.842 (2.84), 2.851 (1.75), 2.859 (2.36), 2.864 (2.04), 2.871 (1.48), 2.879 (1.75), 2.893 (1.22), 2.898 (1.18), 2.912 (0.59), 3.518 (0.59), 3.578 (2.34), 3.593 (4.17), 3.609 (2.06), 4.263 (0.97), 4.268 (1.06), 4.290 (2.09), 4.312 (1.32), 4.318 (1.17), 4.463 (1.12), 4.471 (2.05), 4.482 (1.47), 4.495 (1.70), 4.504 (0.96), 4.679 (1.04), 4.693 (2.35), 4.703 (1.46), 4.707 (1.66), 4.717 (2.24), 4.730 (1.17), 4.821 (1.28), 4.841 (2.61), 4.855 (2.18), 4.861 (1.42), 4.875 (0.97), 5.247 (0.85), 5.267 (2.54), 5.285 (4.28), 5.305 (0.92), 6.315 (3.02), 6.318 (3.04), 6.335 (3.35), 6.338 (3.31), 6.749 (2.06), 6.769 (4.89), 6.789 (3.23), 6.828 (4.25), 6.832 (4.36), 6.848 (2.60), 6.852 (2.32), 7.006 (0.43), 7.374 (1.79), 7.385 (1.75), 7.528 (0.46), 7.644 (4.21), 8.000 (1.30), 8.279 (1.50), 11.155 (1.70).
For the preparation of the racemic title compound see example 64. Separation of enantiomers by preparative chiral HPLC (method see example 64) gave 6.00 mg of the title compound.
Analytical chiral HPLC (method see example 64): Rt=4.34 min.
Optical rotation:[α]D=42.6°+/−2.00° (c=3.3 mg/ml in DMSO)
1H-NMR (400 MHz, CHLOROFORM-d) δ [ppm]: 0.086 (0.48), 0.102 (1.55), 0.181 (2.41), 0.844 (2.19), 0.861 (2.76), 0.888 (2.97), 0.905 (1.57), 0.947 (0.51), 1.061 (0.69), 1.263 (16.00), 1.353 (1.37), 1.371 (1.73), 1.389 (1.13), 1.725 (1.81), 2.045 (0.42), 2.102 (1.89), 2.141 (2.12), 2.583 (0.69), 2.602 (1.47), 2.611 (1.28), 2.625 (2.55), 2.630 (2.33), 2.644 (2.15), 2.653 (2.88), 2.665 (1.32), 2.672 (1.56), 2.705 (0.68), 2.713 (0.60), 2.756 (0.58), 2.772 (1.17), 2.796 (1.95), 2.812 (3.97), 2.829 (2.92), 2.843 (3.91), 2.851 (2.17), 2.860 (3.22), 2.864 (2.66), 2.872 (1.89), 2.880 (2.19), 2.893 (1.49), 2.899 (1.52), 2.913 (0.75), 3.578 (2.95), 3.594 (5.47), 3.604 (2.34), 3.609 (2.74), 4.263 (1.27), 4.268 (1.47), 4.290 (2.74), 4.312 (1.79), 4.318 (1.52), 4.463 (1.48), 4.471 (2.79), 4.482 (1.91), 4.495 (2.24), 4.504 (1.19), 4.679 (1.41), 4.693 (3.18), 4.703 (1.92), 4.708 (2.19), 4.717 (3.13), 4.730 (1.62), 4.822 (1.64), 4.836 (1.79), 4.841 (3.42), 4.856 (2.91), 4.862 (1.95), 4.875 (1.31), 5.248 (1.17), 5.267 (5.26), 5.286 (3.30), 5.305 (1.06), 6.314 (4.07), 6.318 (4.22), 6.335 (4.58), 6.338 (4.49), 6.749 (2.85), 6.770 (6.90), 6.790 (4.54), 6.829 (5.84), 6.832 (5.98), 6.849 (3.63), 6.853 (3.29), 7.006 (0.76), 7.373 (3.30), 7.386 (3.48), 7.528 (0.77), 7.645 (5.51), 7.996 (2.32), 8.007 (2.25), 8.279 (3.39), 11.158 (2.16).
Using an analogous method as described for example 1, N-(3-chloro-5-fluoro-2-methoxyphenyl)-4-{[(3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-65, 260 mg, 513 μmol) as the starting material, 34.5 mg of the title compound were prepared (11% yield) after heating overnight and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 15% B, 0.50-6.00 min 15-65% B).
LC-MS (method 2): Rt=0.99 min; MS (ESIpos): m/z=473 [M+H]+
1H NMR (400 MHz, DMSO-d6) b ppm 2.52-2.59 (m, 3H) 2.79-2.84 (m, 2H) 3.41 (td, J=6.91, 2.66 Hz, 2H) 3.79-3.84 (m, 3H) 4.28 (dd, J=11.15, 3.04 Hz, 1H) 4.35-4.45 (m, 2H) 4.56 (ddd, J=8.49, 7.22, 5.58 Hz, 1H) 5.05-5.10 (m, 1H) 5.84-5.88 (m, 1H) 6.52 (dd, J=8.49, 2.91 Hz, 1H) 7.08 (s, 1H) 7.31-7.33 (m, 1H) 7.55 (d, J=1.52 Hz, 1H) 8.12 (d, J=5.04 Hz, 1H) 8.44-8.48 (s, 1H).
The title compound from example 65 (34.5 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 11.0 mg at Rt=18.2-21.4 min) and enantiomer 2 (13.4 mg at Rt=11.0-14.0 min, see example 67).
Instrument: PrepCon Labomatic HPLC; Column: YMC Amylose SA 5μ, 250×30; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: ethanol; isocratic: 80% A+20% B; flow: 50 ml/min; temperature: UV: 254 nm
Instrument: Waters Alliance 2695; Column: YMC Amylose SA 3μ, 100×4.6; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: ethanol; isocratic: 80% A+20% B; flow: 1.4 ml/min; temperature: 25° C.; UV: 254 nm
Analytical chiral HPLC: Rt=5.76 min.
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.851 (0.48), 1.035 (0.41), 1.052 (0.83), 1.070 (0.48), 1.137 (0.41), 1.232 (1.93), 1.352 (0.48), 2.327 (4.00), 2.331 (2.97), 2.336 (1.38), 2.518 (16.00), 2.523 (10.62), 2.669 (4.21), 2.673 (3.03), 2.678 (1.52), 2.813 (0.76), 2.831 (0.41), 3.407 (0.62), 3.422 (0.41), 3.801 (5.66), 3.816 (3.45), 4.288 (0.41), 4.295 (0.41), 4.353 (0.41), 4.367 (0.41), 5.846 (0.41), 5.853 (0.48), 5.874 (0.41), 5.881 (0.48), 6.509 (0.48), 6.516 (0.55), 6.530 (0.48), 6.537 (0.55), 7.086 (0.48), 7.316 (0.83), 7.329 (0.83), 7.552 (0.69), 8.116 (1.24), 8.129 (1.10), 8.481 (1.52), 11.345 (0.55).
For the preparation of the racemic title compound see example 65. Separation of enantiomers by preparative chiral HPLC (method see example 66) gave 13.4 mg of the title compound.
Analytical chiral HPLC (method see example 69): Rt=3.77 min.
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.852 (0.62), 1.035 (0.55), 1.053 (1.25), 1.071 (0.55), 1.232 (2.22), 1.353 (0.48), 2.332 (2.98), 2.337 (1.32), 2.518 (16.00), 2.523 (10.87), 2.674 (2.98), 2.679 (1.39), 2.814 (0.55), 3.402 (0.48), 3.422 (0.42), 3.802 (4.99), 4.355 (0.48), 4.367 (0.42), 5.846 (0.42), 5.854 (0.48), 5.874 (0.42), 5.882 (0.48), 6.509 (0.48), 6.516 (0.55), 6.530 (0.48), 6.537 (0.48), 7.085 (0.42), 7.317 (0.76), 7.329 (0.76), 7.551 (0.62), 8.117 (1.11), 8.130 (0.97), 8.481 (1.39), 11.343 (0.48).
Using an analogous method as described for example 1, N-(3-chloro-5-fluoro-2-methoxyphenyl)-4-[({3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-68, 190 mg, 365 μmol) as the starting material, 63.1 mg of the title compound were prepared (35% yield) after heating overnight and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 5% B, 0.50-8.00 min 5-60% B).
LC-MS (method 2): Rt=1.04 min; MS (ESIpos): m/z=487 [M+H]+
1H-NMR (400 MHz, DMSO-d6): δ [ppm]=11.62-11.69 (m, 1H), 11.37-11.52 (m, 1H), 8.50 (s, 1H), 8.12 (d, 1H), 7.52 (d, 1H), 7.33 (d, 1H), 7.05-7.09 (m, 1H), 6.51 (dd, 1H), 5.83-5.92 (m, 1H), 4.22-4.44 (m, 3H), 4.06-4.15 (m, 1H), 3.76-3.81 (m, 3H), 3.37-3.50 (m, 2H), 2.68-2.86 (m, 3H), 1.39-1.48 (m, 3H).
The title compound from example 68 (50 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 27.0 mg at Rt=10.2-13.5 min) and enantiomer 2 (31 mg at Rt=13.5-16.6 min, see example 70).
Instrument: Sepiatec: Prep SFC100; Column: Chiralpak IA 5μ 250×30 mm; eluent A: C02; eluent B: 2-propanol+0.4 vol % diethylamin; isocratic: 25% B; flow: 100 ml/min; temperature: 40° C.; BPR: 150bar; UV: 280 nm
Instrument: Agilent: 1260, Aurora SFC-Modul; Saule: Chiralpak IA 5μ 100×4.6 mm; Eluent A: CO2; Eluent B: 2-Propanol+0.4% Diethylamin (99%); Isokratisch: 25% B; FluB: 4 ml/min; Temperatur: 37.5° C.; BPR: 100bar; UV: 280 nm
Analytical chiral HPLC: Rt=2.66 min.
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.152 (0.98), 1.231 (0.85), 1.434 (13.57), 2.331 (0.43), 2.340 (0.45), 2.357 (0.55), 2.362 (0.68), 2.367 (0.62), 2.379 (0.62), 2.384 (0.73), 2.389 (0.62), 2.406 (0.51), 2.518 (2.20), 2.523 (1.43), 2.539 (0.87), 2.673 (0.41), 2.691 (0.47), 2.708 (0.60), 2.713 (0.73), 2.718 (0.58), 2.730 (0.66), 2.735 (0.66), 2.741 (0.51), 2.757 (0.49), 2.774 (1.17), 2.790 (2.41), 2.808 (1.34), 3.379 (1.07), 3.385 (1.07), 3.395 (1.88), 3.402 (1.83), 3.413 (0.92), 3.419 (0.81), 3.796 (16.00), 4.102 (1.86), 4.128 (2.47), 4.246 (2.39), 4.272 (1.86), 4.283 (0.87), 4.291 (0.64), 4.299 (0.75), 4.305 (0.83), 4.322 (0.49), 4.381 (0.53), 4.396 (0.85), 4.403 (0.75), 4.412 (0.64), 4.418 (0.83), 4.433 (0.41), 5.857 (1.37), 5.865 (1.58), 5.885 (1.34), 5.893 (1.43), 6.501 (1.45), 6.508 (1.60), 6.521 (1.45), 6.529 (1.47), 7.077 (1.71), 7.329 (2.28), 7.341 (2.30), 7.521 (2.39), 7.524 (2.33), 8.118 (1.96), 8.130 (1.86), 8.500 (3.14), 11.429 (1.88).
For the preparation of the racemic title compound see example 68. Separation of enantiomers by preparative chiral HPLC (method see example 69) gave 31.0 mg of the title compound.
Analytical chiral HPLC (method see example 69): Rt=3.47 min.
Using an analogous method as described for example 1, N-[3-chloro-2-(difluoromethoxy)phenyl]-4-{[(3-{[(2S)-3,3-dimethyloxetan-2-yl]methoxy}pyridin-4-yl)methyl]amino}-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-71, 42.0 mg, 99% purity, 75.2 μmol) as the starting material, 18.6 mg of the title compound were prepared (46% yield) after heating overnight and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B).
LC-MS (method 6): Rt=0.81 min; MS (ESIpos): m/z=519 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.153 (14.99), 1.274 (16.00), 2.085 (0.52), 2.518 (4.73), 2.523 (3.08), 2.540 (3.22), 2.792 (1.44), 2.809 (3.00), 2.826 (1.76), 3.380 (1.28), 3.391 (2.04), 3.397 (2.19), 3.410 (1.07), 4.207 (2.40), 4.220 (3.68), 4.256 (3.89), 4.270 (2.40), 4.286 (1.17), 4.296 (1.24), 4.312 (1.55), 4.322 (1.55), 4.434 (1.40), 4.452 (1.67), 4.460 (1.10), 4.479 (1.21), 4.700 (1.55), 4.710 (1.69), 4.717 (1.65), 4.728 (1.30), 6.248 (2.10), 6.252 (2.17), 6.268 (2.24), 6.272 (2.17), 6.733 (1.87), 6.737 (2.06), 6.754 (3.09), 6.758 (2.76), 6.804 (2.42), 6.825 (3.45), 6.846 (1.39), 6.979 (1.64), 7.087 (2.35), 7.165 (3.40), 7.295 (4.80), 7.311 (3.00), 7.324 (2.97), 7.350 (1.55), 8.066 (2.04), 8.079 (1.92), 8.461 (3.15), 11.241 (2.58).
The title compound from example 71 (13.0 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 5.0 mg at Rt=12.8-15.7) and enantiomer 2 (5.0 mg at 16.2-19.5, see example 67).
Instrument: PrepCon Labomatic HPLC; Column: YMC Amylose SA 5μ, 250×30; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: ethanol; gradient: 0-20 min 2-60% B; flow: 40 ml/min; temperature: 25° C.; UV: 280 nm Analytical chiral HPLC method:
Instrument: Waters Alliance 2695; Column: YMC Amylose SA 3μ, 100×4.6; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: ethanol; gradient: 0-7 min 2-60% B; flow: 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=4.01 min.
Optical rotation:[α]D=−26.1°+/−5.39° (c=5.4 mg/3 ml in METHANOL)
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.036 (8.36), 1.054 (16.00), 1.071 (8.58), 1.104 (0.71), 1.119 (0.91), 1.138 (1.89), 1.153 (12.46), 1.208 (0.44), 1.233 (1.32), 1.273 (13.03), 2.084 (1.11), 2.116 (0.46), 2.523 (1.01), 2.793 (1.22), 2.810 (2.56), 2.827 (1.59), 3.161 (0.82), 3.171 (0.83), 3.382 (1.30), 3.392 (1.87), 3.398 (1.99), 3.406 (1.56), 3.418 (1.63), 3.423 (2.15), 3.435 (2.15), 3.441 (2.07), 3.452 (1.94), 3.469 (0.64), 4.207 (1.89), 4.220 (2.95), 4.256 (3.06), 4.270 (1.89), 4.287 (0.93), 4.297 (1.01), 4.313 (1.26), 4.323 (1.30), 4.347 (0.96), 4.360 (1.67), 4.373 (0.90), 4.434 (1.14), 4.452 (1.33), 4.461 (0.90), 4.478 (0.95), 4.700 (1.23), 4.711 (1.34), 4.718 (1.32), 4.728 (1.01), 6.249 (1.61), 6.252 (1.71), 6.269 (1.75), 6.273 (1.72), 6.733 (1.34), 6.737 (1.49), 6.753 (2.41), 6.757 (2.17), 6.805 (1.90), 6.825 (2.69), 6.845 (1.08), 6.979 (1.25), 7.086 (1.92), 7.164 (2.61), 7.295 (3.85), 7.312 (1.98), 7.324 (1.99), 7.350 (1.19), 8.067 (1.23), 8.079 (1.18), 8.461 (1.91), 11.243 (2.03).
For the preparation of the racemic title compound see example 71. Separation of enantiomers by preparative chiral HPLC (method see example 72) gave 5.0 mg of the title compound.
Analytical chiral HPLC (method see example 72): Rt=4.99 min.
Optical rotation:[α]D=23.7°+/−3.49° (c=5.6 mg/3 ml in methanol)
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.756 (0.67), 0.776 (0.68), 0.798 (0.97), 0.814 (0.97), 0.821 (1.02), 0.840 (0.77), 0.850 (0.55), 0.886 (0.48), 0.904 (0.76), 0.922 (0.46), 1.035 (1.68), 1.053 (3.23), 1.070 (2.03), 1.080 (1.20), 1.099 (0.86), 1.107 (0.75), 1.138 (3.41), 1.152 (15.78), 1.232 (2.18), 1.273 (16.00), 1.899 (0.60), 2.084 (0.85), 2.116 (1.02), 2.792 (1.61), 2.809 (3.26), 2.826 (1.89), 3.381 (1.79), 3.392 (2.51), 3.397 (2.61), 3.411 (1.47), 3.442 (0.47), 4.206 (2.30), 4.220 (3.56), 4.256 (3.70), 4.270 (2.30), 4.286 (1.15), 4.297 (1.25), 4.312 (1.56), 4.323 (1.56), 4.434 (1.38), 4.452 (1.64), 4.460 (1.13), 4.478 (1.15), 4.700 (1.52), 4.710 (1.68), 4.717 (1.61), 4.728 (1.27), 6.249 (2.02), 6.252 (2.08), 6.269 (2.13), 6.273 (2.11), 6.733 (1.66), 6.737 (1.78), 6.753 (2.79), 6.756 (2.61), 6.805 (2.21), 6.825 (3.14), 6.845 (1.25), 6.979 (1.47), 7.086 (2.48), 7.164 (3.07), 7.295 (4.81), 7.312 (2.63), 7.324 (2.62), 7.350 (1.42), 8.066 (1.80), 8.079 (1.71), 8.461 (2.79), 11.242 (2.44).
Using an analogous method as described for example 1, N-[3-chloro-2-(difluoromethoxy)phenyl]-4-[({3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-74, 35.0 mg, 70% purity, 45.5 μmol) as the starting material, 10.9 mg of the title compound were prepared (47% yield) after heating overnight and purification by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-3.26 min 30-50% B, 3.26-3.68 min 50% B, 3.68-6.40 min 50-70% B).
LC-MS (method 6): Rt=0.75 min; MS (ESIpos): m/z=505 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.232 (0.41), 1.454 (16.00), 2.332 (1.24), 2.336 (0.58), 2.358 (0.48), 2.375 (0.58), 2.380 (0.76), 2.385 (0.69), 2.396 (0.65), 2.401 (0.83), 2.407 (0.69), 2.423 (0.58), 2.518 (8.26), 2.522 (5.20), 2.673 (1.24), 2.678 (0.55), 2.725 (0.52), 2.743 (0.69), 2.748 (0.86), 2.753 (0.83), 2.764 (1.62), 2.770 (1.31), 2.777 (2.75), 2.795 (1.58), 3.376 (1.17), 3.387 (2.06), 3.393 (2.10), 3.404 (1.00), 3.410 (0.93), 4.095 (2.31), 4.121 (2.86), 4.286 (2.96), 4.302 (1.00), 4.312 (2.44), 4.319 (1.00), 4.325 (0.96), 4.341 (0.55), 4.409 (0.58), 4.425 (0.93), 4.431 (0.89), 4.440 (0.76), 4.447 (0.93), 4.462 (0.48), 6.296 (1.82), 6.299 (1.86), 6.316 (1.93), 6.320 (1.93), 6.742 (1.65), 6.746 (1.82), 6.762 (2.62), 6.765 (2.41), 6.826 (2.10), 6.846 (3.06), 6.866 (1.27), 6.993 (1.48), 7.095 (1.93), 7.178 (2.96), 7.322 (5.06), 7.336 (3.13), 7.363 (1.38), 8.059 (3.03), 8.072 (2.89), 8.480 (4.58), 11.386 (2.10).
The title compound from example 74 (9.00 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 3.00 mg at Rt=11.0-12.2 min) and enantiomer 2 (2 mg at Rt=12.5-14.0 min, see example 76).
Instrument: PrepCon Labomatic HPLC; Column: YMC Cellulose SC 5μ, 250×30; eluent A: hexane+0.1 vol % diethylamine; eluent B: ethanol; gradient: 0-20 min 20-50% B; flow: 40 ml/min; temperature: 25° C.; UV: 280 nm
Instrument: Waters Alliance 2695; Column: YMC Cellulose SC 3μ, 100×4.6; eluent A: hexane+0.1 vol % diethylamine; eluent B: ethanol; gradient: 0-7 min 20-50% B; flow: 1.4 ml/min; temperature: 25° C.; UV: 280 nm
Analytical chiral HPLC: Rt=4.29 min.
Optical rotation:[α]D=26.8°+/−6.37° (c=3.3 mg/ml in methanol)
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.746 (0.77), 0.833 (0.43), 0.852 (0.49), 1.028 (0.48), 1.043 (0.51), 1.054 (0.41), 1.138 (5.41), 1.154 (2.32), 1.233 (2.37), 1.259 (0.85), 1.294 (0.51), 1.454 (16.00), 2.085 (3.79), 2.116 (1.92), 2.327 (0.70), 2.332 (0.57), 2.358 (0.49), 2.380 (0.83), 2.385 (0.75), 2.397 (0.75), 2.402 (0.88), 2.424 (0.63), 2.665 (0.60), 2.669 (0.77), 2.726 (0.60), 2.749 (1.05), 2.754 (1.04), 2.765 (1.93), 2.779 (3.22), 2.796 (1.90), 3.377 (1.41), 3.387 (2.31), 3.394 (2.37), 3.405 (1.22), 3.573 (0.99), 4.095 (2.25), 4.122 (2.85), 4.287 (3.03), 4.304 (1.22), 4.313 (2.68), 4.326 (1.13), 4.342 (0.63), 4.410 (0.62), 4.426 (1.08), 4.432 (0.94), 4.440 (0.83), 4.447 (1.08), 4.463 (0.50), 4.560 (0.74), 6.297 (1.82), 6.300 (1.93), 6.318 (1.98), 6.321 (1.91), 6.743 (1.59), 6.746 (1.68), 6.762 (2.54), 6.766 (2.37), 6.827 (2.02), 6.847 (2.99), 6.867 (1.24), 6.993 (1.41), 7.097 (2.17), 7.179 (2.95), 7.322 (5.51), 7.337 (2.66), 7.364 (1.36), 8.060 (1.86), 8.073 (1.78), 8.482 (3.03), 11.387 (2.39).
For the preparation of the racemic title compound see example 77. Separation of enantiomers by preparative chiral HPLC (method see example 78) gave 2.00 mg of the title compound (20% yield).
Analytical chiral HPLC (method see example 78): Rt=4.88 min.
Optical rotation:[α]D=−27.1°+/−2.39° (c=2.7 mg/ml in METHANOL)
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.745 (0.73), 0.765 (0.69), 0.795 (0.49), 0.814 (0.41), 0.836 (0.45), 0.852 (0.52), 1.006 (0.45), 1.027 (1.38), 1.035 (0.46), 1.042 (1.40), 1.085 (0.67), 1.128 (1.11), 1.138 (3.67), 1.145 (2.18), 1.154 (2.02), 1.233 (2.53), 1.259 (1.28), 1.294 (0.50), 1.454 (16.00), 2.085 (0.78), 2.116 (1.18), 2.323 (0.59), 2.327 (0.79), 2.332 (0.61), 2.358 (0.54), 2.375 (0.74), 2.380 (0.88), 2.385 (0.81), 2.397 (0.81), 2.402 (0.95), 2.407 (0.83), 2.424 (0.68), 2.523 (2.68), 2.665 (0.58), 2.669 (0.77), 2.673 (0.58), 2.727 (0.56), 2.743 (0.79), 2.749 (1.00), 2.754 (0.99), 2.765 (1.87), 2.770 (1.56), 2.779 (3.10), 2.796 (1.80), 3.371 (1.25), 3.377 (1.34), 3.387 (2.28), 3.394 (2.29), 3.405 (1.15), 3.573 (1.33), 4.095 (2.30), 4.122 (2.87), 4.287 (3.06), 4.304 (1.18), 4.313 (2.64), 4.319 (1.14), 4.326 (1.10), 4.342 (0.63), 4.409 (0.63), 4.425 (1.05), 4.432 (0.93), 4.440 (0.81), 4.447 (1.04), 4.463 (0.50), 4.560 (0.41), 6.297 (1.84), 6.300 (1.93), 6.318 (1.98), 6.321 (1.92), 6.743 (1.60), 6.746 (1.72), 6.762 (2.59), 6.766 (2.38), 6.827 (2.04), 6.847 (3.01), 6.867 (1.25), 6.993 (1.43), 7.097 (2.15), 7.179 (2.96), 7.322 (5.52), 7.336 (2.76), 7.364 (1.34), 8.060 (2.23), 8.073 (2.10), 8.482 (3.57), 11.387 (2.34).
In analogy to example 1 N-(3-chloro-2-ethylphenyl)-4-[({3-[(oxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-77, 280 mg, 85% purity, 489 μmol) was used to prepare 11.1 mg of the title compound (5% yield) after heating overnight and purification by preparative HPLC (method 7).
LC-MS (method 6): Rt=0.75 min; MS (ESIpos): m/z=453 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.851 (0.50), 1.108 (0.53), 1.184 (6.93), 1.203 (16.00), 1.222 (7.54), 1.231 (3.47), 2.323 (0.54), 2.327 (0.74), 2.331 (0.53), 2.523 (2.26), 2.540 (2.00), 2.556 (0.62), 2.574 (1.16), 2.584 (1.14), 2.601 (2.05), 2.624 (1.75), 2.642 (0.91), 2.664 (1.30), 2.669 (1.09), 2.684 (1.83), 2.699 (1.88), 2.705 (1.81), 2.711 (1.39), 2.720 (1.19), 2.727 (1.24), 2.748 (0.62), 2.793 (3.59), 2.810 (7.86), 2.822 (7.79), 2.841 (5.68), 2.859 (1.92), 3.230 (0.70), 3.249 (0.91), 3.367 (4.91), 3.397 (6.29), 3.409 (7.61), 3.414 (7.50), 3.424 (4.54), 3.504 (0.77), 4.262 (2.28), 4.269 (2.46), 4.289 (3.57), 4.296 (3.49), 4.377 (3.24), 4.390 (3.43), 4.405 (2.24), 4.418 (2.26), 4.455 (1.40), 4.469 (2.99), 4.477 (1.84), 4.484 (2.30), 4.492 (2.86), 4.507 (1.55), 4.578 (1.60), 4.599 (2.73), 4.613 (2.36), 4.616 (2.09), 4.631 (1.24), 5.101 (0.79), 5.107 (0.95), 5.121 (2.00), 5.137 (1.88), 5.151 (0.87), 5.158 (0.67), 6.230 (4.51), 6.234 (4.76), 6.250 (4.83), 6.254 (4.76), 6.676 (2.66), 6.680 (3.43), 6.696 (8.15), 6.700 (7.04), 6.713 (6.75), 6.733 (7.48), 6.753 (2.55), 7.128 (4.65), 7.199 (5.69), 7.211 (5.78), 7.344 (9.22), 7.993 (4.00), 8.005 (3.82), 8.433 (6.42), 11.177 (5.10).
In analogy to example 1 N-(3-chloro-2-ethylphenyl)-4-[({3-[(2-methyloxetan-2-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-78, 145 mg, 260 μmol) was used to prepare 8.40 mg of the title compound (7% yield) after heating for 48 h at 60° C. and purification by preparative HPLC (method 7).
LC-MS (method 6): Rt=0.79 min; MS (ESIpos): m/z=467 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.272 (2.87), 1.291 (6.40), 1.309 (3.16), 1.320 (1.35), 1.560 (16.00), 2.163 (2.83), 2.456 (0.55), 2.473 (0.74), 2.478 (0.94), 2.484 (0.88), 2.495 (0.85), 2.500 (0.99), 2.506 (0.87), 2.522 (0.70), 2.834 (0.71), 2.855 (2.48), 2.872 (4.04), 2.889 (2.71), 2.901 (1.43), 2.910 (2.53), 2.929 (2.37), 2.948 (0.83), 2.959 (0.56), 3.481 (1.51), 3.493 (2.42), 3.499 (2.52), 3.510 (1.35), 4.180 (2.40), 4.207 (2.91), 4.393 (2.79), 4.419 (2.28), 4.429 (0.71), 4.445 (1.18), 4.451 (0.90), 4.460 (1.00), 4.467 (1.19), 4.483 (0.67), 4.535 (0.68), 4.552 (1.14), 4.557 (1.05), 4.567 (0.88), 4.573 (1.11), 4.588 (0.52), 6.343 (1.86), 6.346 (1.95), 6.362 (2.04), 6.366 (2.03), 6.770 (1.25), 6.773 (1.48), 6.789 (3.00), 6.792 (2.78), 6.813 (2.45), 6.834 (2.90), 6.853 (1.06), 7.214 (2.23), 7.290 (2.68), 7.302 (2.71), 7.396 (3.77), 8.085 (2.14), 8.098 (2.05), 8.549 (3.46), 11.398 (2.45).
To a solution of 2-(3-{[(2R)-azetidin-2-yl]methoxy}pyridin-4-yl)-3-(3-fluoro-2-methoxyanilino)-1,5,6,7-tetrahydro-4H-pyrrolo[3,2-c]pyridin-4-one (intermediate 79-2, 40.0 mg, 91.4 μmol) in methanol (750 μl) were added formaldehyde (13.7 μl, 37% purity, 183 μmol) and acetic acid (5.2 μl, 91 μmol) and the mixture was stirred for 15 min. at RT. After that sodium trisacetoxyborohydride (29.1 mg, 137 μmol) was added and the mixture was stirred for 1.5 h at RT. The mixture was basified with aqueous sodium hydroxid solution (1 M, 20 μL) and purified by preparative HPLC (method 10, gradient: 0.00-0.50 min 30% B, 0.50-6.00 min 30-70% B) to give 20.0 mg of the title compound (47% yield).
LC-MS (method 6): Rt=0.47 min; MS (ESIpos): m/z=452 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 2.012 (0.79), 2.032 (0.84), 2.223 (0.69), 2.246 (0.94), 2.270 (0.65), 2.337 (16.00), 2.518 (0.93), 2.522 (0.61), 2.781 (1.01), 2.786 (1.05), 2.797 (1.91), 2.813 (1.05), 2.821 (0.96), 2.913 (0.46), 2.933 (0.92), 2.954 (0.94), 2.974 (0.41), 3.384 (0.52), 3.391 (0.52), 3.399 (0.84), 3.416 (2.05), 3.435 (2.43), 3.450 (1.20), 3.455 (1.10), 3.469 (0.74), 3.492 (1.05), 3.513 (0.53), 3.938 (15.54), 3.952 (0.48), 4.044 (1.08), 4.051 (1.14), 4.070 (1.27), 4.077 (1.14), 4.453 (1.41), 4.457 (1.49), 4.480 (1.30), 4.484 (1.23), 6.060 (1.82), 6.080 (1.87), 6.507 (0.84), 6.510 (0.89), 6.528 (1.17), 6.531 (1.25), 6.534 (1.07), 6.537 (0.94), 6.555 (1.09), 6.558 (1.05), 6.659 (0.85), 6.674 (1.00), 6.680 (1.53), 6.695 (1.50), 6.701 (0.75), 6.716 (0.64), 7.162 (2.00), 7.306 (3.28), 7.319 (3.34), 7.622 (4.42), 7.970 (3.55), 7.983 (3.33), 8.429 (5.22), 12.844 (1.70).
Using an analogous method as described for example 1, N-(3-chloro-2-methylphenyl)-4-{[(3-{[2-methyloxetan-2-yl]methoxy}pyridin-4-yl)methyl]amino}-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (160 mg, 329 μmol) as the starting material, 48 mg of the title compound were prepared (26% yield) after heating for 16 h at 60° C. and purification by chromatography on silica gel (DCM/ethanol).
LC-MS (method 7): Rt=0.79 min; MS (ESIpos): m/z=453.5 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 0.801 (0.69), 0.817 (0.75), 0.824 (0.74), 0.907 (0.81), 1.156 (0.64), 1.175 (1.23), 1.193 (0.73), 1.234 (0.66), 1.367 (0.94), 1.377 (2.72), 1.471 (16.00), 1.990 (2.26), 2.256 (2.46), 2.330 (14.32), 2.368 (0.53), 2.384 (0.62), 2.390 (0.92), 2.395 (0.84), 2.407 (0.74), 2.412 (0.87), 2.417 (0.78), 2.434 (0.61), 2.520 (2.88), 2.525 (1.79), 2.667 (0.50), 2.671 (0.68), 2.676 (0.50), 2.741 (0.58), 2.764 (2.03), 2.782 (3.38), 2.799 (1.70), 2.807 (0.72), 2.998 (0.43), 3.218 (4.25), 3.383 (1.02), 3.389 (1.16), 3.400 (2.04), 3.406 (2.10), 3.417 (1.00), 3.484 (0.53), 4.020 (0.51), 4.037 (0.50), 4.091 (2.38), 4.118 (2.84), 4.158 (0.73), 4.172 (0.68), 4.304 (2.69), 4.330 (2.28), 4.342 (0.70), 4.358 (1.18), 4.365 (0.82), 4.374 (0.97), 4.380 (1.13), 4.396 (0.63), 4.446 (0.62), 4.463 (0.97), 4.469 (0.89), 4.477 (0.75), 4.484 (0.94), 4.500 (0.46), 4.813 (1.36), 5.761 (4.18), 6.234 (1.62), 6.237 (1.67), 6.254 (1.78), 6.256 (1.70), 6.697 (1.13), 6.700 (1.29), 6.717 (2.56), 6.720 (2.28), 6.746 (1.80), 6.766 (2.28), 6.785 (0.82), 7.135 (1.95), 7.229 (3.94), 7.237 (3.55), 7.250 (3.50), 7.269 (0.59), 7.440 (0.42), 7.739 (0.42), 8.019 (3.97), 8.032 (3.62), 8.284 (0.57), 8.296 (0.69), 8.466 (5.79), 8.494 (0.89), 11.313 (2.04).
The title compound from example 80 (45 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 18 mg at Rt=17.8-21.3 min) and enantiomer 2 (16 mg at Rt=24.9-30.2 min, see example 82).
Instrument: PrepCon Labomatic HPLC; Column: YMC Amylose SA 5μ, 250×30; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: acetonitrile+0.1 vol % diethylamine; isocratic: 50% A+50% B; flow: 50 ml/min; temperature: 25° C.; UV: 254 nm
Instrument: Waters Alliance 2695; Column: YMC Amylose SA 3μ, 100×4.6; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: acetonitrile; isocratic: 50% A+50% B; flow: 1.4 ml/min; temperature: 25° C.; UV: 254 nm
Analytical chiral HPLC: Rt=6.53 min.
Optical rotation:[α]D=−63.6°+/−0.85° (c=1.0 g/100 ml in DMSO)
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.235 (0.66), 1.471 (16.00), 2.076 (5.65), 2.329 (14.22), 2.368 (0.45), 2.384 (0.55), 2.390 (0.74), 2.395 (0.67), 2.407 (0.62), 2.412 (0.77), 2.417 (0.69), 2.434 (0.52), 2.520 (2.85), 2.525 (1.69), 2.667 (0.52), 2.671 (0.73), 2.676 (0.53), 2.741 (0.54), 2.764 (1.96), 2.782 (3.28), 2.799 (1.66), 3.389 (1.11), 3.400 (1.98), 3.405 (2.14), 3.422 (1.08), 3.883 (2.20), 4.091 (2.38), 4.117 (2.88), 4.303 (2.69), 4.330 (2.17), 4.342 (0.54), 4.357 (0.95), 4.365 (0.71), 4.373 (0.84), 4.379 (0.96), 4.396 (0.57), 4.446 (0.60), 4.462 (0.92), 4.468 (0.89), 4.477 (0.74), 4.484 (0.91), 4.499 (0.47), 6.234 (1.57), 6.237 (1.64), 6.254 (1.74), 6.256 (1.72), 6.680 (0.51), 6.691 (0.63), 6.697 (1.12), 6.700 (1.34), 6.717 (2.54), 6.720 (2.30), 6.745 (1.81), 6.766 (2.30), 6.785 (0.82), 7.134 (1.87), 7.229 (3.87), 7.237 (3.31), 7.250 (3.13), 7.551 (0.48), 8.018 (3.11), 8.031 (2.99), 8.046 (0.43), 8.451 (0.58), 8.466 (4.81), 11.313 (1.98).
For the preparation of the racemic title compound see example 80. Separation of enantiomers by preparative chiral HPLC (method see example 81) gave 16 mg of the title compound (10% yield).
Analytical chiral HPLC (method see example 81): Rt=9.13 min.
Optical rotation:[α]D=33.0°+/−0.53° (c=1.0 g/100 ml in DMSO)
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.168 (0.48), 1.235 (0.71), 1.471 (16.00), 2.077 (4.89), 2.329 (14.32), 2.368 (0.48), 2.385 (0.58), 2.390 (0.75), 2.395 (0.69), 2.407 (0.64), 2.412 (0.78), 2.417 (0.67), 2.434 (0.52), 2.520 (2.40), 2.525 (1.52), 2.666 (0.48), 2.671 (0.64), 2.676 (0.47), 2.740 (0.57), 2.764 (2.02), 2.782 (3.37), 2.799 (1.67), 3.382 (1.00), 3.388 (1.11), 3.400 (1.98), 3.405 (2.11), 3.417 (1.02), 3.422 (1.04), 3.883 (1.57), 4.091 (2.34), 4.118 (2.80), 4.304 (2.66), 4.330 (2.10), 4.342 (0.52), 4.357 (0.97), 4.365 (0.70), 4.373 (0.81), 4.380 (0.98), 4.396 (0.54), 4.446 (0.60), 4.463 (0.94), 4.468 (0.86), 4.477 (0.72), 4.484 (0.92), 4.499 (0.45), 6.234 (1.62), 6.236 (1.65), 6.254 (1.78), 6.680 (0.40), 6.692 (0.53), 6.697 (1.15), 6.700 (1.31), 6.717 (2.55), 6.720 (2.26), 6.745 (1.80), 6.766 (2.31), 6.785 (0.82), 7.136 (1.93), 7.229 (3.95), 7.237 (3.19), 7.250 (2.95), 8.019 (2.80), 8.031 (2.64), 8.451 (0.47), 8.466 (4.36), 11.314 (2.04).
Using an analogous method as described for example 1, N-(3-fluoro-2-methylphenyl)-4-{[(3-{[2-methyloxetan-2-yl]methoxy}pyridin-4-yl)methyl]amino}-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (160 mg, 340 μmol) as the starting material, 34 mg of the title compound were prepared (22% yield) after heating for 16 h at 60° C. and purification by chromatography on silica gel (DCM/ethanol).
LC-MS (method 7): Rt=0.73 min; MS (ESIpos): m/z=437.5 [M+H]+
1H-NMR (400 MHz, DMSO-d6) delta [ppm]: −0.008 (1.00), 0.008 (1.15), 1.383 (0.82), 1.473 (16.00), 1.990 (0.55), 2.163 (7.67), 2.166 (7.74), 2.369 (0.51), 2.385 (0.61), 2.391 (0.83), 2.396 (0.76), 2.407 (0.69), 2.412 (0.85), 2.418 (0.74), 2.434 (0.61), 2.520 (3.92), 2.525 (2.37), 2.743 (0.54), 2.764 (1.87), 2.770 (1.22), 2.781 (3.28), 2.797 (1.78), 2.810 (0.60), 3.217 (1.22), 3.384 (0.97), 3.390 (1.11), 3.400 (1.99), 3.407 (2.06), 3.418 (0.97), 4.091 (2.34), 4.118 (2.88), 4.305 (2.69), 4.332 (2.16), 4.347 (0.56), 4.362 (1.00), 4.370 (0.78), 4.378 (0.87), 4.385 (1.01), 4.401 (0.59), 4.448 (0.60), 4.465 (0.94), 4.470 (0.88), 4.479 (0.72), 4.486 (0.92), 4.502 (0.46), 5.761 (1.52), 6.099 (1.83), 6.119 (1.91), 6.417 (0.85), 6.439 (1.55), 6.461 (0.96), 6.730 (0.58), 6.749 (1.23), 6.767 (1.19), 6.787 (0.49), 7.137 (1.90), 7.217 (3.75), 7.239 (3.19), 7.251 (3.22), 8.013 (4.29), 8.026 (3.97), 8.465 (5.92), 11.297 (1.96).
The title compound from example 83 (30 mg) was separated into enantiomers by preparative chiral HPLC to give title compound (enantiomer 1, 13 mg at Rt=13.8-16.7 min) and enantiomer 2 (10 mg at Rt=19.0-23.2 min, see example 85).
Instrument: PrepCon Labomatic HPLC; Column: YMC Amylose SA 5μ, 250×30; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: acetonitrile+0.1 vol % diethylamine; isocratic: 50% A+50% B; flow: 50 ml/min; temperature: 25° C.; UV: 254 nm
Instrument: Waters Alliance 2695; Column: YMC Amylose SA 3μ, 100×4.6; eluent A: methyl tert-butyl ether+0.1 vol % diethylamine; eluent B: acetonitrile; isocratic: 50% A+50% B; flow: 1.4 ml/min; temperature: 25° C.; UV: 254 nm Analytical chiral HPLC: Rt=5.11 min.
Optical rotation:[α]D=−29.8°+/−0.39° (c=1.0 g/100 ml in DMSO)
1H-NMR (400 MHz, DMSO-d6) δ[ppm]: 1.234 (0.48), 1.473 (16.00), 2.076 (2.27), 2.163 (7.44), 2.166 (7.40), 2.329 (1.45), 2.369 (0.46), 2.385 (0.59), 2.390 (0.76), 2.395 (0.69), 2.407 (0.67), 2.412 (0.80), 2.417 (0.68), 2.434 (0.59), 2.520 (2.35), 2.525 (1.53), 2.666 (0.50), 2.671 (0.65), 2.676 (0.48), 2.743 (0.56), 2.764 (1.89), 2.780 (3.34), 2.797 (1.76), 2.809 (0.56), 3.383 (1.01), 3.389 (1.12), 3.400 (2.01), 3.407 (2.04), 3.417 (0.99), 4.091 (2.40), 4.118 (2.91), 4.305 (2.66), 4.332 (2.13), 4.347 (0.49), 4.362 (0.96), 4.370 (0.71), 4.378 (0.84), 4.384 (0.95), 4.400 (0.54), 4.448 (0.61), 4.465 (0.94), 4.470 (0.86), 4.480 (0.72), 4.486 (0.93), 4.501 (0.46), 6.098 (1.81), 6.119 (1.87), 6.417 (0.83), 6.440 (1.52), 6.461 (0.95), 6.729 (0.56), 6.749 (1.22), 6.766 (1.25), 6.787 (0.52), 7.139 (1.90), 7.217 (3.72), 7.239 (2.79), 7.252 (2.77), 8.014 (2.49), 8.026 (2.31), 8.465 (4.03), 11.299 (1.91).
For the preparation of the racemic title compound see example 80. Separation of enantiomers by preparative chiral HPLC (method see example 81) gave 16 mg of the title compound (6% yield).
Analytical chiral HPLC (method see example 84): Rt=7.06 min.
Optical rotation:[α]D=26.9°+/−0.73° (c=1.0 g/100 ml in DMSO)
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.174 (0.58), 1.234 (0.77), 1.473 (16.00), 2.076 (2.72), 2.163 (7.24), 2.166 (7.25), 2.329 (2.31), 2.369 (0.46), 2.385 (0.60), 2.391 (0.77), 2.396 (0.70), 2.407 (0.65), 2.412 (0.79), 2.417 (0.66), 2.434 (0.55), 2.520 (3.23), 2.524 (1.95), 2.666 (0.50), 2.671 (0.71), 2.676 (0.53), 2.743 (0.57), 2.764 (2.00), 2.781 (3.49), 2.797 (1.82), 2.809 (0.59), 3.384 (1.06), 3.390 (1.19), 3.400 (2.11), 3.407 (2.16), 3.418 (1.05), 4.091 (2.48), 4.118 (3.02), 4.305 (2.68), 4.331 (2.17), 4.347 (0.50), 4.362 (0.96), 4.370 (0.71), 4.378 (0.89), 4.385 (0.96), 4.401 (0.55), 4.448 (0.60), 4.465 (0.98), 4.470 (0.89), 4.479 (0.76), 4.486 (0.96), 4.501 (0.47), 6.099 (1.74), 6.119 (1.82), 6.417 (0.81), 6.439 (1.48), 6.461 (0.93), 6.729 (0.55), 6.749 (1.21), 6.766 (1.34), 6.787 (0.53), 7.137 (1.95), 7.217 (3.63), 7.229 (0.68), 7.239 (2.67), 7.252 (2.65), 8.013 (2.16), 8.026 (2.08), 8.465 (3.73), 11.297 (1.87).
Using an analogous method as described for example 1, N-(2-chloro-3-methylphenyl)-4-[({3-[(3-ethyloxetan-3-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-86, 198 mg, 95% purity, 375 μmol) as the starting material, 45 mg of the title compound were prepared (24% yield) after heating for 16 h and purification by prep. reversed phase HPLC (basic conditions).
LC-MS (method 1): Rt=0.76 min; MS (ESIpos): m/z=467 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.850 (4.33), 0.868 (10.69), 0.887 (4.74), 1.688 (1.18), 1.706 (3.69), 1.725 (3.47), 1.743 (1.02), 2.084 (0.60), 2.313 (16.00), 2.327 (0.89), 2.331 (0.57), 2.518 (2.45), 2.523 (1.59), 2.665 (0.45), 2.669 (0.64), 2.673 (0.45), 2.745 (1.88), 2.762 (4.07), 2.779 (2.10), 3.159 (2.89), 3.172 (3.21), 3.362 (2.48), 3.368 (2.04), 3.379 (2.86), 3.385 (2.83), 3.397 (1.40), 3.402 (1.30), 4.104 (0.64), 4.117 (0.60), 4.266 (9.26), 4.515 (4.42), 4.531 (7.25), 4.561 (7.51), 4.576 (4.29), 6.179 (1.94), 6.197 (2.00), 6.635 (1.75), 6.652 (2.29), 6.748 (2.23), 6.768 (3.02), 6.787 (1.53), 7.152 (2.51), 7.261 (4.07), 7.274 (4.10), 7.511 (5.47), 8.013 (4.39), 8.025 (4.04), 8.473 (6.04), 11.349 (2.61).
Using an analogous method as described for example 1, N-(2,3-dichlorophenyl)-4-[({3-[(3-ethyloxetan-3-yl)methoxy]pyridin-4-yl}methyl)amino]-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-87, 174 mg, 334 μmol) as the starting material, 57 mg of the title compound were prepared (33% yield) after heating for 16 h at 60° C. and purification by prep. reversed phase HPLC (basic conditions).
LC-MS (method 1): Rt=0.79 min; MS (ESIpos): m/z=487 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.837 (4.02), 0.856 (10.33), 0.875 (4.30), 1.681 (1.05), 1.700 (3.32), 1.719 (3.18), 1.738 (0.91), 2.074 (0.59), 2.331 (0.41), 2.518 (2.47), 2.522 (1.57), 2.539 (16.00), 2.673 (0.43), 2.755 (1.69), 2.772 (3.65), 2.789 (1.88), 3.364 (1.24), 3.370 (1.33), 3.381 (2.40), 3.386 (2.33), 3.398 (1.17), 3.404 (1.06), 4.252 (8.50), 4.479 (4.23), 4.495 (6.67), 4.529 (6.84), 4.544 (4.17), 6.254 (2.26), 6.259 (2.34), 6.273 (2.40), 6.278 (2.25), 6.844 (1.08), 6.849 (1.58), 6.863 (4.52), 6.868 (3.83), 6.874 (3.57), 6.894 (3.40), 6.913 (1.08), 7.132 (2.26), 7.338 (3.78), 7.351 (3.82), 7.585 (5.28), 8.075 (4.16), 8.088 (3.90), 8.491 (5.51), 11.404 (2.36).
Using an analogous method as described for example 1, 4-[({3-[(3-ethyloxetan-3-yl)methoxy]pyridin-4-yl}methyl)amino]-N-(3-fluoro-2-methoxyphenyl)-2-oxo-1,2,5,6-tetrahydropyridine-3-carbothioamide (intermediate 6-88, 105 mg, 210 μmol) as the starting material, 39 mg of the title compound were prepared (39% yield) after heating for 16 h at 50° C. and purification by prep. reversed phase HPLC (basic conditions).
LC-MS (method 1): Rt=0.72 min; MS (ESIpos): m/z=467 [M+H]+
1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 0.855 (3.76), 0.874 (9.54), 0.892 (4.00), 1.691 (0.99), 1.710 (3.14), 1.728 (2.97), 1.747 (0.86), 2.074 (0.79), 2.332 (0.66), 2.518 (3.80), 2.523 (2.41), 2.673 (0.69), 2.736 (1.60), 2.753 (3.47), 2.769 (1.78), 3.354 (1.25), 3.360 (1.29), 3.372 (2.31), 3.377 (2.26), 3.388 (1.12), 3.394 (1.02), 3.910 (16.00), 4.292 (7.89), 4.526 (3.68), 4.541 (6.27), 4.569 (6.39), 4.584 (3.62), 5.758 (3.43), 5.990 (1.87), 6.010 (1.93), 6.470 (0.88), 6.474 (0.89), 6.491 (1.24), 6.494 (1.24), 6.497 (1.07), 6.500 (0.94), 6.518 (1.17), 6.521 (1.09), 6.612 (0.89), 6.627 (1.01), 6.633 (1.60), 6.648 (1.55), 6.653 (0.78), 6.668 (0.66), 7.119 (2.13), 7.378 (3.76), 7.391 (3.81), 7.437 (4.76), 8.042 (4.71), 8.055 (4.34), 8.491 (6.08), 11.323 (2.16).
The pharmacological activity of the compounds according to the invention can be assessed using in vitro- and/or in vivo-assays, as known to the person skilled in the art. The following examples describe the biological activity of the compounds according to the invention, without the invention being limited to said examples.
Example compounds according to the invention were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein
Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch.
The in vitro activity of the compounds of the present invention can be demonstrated in the following assays:
The different EGFR proteins used in the biochemical kinase activity inhibition assays were generated in house by expression in insect cells using Baculo Virus system and subsequent purification as described in the following paragraphs.
The cDNAs encoding the various protein sequences from human EGFR human (P00533) were optimized for expression in eukaryotic cells and synthesized by the GeneArt Technology at Life Technologies.
These DNA sequences encoded the following sequence:
Additionally all constructs EGFR #1 to #3 encoded: at the N-terminus a TEV (Tobacco etch virus) protease cleavage site (DYDIPTTENLYFQG), at the C-terminus two stop codons and additionally 5′ and 3′ att-DNA sequences for Gateway Cloning.
Each of the three EFGR constructs was subcloned using the Gateway Technology into the Destination vector pD-Ins1. The vector pD-Ins1 is a Baculovirus transfer vector (based on vector pVL1393, Pharmingen) which provides a N-terminal fusion of a GST-tag to the integrated gene construct. The respective transfer vectors were termed pD-Ins1_EGFR #1, pD-Ins1_EGFR #2, pD-Ins1_EGFR #3.
In separate approaches each of the three transfer vectors was co-transfected in Sf9 cells with Baculovirus DNA (Flashbac Gold DNA, Oxford Expression Technologies) using Fugene HD (Roche). After 5 days the supernatant of the transfected cells containing the recombinant Baculovirus encoding the various EGFR proteins was used for further infection of Sf9 cells for virus amplification whereby the virus titer was monitored using qPCR.
Sf9 cells cultured (Insect-xpress medium, Lonza, 27° C.) in a Wave-bioreactor with a disposable culture bag were infected at a cell density of 106 cells/ml with one of the recombinant baculovirus stocks at a multiplicity of infection of 1 and incubated for 48 h. Subsequently the cells were harvested by centrifugation and the cell pellet frozen at −80° C.
Purification of the GST-EGFR fusion proteins was achieved by affinity chromatography using Glutathion Sepharose 4B matrix (GE Healthcare Life Sciences).
The pelleted cells (from 4 I cell culture) were resuspended in Lysis-Buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 5% Glycerol, 1 mM MgCl2, 1 mM MnCl2, 0.5 mM Na3VO4) and lysed by a freeze-thaw cycle followed by an incubation on ice for 60 min. The supernatant was centrifuged at 4000×g for 30 min. at 4° C. The supernatant was than incubated with Glutathion Sepharose 4B matrix (in a glass bottle rotating for 16 h, at 4° C.) for binding of the GST EGFR fusion protein, rinsed with Wash-Buffer and finally the bound protein was eluted using Elusion-Buffer (Lysis Buffer plus 25 mM Glutathione) and shock frozen with liquid nitrogen.
Inhibitory activity of compounds of the present invention against wild-type Epidermal Growth Factor Receptor (EGFR) was quantified employing the TR-FRET based EGFR assay as described in the following paragraphs.
Recombinant fusion protein of N-terminal Glutathion-S-Transferase (GST) and a fragment of human EGFR (amino acids R669 to A1210), expressed in Sf9 insect cells and purified via affinity chromatography using Glutathion Sepharose as described above, was used as a kinase. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-AEEEEYFELVAKKK—SEQ ID 6 (C-terminus in amide form) was used, which can be purchased e.g. form the company Biosynthan GmbH (Berlin-Buch, Germany).
For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384 well microtiter plate or a black 1536 well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of EGFR in aqueous assay buffer [50 mM Hepes pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EGTA, 0.3 mM activated sodium ortho-vanadate, 0.005% (w/v) bovine serum albumin, 0.005% (v/v) Tween-20] were added and the mixture was incubated for 15 min at 22° C. to allow pre binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine tri phosphate (ATP, 3.33 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (1.67 μM=>final conc. in the 5 μL assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of EGFR was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentration was 7.6 μg/μl. The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (83.3 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1.67 nM PT66-Tb-Cryptate, an terbium-cryptate labelled anti-phospho-tyrosine antibody from Cisbio Bioassays [instead of the PT66 Tb cryptate PT66 Eu Chelate from Perkin Elmer can also be used]) in an aqueous EDTA-solution (133.3 mM EDTA, 0.2% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
The resulting mixture was incubated 1 h at 22° C. to allow the binding of the biotinylated phosphorylated peptide to the streptavidine-XL665 and the PT66-Tb-Cryptate. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the PT66-Tb-Cryptate to the streptavidine-XL665. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 337 nm were measured in a HTRF reader, e.g. a Pherastar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.07 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100-fold concentrated solutions in DMSO by serial dilutions, exact concentrations may vary depending pipettors used) in duplicate values for each concentration and IC50 values were calculated using Genedata Screener™ software.
Inhibitory activity of compounds of the present invention against an Epidermal Growth Factor Receptor (EGFR) with an insertion of the amino acids sequence SVD between D770 and N771 was quantified employing the TR-FRET based kinase activity assay as described in the following paragraphs.
A recombinant fusion protein of N-terminal Glutathion-S-Transferase (GST) and a fragment of human EGFR variant (amino acids R669 to A1210 with insertion of the amino acids sequence SVD between D770 and N771 (“EGFR ins SVD”), expressed in Sf9 insect cells and purified via affinity chromatography using Glutathion Sepharose as described above, was used as a kinase. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-AEEEEYFELVAKKK—SEQ ID 6 (C-terminus in amide form) was used which can be purchased e.g. form the company Biosynthan GmbH (Berlin-Buch, Germany).
For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384 well microtiter plate or a black 1536 well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of EGFR in aqueous assay buffer [50 mM Hepes pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EGTA, 0.3 mM activated sodium ortho-vanadate, 0.005% (w/v) bovine serum albumin, 0.005% (v/v) Tween-20] were added and the mixture was incubated for 15 min at 22° C. to allow pre binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine tri phosphate (ATP, 3.33 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (1.67 μM=>final conc. in the 5 μL assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of EGFR was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentration was 15 μg/μl. The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (83.3 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1.67 nM PT66-Tb-Cryptate, a terbium-cryptate labelled anti-phospho-tyrosine antibody from Cisbio Bioassays [instead of the PT66 Tb cryptate PT66 Eu Chelate from Perkin Elmer can also be used]) in an aqueous EDTA-solution (133.3 mM EDTA, 0.2% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
The resulting mixture was incubated 1 h at 22° C. to allow the binding of the biotinylated phosphorylated peptide to the streptavidine-XL665 and the PT66-Tb-Cryptate. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the PT66-Tb-Cryptate to the streptavidine-XL665. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 337 nm were measured in a HTRF reader, e.g. a Pherastar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.07 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100fold concentrated solutions in DMSO by serial dilutions, exact concentrations may vary depending pipettors used) in duplicate values for each concentration and IC50 values were calculated using Genedata Screener™ software.
Inhibitory activity of compounds of the present invention against an Epidermal Growth Factor Receptor (EGFR) with an insertion of the amino acids sequence ASV between V769 and D770 was quantified employing the TR-FRET based kinase activity assay as described in the following paragraphs.
A recombinant fusion protein of N-terminal Glutathion-S-Transferase (GST) and a fragment of human EGFR variant (amino acids R669 to A1210 with insertion of the amino acids sequence ASV between V769 and D770; (“EGFR ins ASV”), expressed in Sf9 insect cells and purified via affinity chromatography using Glutathion Sepharose as described above, was used as kinase. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-AEEEEYFELVAKKK—SEQ ID 6 (C-terminus in amide form) was used which can be purchased e.g. form the company Biosynthan GmbH (Berlin-Buch, Germany).
For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384well microtiter plate or a black 1536 well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of EGFR in aqueous assay buffer [50 mM Hepes pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EGTA, 0.3 mM activated sodium ortho-vanadate, 0.005% (w/v) bovine serum albumin, 0.005% (v/v) Tween-20] were added and the mixture was incubated for 15 min at 22° C. to allow pre binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine tri phosphate (ATP, 3.33 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (1.67 μM=>final conc. in the 5 μL assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of EGFR was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentration was 2.5 μg/μl. The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (83.3 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1.67 nM PT66-Tb-Cryptate, an terbium-cryptate labelled anti-phospho-tyrosine antibody from Cisbio Bioassays [instead of the PT66 Tb cryptate PT66 Eu Chelate from Perkin Elmer can also be used]) in an aqueous EDTA-solution (133.3 mM EDTA, 0.2% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).
The resulting mixture was incubated 1 h at 22° C. to allow the binding of the biotinylated phosphorylated peptide to the streptavidine-XL665 and the PT66-Tb-Cryptate. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the PT66-Tb-Cryptate to the streptavidine-XL665. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 337 nm were measured in a HTRF reader, e.g. a Pherastar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.07 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100-fold concentrated solutions in DMSO by serial dilutions, exact concentrations may vary depending pipettors used) in duplicate values for each concentration and IC50 values were calculated using Genedata Screener™ software.
Table 2 shows the results of the inhibition in mutant EGFR biochemical assay.
Bub1-inhibitory activity of compounds of the present invention at a high ATP concentration was quantified employing the Bub1 TR-FRET high ATP kinase assay as described in the following paragraphs.
N-terminally His6-tagged recombinant catalytic domain of human Bub1 (amino acids 704-1085), expressed in insect cells (Hi5) and purified by Ni-NTA affinity chromatography and subsequent size exclusion chromatography, was used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-VLLPKKSFAEPG—SEQ ID 7 (C-terminus in amid form) was used which can be purchased e.g. form the company Biosyntan (Berlin, Germany).
For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384 well microtiter plate or a black 1536well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 3 μl of a solution of adenosine-tri-phosphate (ATP, 3.33 mM=>final conc. in the 5 μl assay volume is 2 mM) and substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in aqueous assay buffer [50 mM Tris/HCl pH 7.5, 10 mM magnesium chloride (MgCl2), 200 mM potassium chloride (KCl), 1.0 mM dithiothreitol (DTT), 0.1 mM sodium ortho-vanadate, 1% (v/v) glycerol, 0.01% (w/v) bovine serum albumine (BSA), 0.005% (v/v) Trition X-100 (Sigma), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added. Then the kinase reaction was started by the addition of 2 μl of a solution of Bub1 in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Bub1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, a typical concentration is about 200 ng/ml. The reaction was stopped by the addition of 3 μl of a solution of TR-FRET detection reagents (0.167 μM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1.67 nM anti-phosho-Serine antibody [Merck Millipore, cat. #35-002] and 0.67 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077, as an alternative a Terbium-cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used]) in an aqueous EDTA-solution (83.3 mM EDTA, 0.2% (w/v) bovine serum albumin in 100 mM HEPES pH 7.5).
The resulting mixture was incubated 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a TR-FRET reader, e.g. a Pherastar or Pherastar FS (both from BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.7 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100fold concentrated solutions in DMSO by serial dilutions, exact concentrations may vary depending pipettors used) in duplicate values for each concentration and IC50 values were calculated by a 4 parameter fit.
Table 3 shows the results of the inhibition in Bub1 high ATP kinase assay.
Compounds of the present invention may show additional advantageous properties, such as, more potent inhibition of mutant EGFR with exon20 insertions than inhibition of wild-type EGFR, which may be useful to reduce potential toxicity arising from excessive inhibition of wild-type EGFR.
Cellular Data Description (WT, insSVD)
293T cells from ATCC were transfected with pBABEpuro expression constructs for WT EGFR or EGFR-insSVD and pCL-Eco packaging vector using Fugene-6 transfection reagent from Promega. Plates were incubated at at 37° C. for 48 h. Retrovirus was harvested by filtering the media supernatant through a 0.45 μm filter.
Ba/F3 cells purchased from DSMZ were grown in RPMI+10% FBS+10 ng/mL IL-3 and infected with filtered retroviral supernatant at a 1:2 dilution. Polybrene was added to a concentration of 8 μg/mL, plates were spun for 90 min, and incubated overnight at 37° C. 2 μg/mL puromycin was added to the infected cells 24 h after infection and cells were continually grown in the presence of puromycin and 10 ng/mL IL-3. Following stably expressing Ba/F3 cell lines were generated: Ba/F3-EGFR-WT, Ba/F3-EGFR-insSVD, (Ba/F3-vector-control).
For cell survival assays, Ba/F3 cells were grown to a density of 1-2 million cells per mL, spun down and resuspended in media without IL-3, and replated at a concentration 200,000-500,000 cells per mL. The cells ectopically expressing WT EGFR or EGFR-insSVD were plated with 10 ng/mL Millipore Culture grade EGF. The cells ectopically expressing pBABEpuro empty vector were plated with 10 ng/mL IL-3.
2 days later, cells were plated in 50 μL in a 384 well plate at a concentration of 4000 cells per well for cells assayed in the absence of IL-3 and 2000 cells per well for cells assayed in the presence of IL-3. 100 nL of compound was added to each well using a 100 nL pin head, and plates were incubated at 37° C. for 48 h.
Cell viability was measured by adding 20 μL of Cell Titer-Glo Luminescent Cell Viability Reagent diluted 1:3 in PBS. Plates were sealed with Perkin Elmer Top-Seal, inverted several times to mix, and immediately centrifuged at 1000 rpm for 2 min. Plates were incubated in low light conditions for 8-10 min and luminescence was measured. The IC50 values for the examples are shown in Table 4.
In contrast to the claimed compounds of this invention, the compounds claimed in WO 2016/120196 do not show the advantageous combined properties described above. This can be seen in Table 5.
Cellular Data Description (Ba/F3 Cells Overexpressing Mutant EGFR Different from insSVD)
293T cells from ATCC were transfected with pBABEpuro expression constructs for mutant EGFR (V769_D770insASV, D770_N771insNPG, N771_P772insH, H773_V774insNPH, E746_A750del, L858R, D770_N771 insSVD C797S, E746_A750del C797S, L858R C797S, L861Q) or mutant ERBB2 (A775_G776insYVMA) and pCL-Eco packaging vector using Fugene-6 transfection reagent from Promega. Plates were incubated at at 37° C. for 48 h. Retrovirus was harvested by filtering the media supernatant through a 0.45 μm filter.
Ba/F3 cells purchased from DSMZ were grown in RPMI+10% FBS+10 ng/mL IL-3 and infected with filtered retroviral supernatant at a 1:2 dilution. Polybrene was added to a concentration of 8 μg/mL, plates were spun for 90 min, and incubated overnight at 37° C. 2 μg/mL puromycin was added to the infected cells 24 h after infection and cells were continually grown in the presence of puromycin and 10 ng/mL IL-3. Following stably expressing Ba/F3 cell lines were generated: Ba/F3-EGFR-V769_D770insASV, Ba/F3-EGFR-D770_N771insNPG, Ba/F3-EGFR-N771_P772insH, Ba/F3-EG, Ba/F3-EGFR-H773_V774insNPH, Ba/F3-EGFR-E746_A750del, Ba/F3-EGFR-L858R, Ba/F3-EGFR-D770_N771 insSVD C797S, Ba/F3-EGFR-E746_A750del C797S, Ba/F3-EGFR-L858R C797S, Ba/F3-EGFR L861Q and Ba/F3-ERBB2-A775_G776insYVMA (Ba/F3—vector-control).
For cell survival assays, Ba/F3 cells were grown to a density of 1-2 million cells per mL, spun down and resuspended in media without IL-3, and replated at a concentration 200,000-500,000 cells per mL. The cells ectopically expressing WT EGFR plated with 10 ng/mL Millipore Culture grade EGF and Ba/F3 cells containing mutant EGFR or Mutant ERBB2 were cultivated without EGF. The cells ectopically expressing pBABEpuro empty vector were plated with 10 ng/mL IL-3.
2 days later, cells were plated in 50 μL in a 384 well plate at a concentration of 4000 cells per well for cells assayed in the absence of IL-3 and 2000 cells per well for cells assayed in the presence of IL-3. 100 nL of compound was added to each well using a 100 nL pin head, and plates were incubated at 37° C. for 48 h.
Cell viability was measured by adding 20 μL of Cell Titer-Glo Luminescent Cell Viability Reagent diluted 1:3 in PBS. Plates were sealed with Perkin Elmer Top-Seal, inverted several times to mix, and immediately centrifuged at 1000 rpm for 2 min. Plates were incubated in low light conditions for 8-10 min and luminescence was measured. The IC50 values for the examples are shown in Tables 6, 7, 8 and 9.
PC9 cells were purchased from ATCC. 400 PC9 cells per well were seeded in growth medium (DMEM, 10% FCS) in a 384-well plate (CORNING #3571). Seed reference plate for time zero determination on the same day. All plates were incubated overnight at 37° C. After 24 hours, test compound were added in 7-step dilution using HP Compound printer and incubated at 37° C. for 72 h. After 3 days, 30 μL/well CTG solution (Promega Cell Titer Glo solution; catalog #G755B and G756B) were added to each well, incubated for 30 minutes and the plate were read on PheraStar. Proliferation is calculated after subtracting time zero luminescence values from day 4 values and comparing to untreated wells. The IC50 values were determined using the four parameter fit. The IC50 values for the examples are shown in Table 9.
HCC-827 cells were purchased from ATCC. 400 HCC-829 cells per well were seeded in growth medium (RPMI1640, 10% FCS) in a 384-well plate (CORNING #3571). Seed reference plate for time zero determination on the same day. All plates were incubated overnight at 37° C. After 24 hours, test compound were added in 7-step dilution using HP Compound printer and incubated at 37° C. for 72 h. After 3 days, 30 μL/well CTG solution (Promega Cell Titer Glo solution; catalog #G755B and G756B) were added to each well, incubated for 30 minutes and the plate were read on PheraStar. Proliferation is calculated after subtracting time zero luminescence values from day 4 values and comparing to untreated wells.
The IC50 values were determined using the four parameter fit. The IC50 values for the examples are shown in Table 9.
This application is a National Phase Application of Int'l App. No. PCT/EP2020/061166, filed Apr. 22, 2020, which claims priority to and the benefit of U.S. App. No. 62/838,043, filed Apr. 24, 2019, each of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/061166 | 4/22/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62838043 | Apr 2019 | US |
