Imidazolonylquinoline compounds and their therapeutic uses

Abstract

The present invention relates to atropisomers, solid forms, salt forms, and deuterated derivatives of the ATM inhibitor 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5c]quinolin-2-one, and pharmaceutical compositions thereof. Stable atropisomers do not interconvert and are represented by the following formula:

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention provides atropisomers and deuterated derivatives of the ATM inhibitor 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(3-fluoro-5-methoxypyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5c]quinolin-2-one, as well as pharmaceutically acceptable salts and solid forms thereof. These compounds are useful in inhibiting, regulating, and / or modulating signal transduction by TM kinase. The present invention also provides compositions comprising the atropisomers, solid forms, pharmaceutically acceptable salts, and deuterated derivatives of the present invention, as well as methods of using these compositions in the treatment of various disorders related to ATM kinase, particularly cancer. [Background technology]

[0002] Background of the Invention The serine / threonine protein kinase ATM (ataxia telangiectasia mutated kinase) belongs to the PIKK family of kinases with catalytic domains that share homology with phosphoinositide-3 kinases (PI3 kinases, PI3Ks). These kinases are involved in a variety of key cellular functions, including cell growth, proliferation, migration, differentiation, survival, and cell adhesion. Specifically, these kinases respond to DNA damage by activating cell cycle arrest and the DNA repair program (DDR: DNA damage response). ATM, the product of the ATM gene, plays a key role in repairing DNA double-strand breaks (DSBs) through homologous recombination and nonhomologous end-to-end joining (NHEJ). This type of double-strand break is particularly cytotoxic.

[0003] One of the major features of human tumors is their genomic instability, which is accompanied by specific defects in DNA repair mechanisms that are not yet known in most cancers. This instability represents a therapeutic starting point for chemotherapy, which has been a priority for some time. In addition, there are several syndromes in which the underlying genetic factor is a loss of function-related mutation in a gene that regulates the response to DNA double-strand breaks. This includes ataxia telangiectasia, which is caused by a defective ATM gene. A common feature of all these syndromes is that they cause extreme radiosensitivity (Lavin & Shiloh (1997) Annu. Rev. Immunol. 15: 177; Rotman & Shiloh (1998) Hum. Mol. Genet. 7: 1555, incorporated herein by reference in their entirety). Thus, ATM-deficient cells are sensitive to agents and other means that cause damage to the DNA double helix, making ATM an attractive target for chemo- and radiosensitivity in cancer treatment.

[0004] In summary, ATM (ataxia telangiectasia mutated kinase) is a key regulator of DNA double-strand break repair, which is induced by widely used radiation and chemotherapy. ATM relays a wide range of signals to a multitude of downstream effectors, including p53. Unrepaired double-strand breaks lead to activation of checkpoint responses, cell cycle arrest, and ultimately tumor cell death. Thus, ATM has become an attractive intervention site to inhibit repair of induced double-strand breaks.

[0005] The compound wortmannin was one of the first to be investigated in this context and showed radiosensitizing effects, which could be attributed to the inhibition of ATM. However, due to its in vivo toxicity, it was not suitable for therapeutic use. Starting from the chemical structure of the PI3K inhibitor LY294002, KuDOS Pharmaceuticals identified an ATM inhibitor: Ku-55933 (2-morpholino-6-(thianthren-1-yl)-4H-pyran-4-one). This compound achieved sensitivity to ionizing radiation and DNA double-strand damage chemotherapy agents (Hickson, I., et al. (2004), Cancer Res 64, 9152-9159, the entire contents of which are incorporated herein by reference). However, Ku-55933 proved unsuitable for in vivo use, likely due to its high lipophilicity. Ku-60019 (2-((2S,6R)-2,6-dimethylmorpholino)-N-(5-(6-morpholino-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-2-yl)-acetamide) and Ku-559403 (2-(4-methylpiperazin-1-yl)N-acetamide [5-thioxanthen-(6-morpholino-4-oxopyran-2-yl)2-yl]) were subsequently developed, and Ku-559403 was hailed as promising enough to enter clinical trials for the treatment of advanced solid tumors.

[0006] There are further ATM inhibitors that support the above notion in that they are currently in clinical development, including clinical trials involving combination with radiation therapy, such as AZD0156, AZD1390, and M3541.

[0007] While much progress has been made in the field of ATM inhibitors, there remains a need to provide compounds that not only have high inhibitory potency of ATM kinase, but also advantageous selectivity over other kinases, advantageous bioavailability, and / or reduced off-target effects. Summary of the Invention

[0008] It is desirable to provide small molecules that effectively inhibit, regulate, and / or modulate signal transduction by ATM kinase, and this is one object of the present invention. It is further desirable to provide ATM inhibitors that are selective, i.e., have no or significantly less activity against other kinases. It is further desirable to provide ATM inhibitors that exhibit advantageous properties with respect to targets known to cause undesirable side effects. One object, therefore, is to provide compounds that reduce off-target effects and / or associated toxicity. It is still further an object of the present invention to provide ATM inhibitors with good bioavailability. It is a further or alternative object of the present invention to provide ATM inhibitors with advantageous solid-state properties, such as advantageously low hygroscopicity and / or other physical properties.

[0009] The above outlined and at least one of the further objects have been solved by atropisomers and deuterated derivative solid forms of the ATM inhibitor 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(3-fluoro-5-methoxypyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5c]quinolin-2-one (compound Y), as well as pharmaceutically acceptable salts and compositions thereof.

[0010] One aspect of the present invention provides two compounds, which are atropisomers of compound Y and are represented by the following formula: [ka] and pharmaceutically acceptable salts thereof.

[0011] Another aspect of the present invention provides a solid form of Compound 1: [ka]

[0012] Another aspect relates to certain particularly advantageous pharmaceutically acceptable salts of Compound 1, particularly Compound 1 fumarate, Compound 1 edisylate, and Compound 1 napsylate, which may hereinafter be collectively referred to as "Compound 1-a." Another aspect of the present invention provides deuterated compounds 3, 4, and 5, which are represented by the following formulas:

[0013] [ka] or an atropisomer or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 depicts the annotated 1H NMR spectrum of compound 1. [Figure 2] FIG. 2 depicts the annotated 13C NMR spectrum of compound 1. [Figure 3] FIG. 3 depicts the annotated 19F NMR spectrum of compound 1. [Figure 4] FIG. 4 depicts the UV-Vis spectrum of compound 1 in methanol. [Figure 5] FIG. 5 depicts the HPLC chromatograms of compounds 1 and 2. [Figure 6] FIG. 6 depicts a flow chart for the preparation of Compound 1. [Figure 7a] FIG. 7 depicts the crystal structure of the compound 1-dibenzoyl-D-tartrate (A) and its XRPD (B). [Figure 7b] FIG. 7 depicts the crystal structure of the compound 1-dibenzoyl-D-tartrate (A) and its XRPD (B). [Figure 8] FIG. 8 depicts the crystal structure of the compound 2-dibenzoyl-L-tartrate. [Figure 9] FIG. 9 depicts a chart of the non-sink dissolution behavior of Compound 1 and certain of its salts in FaSSIF. [Figure 10]FIG. 10 depicts the X-ray powder diffraction (XRPD) pattern of the solid form of Compound 1 fumarate. [Figure 11] FIG. 11 depicts the X-ray powder diffraction (XRPD) pattern of the solid form of Compound 1 napsylate. [Figure 12] FIG. 12 depicts the X-ray powder diffraction (XRPD) pattern of the solid form of the edisylate of Compound 1. [Figure 13] FIG. 13 depicts the X-ray powder diffraction (XRPD) pattern of solid form A2 of Compound 1. [Figure 14] FIG. 14 depicts the X-ray powder diffraction (XRPD) pattern of solid form A1 of Compound 1. [Figure 15] FIG. 15 depicts the X-ray powder diffraction (XRPD) pattern of solid form A3 of Compound 1. [Figure 16] FIG. 16 depicts the X-ray powder diffraction (XRPD) pattern of solid form NF9 of Compound 1. [Figure 17] FIG. 17 depicts the X-ray powder diffraction (XRPD) pattern of the hydrate solid form H1 of Compound 1. [Figure 18] FIG. 18 depicts the X-ray powder diffraction (XRPD) pattern of the hydrate solid form H2 of Compound 1. [Figure 19] FIG. 19 depicts the X-ray powder diffraction (XRPD) pattern of the solid form of Compound 1, NF19. [Figure 20] FIG. 20 shows the strong tumor growth inhibition induced by irradiation (IR) and concomitant administration of oral Compound 1 (6×5 days, 2 Gy; FaDu SCCHN tumor model). [Figure 21] FIG. 21 shows the results of an in vivo evaluation of the antitumor activity of Compound 1 and a comparative ATM inhibitor in combination with olaparib in the HBCx-10 patient-derived triple-negative breast cancer xenograft model. [Figure 22] FIG. 22 shows the DSC heating curve of Compound 1, Form A2. [Figure 23] FIG. 23 shows the TGA heating curve of Compound 1 Form A2. [Figure 24]FIG. 24 shows the DVS water uptake isotherm of Compound 1, Form A2 (25° C.). [Figure 25] FIG. 25 shows the DSC heating curve of Compound 1 Form A1. [Figure 26] FIG. 26 depicts the TGA heating curve of Compound 1 Form A1. [Figure 27] FIG. 27 shows the DVS water uptake isotherm of Compound 1, Form A3 (25° C.). [Figure 28] FIG. 28 depicts the DSC heating curve of Form H2 of Compound 1 (hydrate). [Figure 29] FIG. 29 shows the TGA heating curve of Form H2 of Compound 1 (hydrate). [Figure 30] FIG. 30 shows the DVS water uptake isotherm (25° C.) of Form H2 of Compound 1 (hydrate). [Figure 31] FIG. 31 depicts the DSC heating curve of Compound 1 fumarate (form NF6). [Figure 32] FIG. 32 shows the TGA heating curve of Compound 1 fumarate (form NF6). [Figure 33] FIG. 33 shows the DVS water uptake isotherm (25° C.) of Compound 1 fumarate (Form NF6). [Figure 34] FIG. 34 depicts the DSC heating curve of compound one, napsylate (NF7). [Figure 35] FIG. 35 shows the TGA heating curve of Compound 1 napsylate (NF7). [Figure 36] FIG. 36 depicts the DVS water uptake isotherm (25° C.) of Compound 1 napsylate (NF7). [Figure 37] FIG. 37 depicts the DSC heating curve of the edisylate of Compound 1 (NF8). [Figure 38] FIG. 38 shows the TGA heating curve of the edisylate of Compound 1 (NF8). [Figure 39] FIG. 39 depicts the DVS water uptake isotherm (25° C.) of Compound 1 edisylate (NF8). [Figure 40]FIG. 40 shows the XRPD of the methanolate of Compound 1 in solid form S1. [Figure 41] FIG. 41 depicts the XRPD of the mixed hydrate / methanolate of Compound 1 in solid form S2. [Figure 42] FIG. 42 shows the XRPD of the THF solvate of Compound 1 in solid form S3. [Figure 43] FIG. 43 depicts the XRPD of the dioxane solvate of Compound 1 in solid form NF11. [Figure 44] FIG. 44 shows the XRPD of the chloroform solvate of Compound 1 in solid form NF15. [Figure 45] FIG. 45 depicts the XRPD of the acetic acid solvate of Compound 1 in solid form NF16. [Figure 46] FIG. 46 shows the XRPD of the acetic acid solvate of Compound 1 in solid form NF18. [Figure 47] FIG. 47 depicts the XRPD of the 1,4-dioxane solvate of Compound 1 in the solid form NF29. [Figure 48] FIG. 48 shows the XRPD of the dichloromethane solvate of Compound 1 in the solid form NF32. [Figure 49] FIG. 49 depicts the XRPD of the NMP (N-methyl-2-pyrrolidone) solvate of Compound 1 in the solid form NF33. [Figure 50] FIG. 50 shows the XRPD of the acetonitrile solvate of Compound 1 in the solid form NF35. [Figure 51] FIG. 51 depicts the XRPD of the 1,4-dioxane solvate of Compound 1 in the solid form NF36. [Figure 52] FIG. 52 shows the XRPD of the dimethylacetamide solvate of Compound 1 in the solid form NF37.

[0015] Detailed Description of the Invention International Patent Application WO2016 / 155844, the entirety of which is hereby incorporated by reference, describes imidazolonylquinoline compounds that effectively inhibit, regulate, and / or modulate signaling by ATM kinase. Such compounds include compound Y:

[0016] [ka]

[0017] Compound Y is active in a variety of assays and therapeutic models as defined in Example 4 of WO2016 / 155844, demonstrating selective inhibition of ATM kinase over PI3Kalpha, PI3Kbeta, PI3Kdelta, PI3Kgamma, and mTOR (in enzymatic and cellular assays). The terminology used herein for the definition of compounds is based on the rules of the IUPAC organization for chemical compounds in general and organic compounds in particular.

[0018] Surprisingly, it has now been found that compound Y exists in two atropisomer forms, which can be isolated and are advantageously stable, and that said atropisomers exhibit surprising and highly desirable properties.

[0019] According to one aspect, the present invention provides the following two compounds, which are atropisomers of compound Y: 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (compound 1) and

[0020] 8-(1,3-Dimethyl-1H-pyrazol-4-yl)-1-(Ra)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (Compound 2), as well as pharmaceutically acceptable salts thereof.

[0021] Compounds 1 and 2 are represented by the following formula: [ka] Here, the bold and dashed lines in compounds 1 and 2 represent partial rotation of the pyridine ring out of the plane in which the tricyclic ring lies.

[0022] Those skilled in the art will recognize that the term "atropisomer," as used herein, refers to a stereoisomer resulting from restricted rotation around a single bond that constitutes a chiral axis. It will further be recognized that the rotational barrier around the single bond must be sufficiently high to allow isolation of a single atropisomer. The rotational barrier can result, for example, from steric interactions with other residues of the same molecule, thereby restricting the rotation around the single bond. Stereochemical and electronic factors come into play and may reinforce or oppose each other.

[0023] The use of chiral compounds containing asymmetric carbon atoms is well established in principle in drug discovery.In particular, it is known in the art that a racemic mixture of two chiral compounds usually consists of one more active and one less active enantiomer compared to the racemic mixture.Therefore, the use of only one of the two enantiomers can be advantageous for improving the overall efficacy of the compound.

[0024] However, atropisomers are stereoisomers that arise solely due to rotational hindrance around a single bond, and their use is generally considered undesirable. Because the stability of these isomers depends on the energy difference resulting from steric distortion or other factors that create a barrier to rotation around the single bond, atropisomers are generally considered an obstacle in drug discovery. In contrast to chiral compounds resulting from asymmetric carbon atoms, atropisomeric states cannot be easily predicted. It is generally not possible to easily predict the stability of atropisomers. The height of the energy barrier determines the time required for the interconversion of two corresponding atropisomers. The interconversion of a biologically active atropisomer into its corresponding counterpart can thus reduce its biological activity and introduce off-target or other unwanted effects. Therefore, only stable atropisomers possessing a sufficiently high energy barrier may be suitable for drug discovery.

[0025] Surprisingly, it was found that the atropisomers Compound 1 and Compound 2 do not significantly interconvert to their respective other atropisomers, even after extensive periods of more than 10 years (rotational half-lives of >10 years were determined by computer simulation) and even at temperatures above room temperature.

[0026] The inversion temperatures of the atropisomers have been estimated to be greater than 100°C in solution. This very good stability has been experimentally confirmed, making them suitable for easy pharmaceutical application, manufacturing, and formulation, and providing sufficient shelf life.

[0027] The absolute structure of Compound 1 has been determined based on the (2S,3S)-dibenzoyl-D-tartrate salt and from X-ray diffraction of solid form A2, which will be described in more detail below. The structure of Compound 1 was confirmed by the results of spectroscopy (NMR, MS, IR, and UV), X-ray diffraction, elemental analysis, and polarimetry. The H-C and F-NMR spectra of Compound 1 are shown in Figures 1-3, and the UV-Vis spectrum is shown in Figure 4. The XRPD of solid form A2 of Compound 1 is illustrated in Figure 13. The crystal structure and XRPD of Compound-1-dibenzoyl-D-tartrate are shown in Figures 7A and 7B, and the crystal structure of Compound-2-dibenzoyl-L-tartrate is illustrated in Figure 8.

[0028] Compounds 1 and 2 are highly potent inhibitors of ATM kinase. As illustrated by Table 1, Compound 1 has clearly superior values ​​for ATM inhibition in all assays compared to Compound Y, which is a mixture of Compounds 1 and 2:

[0029] [Table 1]

[0030] Furthermore, the compounds are selective even over related kinases, including mTOR (>30,000 nm), DNA-PK, and, most notably, ATR. Surprisingly, both compounds 1 and 2 are less potent inhibitors of ATR kinase than compound Y, i.e., more advantageous from a selectivity standpoint.

[0031] [Table 2]

[0032] While compound 2 is not different from compound Y as far as ATM inhibition is concerned (see Table 1), it has significantly better selectivity than compound Y across both ATR and DNA-PK, as is evident from Table 2 above.

[0033] The compounds of the present invention can therefore be used to particular advantage to specifically target DNA double-strand breaks and selectively address certain DNA repair mechanisms, especially homologous recombination.

[0034] A further advantage of the selective ATM inhibitors Compounds 1 and 2 is their reduced toxicity, particularly with regard to off-target effects, and thus the tolerability of higher compound dosages. Thus, the compounds according to the present invention open up new possibilities in cancer therapy, for example, Compounds 1 and 2, most preferably Compound 1, may be used in targeted combination therapy, for example, with a potent and selective ATM inhibitor and another potent and selective inhibitor (e.g., an ATR inhibitor).

[0035] In general, compound 1 was found to have the most advantageous overall combination of properties.Surprisingly, it not only has the best ATM inhibitory properties, but also the best microsomal clearance value and the lowest phosphodiesterase (PDE) 2A1, PDE4A1A and PDE4D2 inhibitory properties.Phosphodiesterase inhibition itself is associated with various pharmacological effects, and PDE inhibitors are available as medicines for the treatment of various conditions, including depression, multiple sclerosis and chronic obstructive pulmonary disease, to name just a few, all of which constitute off-target effects in this case.It is also known that PDE4 inhibition is associated with the risk of inducing nausea and should therefore be avoided.Therefore, a high IC 50 As is evident from Table 3 below, Compound 1 has an IC value for PDE4 inhibition that is about a factor of 5-fold higher than that of Compound Y. 50 It has a value.

[0036] [Table 3]

[0037] Table 4 illustrates further advantageous parameters of Compound 1, including a favorably high bioavailability of approximately 80% and favorable properties with respect to CYP and hERG (cardiac ion channels), the latter indicating that no safety-related interactions with the cardiac Kv11.1 hERG ion channel are expected.

[0038] [Table 4]

[0039] Surprisingly, it was further found that both Compounds 1 and 2 exhibit significantly improved solubility in biological buffers (see Table 5 below) compared to Compound Y. The predicted slow human plasma clearance and high bioavailability of Compound 1 contribute to the appropriately low dose requirement.

[0040] [Table 5]

[0041] The structures depicted for compounds 1 or 2 are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, H, C, and N also include in each case the heavier isotopes of these atoms. This applies especially to H, where deuterium or tritium can be advantageously used, but the replacement of carbon by a C- or C-enriched carbon is also within the scope of the present invention. In certain preferred embodiments, isotopically enriched atoms are not used, and instead atoms are used in their naturally occurring form with respect to isotope distribution.

[0042] Reference to the compounds or salts according to the present invention will be considered to include their solvated forms, i.e., their solvates, which mean both solvates in free form or salts. Solvates are taken to mean the addition products of inert solvent molecules on the compounds that form due to their mutual attractive force. Solvates are, for example, mono- or dihydrates or alcoholates. Exemplary embodiments of solvates are disclosed in more detail below.

[0043] As presented above, the present invention provides two stable atropisomers, Compounds 1 and 2. Preparation of these two atropisomers is typically based on separation and purification techniques, as described in detail below. Those skilled in the art will recognize that this may not produce a completely pure product. However, the present invention provides Compound 1 that is substantially free of Compound 2 and Compound 2 that is substantially free of Compound 1. "Substantially free" in this context preferably means that substantially pure Compound 1 may contain at most 20% by weight, preferably at most 15% by weight, more preferably at most 10% by weight, e.g., at most 5% by weight, at most 2.5% by weight, at most 1% by weight, at most 0.5% by weight, or at most 0.1% by weight of Compound 2 or a salt thereof, with the remainder to 100% being made up of Compound 1.

[0044] In one example, substantially pure Compound 1 may consist of 99% by weight of Compound 1 and 1% by weight of Compound 2. The reverse is also true for Compound 2, i.e., Compound 2 substantially free of Compound 1, which would preferably mean that pure Compound 2 may contain at most 20% by weight of Compound 1 or a salt thereof, with the preferred ranges disclosed for Compound 1 being equally applicable by analogy (and vice versa). References to Compound 1 or a salt thereof, respectively Compound 2 or a salt thereof, also include all solvates and solid forms, such as those disclosed herein below, even if not specifically mentioned or otherwise explicitly described.

[0045] In other embodiments, the present invention provides Compound 1 or 2 that is substantially free of impurities. As used herein, the term "substantially free of impurities" means that the compound will not contain any significant amount of foreign matter. Such foreign matter may include residual Compound 1 or a salt thereof, residual Compound 2 or a salt thereof, residual solvent, or any other impurities that may result from the preparation and / or isolation of Compound 1 or 2. In certain embodiments, Compound 1 may contain no more than 30% by weight of foreign matter, the remainder of 100% by weight being constituted by Compound 1, preferably no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 7.5%, no more than 5%, no more than 1%, no more than 0.5%, or no more than 0.1% by weight.

[0046] The same exemplary embodiments are valid, by analogy, for compound 2. Also, as before, reference to compound 1 or a salt thereof, respectively compound 2 or a salt thereof, will further include all solvates and solid forms such as those disclosed herein below, unless expressly stated otherwise.

[0047] According to another embodiment, Compound 1 or 2, respectively, contains no more than about 5.0 area percent HPLC of total organic impurities, and in certain embodiments, no more than about 3.0 area percent HPLC of total organic impurities, and in certain embodiments, no more than 1.5 area percent HPLC of total organic impurities, relative to the total area of ​​the HPLC chromatogram. In other embodiments, Compound 1 or 2 contains no more than about 1.0 area percent HPLC of any single impurity; no more than about 0.6 area percent HPLC of any single impurity, and in certain embodiments, no more than about 0.5 area percent HPLC of any single impurity, relative to the total area of ​​the HPLC chromatogram. By way of illustration, the method described in Example 3.3 for analyzing the purity of each atropisomeric impurity for HPLC chromatograms may be used. Again, reference to Compound 1 or a salt thereof, respectively Compound 2 or a salt thereof, will include all solvates and solid forms, such as those disclosed further below, unless otherwise expressly stated.

[0048] According to another embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof. In an alternative embodiment, the pharmaceutical composition comprises an effective amount of Compound 2 or a pharmaceutically acceptable salt thereof. According to another embodiment, the present invention provides a method for preparing such a composition as described herein (e.g., a composition that can include an effective amount of either Compound 1 or 2). Reference to Compound 1 or 2 will be read in this context as including any amount of the respective other atropisomer, such that the respective other atropisomer is added to the amount of the aforementioned compound, i.e., rather than being present separately, in accordance with the respective definitions of "substantially free," "substantially free of impurities," or the total amount of impurities. The same applies to the respective salts. As previously stated, reference to Compound 1 or 2 or a salt thereof will include all solvates and solid forms, such as those disclosed further below, unless otherwise expressly stated. In an exemplary embodiment, Compound 1 is contained in the pharmaceutical composition in its free, i.e., non-salt, form.

[0049] Yet another aspect provides a method of treating cancer using a pharmaceutical composition of the compound or composition, respectively, or a pharmaceutically acceptable salt thereof, as described herein. According to another aspect, the present invention provides the use of a compound, a pharmaceutically acceptable salt thereof, or a (pharmaceutical) composition according to the present invention in the manufacture of a medicament for treating cancer. In another aspect, the present invention provides a compound or pharmaceutical composition as described herein for use as a medicament, particularly for the treatment of cancer. Again, reference to a compound or salt thereof will include all solvates or solid forms of those compounds or salts, such as those disclosed further herein below, even if not expressly stated otherwise.

[0050] Free form and salts The compounds according to the present invention can be used in their free form, i.e., as shown in the formula above. Illustratively, the free form of Compound 1 is found to be particularly useful, and exemplary solid forms thereof, including preferred solid forms, will be described in more detail below.

[0051] On the other hand, the present invention encompasses the use of these compounds in the form of their pharmaceutically acceptable salts, which can be derived from various organic and inorganic acids by procedures known in the art. Suitable pharmaceutically acceptable salts of the compounds according to the present invention can be prepared by conventional methods. The compounds according to the present invention can be converted to the relevant acid addition salts using an acid by reacting the compound and an equivalent or excess amount of acid in a suitable solvent, such as THF or acetone, followed by cooling and crystallizing the saturated solution thus formed. Alternatively, anti-solvent crystallization or evaporative crystallization can be used.

[0052] Examples of suitable pharmaceutically acceptable salts of compounds according to the invention, especially Compound 1, include the HCl salt, sulfate salt, tosylate salt, besylate salt, lactate salt, especially the L-lactate salt. Preferred salts of Compound 1 include the fumarate, napsylate, and edisylate salts, which may be collectively referred to below as Compound 1a.

[0053] The napsylate of Compound 1 can be prepared from Compound 1 using naphthalenesulfonic acid and either THF or acetone as the solvent. Good crystallinity was obtained. The preferred ratio of Compound 1:napsylate is about 1:1.

[0054] The napsylate of Compound 1 can be obtained by cooling crystallization from acetone using ethanedisulfonic acid. The preferred ratio of Compound 1 to napsylate is about 1:1. Good crystallinity was obtained.

[0055] The fumarate of Compound 1 can be obtained by anti-solvent vapor diffusion in THF using n-pentane as the anti-solvent and fumaric acid as the acid. The ratio of Compound 1 to fumarate was shown to be approximately 1:0.9. The resulting salt has very favorable overall physical properties and good crystallinity. The fumarate salt is a preferred embodiment among the salts, in part due to the absence of desirable hygroscopic properties. Detailed examples of suitable methods for preparing the fumarate, napsylate, and edisylate salts of Compound 1 according to the present invention are disclosed in Example 5.

[0056] These salts of Compound 1 were found to exhibit faster initial dissolution rates than the parent Compound 1. The non-sink dissolution behavior of Compound 1 and certain of its salts in FaSSIF buffer solution (pH 6.5) is illustratively depicted in Figure 9.

[0057] Salts with favorable dissolution behavior are Compound 1 edisylate, Compound 1 fumarate, and Compound 1 napsylate. Parent Compound 1 was used in the form of a mixture of various crystal and solvate forms. Those skilled in the art will recognize that the anionic moiety from the acid and Compound 1 is ionically bound to form Compound 1-a. It is contemplated that Compound 1-a can exist in various physical forms. For example, Compound 1-a can be in solution, suspension, or solid form. In some embodiments, Compound 1-a is in solid form. When Compound 1-a is in solid form, the compound may be amorphous, crystalline, or a mixture thereof. As used herein, the term "polymorph" refers to the various crystal structures into which a compound or its salt can crystallize.

[0058] In some embodiments, compound 1-a is a crystalline solid that is substantially free of amorphous compound 1-a. As used herein, the term "substantially free of amorphous compound 1-a" means that the salt does not contain a significant amount of amorphous compound 1-a. In some embodiments, at least about 90% by weight of crystalline compound 1-a is present, or at least about 95% by weight of crystalline compound 1-a is present. Still in other embodiments of the present invention, at least about 99% by weight of crystalline compound 1-a is present. These percentages are relative to the absolute weight of compound 1-a (100% by weight).

[0059] In an exemplary embodiment, the invention provides a solid form of fumarate of Compound 1, characterized by an X-ray powder diffraction pattern substantially in accordance with that depicted in FIG. 10, and / or characterized by one or more peaks in its X-ray powder diffraction pattern selected from the following:

[0060] [Table 6]

[0061] The fumarate salt of Compound 1 is anhydrous. This solid form may be referred to as fumarate-NF6. The DSC heating curve, TGA heating curve, and DVS water uptake isotherm (25°C) of fumarate-NF6 are depicted in Figures 31, 32, and 33. The results from non-sink dissolution measurements are provided in the table below (non-sink dissolution data in FaSSIF at pH 6.5, method described in the experimental section):

[0062] [Table 7]

[0063] As used herein, the term "about," when used in reference to a 2-theta value, refers to the stated value ±0.3 degrees two-theta (°2θ). In some embodiments, "about" refers to ±0.2 degrees two-theta or ±0.1 degrees two-theta, most preferably ±0.2 degrees two-theta.

[0064] Polymorphs of any solid form described herein may be characterized by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more XRD or XRPD peaks (°2θ), respectively. Polymorphs of any solid form described herein are preferably characterized by at least 6 XRD peaks (°2θ, preferably ±0.2°2θ), respectively. Preferred peaks for characterizing polymorphs of solid forms are indicated by bold type and an asterisk, respectively, in the respective peak lists.

[0065] In a further exemplary embodiment, the present invention provides a solid form of the napsylate of Compound 1, characterized by an X-ray powder diffraction pattern substantially in accordance with that depicted in FIG. 11, and / or characterized by one or more peaks in that X-ray powder diffraction pattern selected from the following:

[0066] [Table 8]

[0067] The thermal and water sorption properties of the napsylate salt of Compound 1 are illustrated in Figures 34, 35, and 36. The resulting solid form of the napsylate salt may be referred to as Napsylate NF7. Furthermore, the dissolution behavior is represented by the following experimental non-sink dissolution data (FaSSIF, pH 6.5):

[0068] [Table 9]

[0069] In a further exemplary embodiment, the present invention provides a solid form of the edisylate of Compound 1, characterized by an X-ray powder diffraction pattern substantially in accordance with that depicted in FIG. 12, and / or characterized by one or more peaks in that X-ray powder diffraction pattern selected from the following:

[0070] [Table 10] The heat resistance and dissolution data of the edisylate salt of Compound 1 are illustrated by Figures 37, 38, and 39. The solid form of the edisylate salt may be referred to as NF8. It is the anhydrous form / salt. The non-sink dissolution data (FaSSIF, pH 6.5) is provided in the table below:

[0071] [Table 11]

[0072] Consistent with what has been set forth above with respect to Compounds 1 and 2, the present invention provides Compound 1-a, or other salts of Compound 1, that are substantially free of Compound 2 or salts thereof. In a further embodiment, Compound 1-a or other salts of Compound 1 are provided substantially free of impurities. According to another embodiment, Compound 1-a or any other salt of Compound 1 contains no more than 5.0 area percent HPLC of total organic impurities compared to the total area of ​​the HPLC chromatogram. The exemplary and preferred ranges disclosed above for Compound 1 with respect to "substantially free of Compound 2 or salts thereof," "free of impurities," and area percent of total organic impurities are equally applicable here. By analogy, the same applies to any compound according to the present invention, and in particular any salt of the atropisomeric compound.

[0073] According to another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of compound 1-a. According to another aspect, the present invention provides a method for preparing such a composition described herein (e.g., a composition that can include an effective amount of compound 1-a). Yet another aspect provides a method for treating cancer using such a composition of compound 1-a, respectively, according to the present invention. According to another aspect, the present invention provides the use of a composition described herein in the manufacture of a medicament for treating cancer. Compound 1-a may be present in the (pharmaceutical) composition in the same amount as disclosed for compound 1. With regard to the presence of other atropisomers or salts thereof in the composition, respectively, the considerations listed above for compound 1 apply equally by analogy. Solid Forms and Solvates According to another aspect, the present invention provides solid forms of Compound 1 or 2, particularly Compound 1.

[0074] It will be appreciated by those skilled in the art that compounds according to the present invention can exist in various physical forms, for example, they can be in solution, suspension, or solid form.

[0075] In certain preferred embodiments, Compound 1 is in a solid form. Solid forms are generally preferred herein because they allow for the provision of solid pharmaceutical compositions. When Compound 1 is in a solid form, the compound may be amorphous, crystalline, or a mixture thereof. Exemplary solid forms of Compound 1 are described in more detail below.

[0076] According to one embodiment, the present invention provides Compound 1 as an amorphous solid. Amorphous solids are well known to those skilled in the art and are typically prepared by methods such as freeze drying, spray drying or impact precipitation. In other embodiments, Compound 1 is a crystalline solid. As used herein, the term "polymorph" refers to the different crystalline structures in which a compound can crystallize.

[0077] In some embodiments, Compound 1 is a crystalline solid that is substantially free of amorphous Compound 1. As used herein, the term "substantially free of amorphous Compound 1" means that the compound does not contain a significant amount of amorphous Compound 1. In certain embodiments, at least about 90% by weight of crystalline Compound 1 is present, or at least about 95% by weight of crystalline Compound 1 is present. In yet other embodiments of the invention, at least about 99% by weight of crystalline Compound 1 is present. These percentages relate to the absolute weight (100% by weight) of Compound 1. The same applies mutatis mutandis to the allowable amorphous content in any crystalline form of each and every polymorph of the compounds disclosed herein, including those described herein for salts, anhydrous forms, solvates, and other forms.

[0078] According to one aspect, the present invention provides a solid form of Compound 1, which is a solid form of anhydrous Compound 1, preferably crystalline anhydrous Compound 1. Five different polymorphic forms of anhydrous Compound 1 are described herein. In the context of the specific solid forms described in this section relating to anhydrous forms and solvates, reference to Compound 1 will be understood as a reference to the compound itself, i.e., its free (non-salt) form.

[0079] The first anhydrous crystalline form of Compound 1, hereinafter referred to as "Form A2," is a polymorph characterized by an X-ray powder diffraction (XRPD) pattern substantially in accordance with that depicted in FIG. 13 and has been found to be highly advantageous.

[0080] According to one embodiment, Form A2 is characterized by one or more peaks in its X-ray powder diffraction pattern selected from those at about 7.3, about 9.6, about 11.1, about 12.0, about 12.7, and about 16.2 degrees 2-theta. In some embodiments, Form A2 is characterized by two or more peaks in its X-ray powder diffraction pattern selected from those at about 7.3, about 9.6, about 12.7, about 16.2, about 22.6, and about 25.1 degrees 2-theta. In some embodiments, Form A2 is characterized by three or more peaks in its X-ray powder diffraction pattern selected from those at about 7.3, about 9.6, about 12.7, about 16.2, about 22.6, and about 25.1 degrees 2-theta. In some embodiments, Form A2 is characterized by four, five, or substantially all of the peaks in its X-ray powder diffraction pattern selected from those at about 7.3, about 9.6, about 12.7, about 16.2, about 22.6, and about 25.1 degrees two-theta. In certain embodiments, Form A2 is characterized by six or more, or substantially all, of the peaks in its X-ray powder diffraction pattern selected from those at about 7.3, 9.6, 11.1, 12.0, 12.7, 14.7, 16.2, 17.3, 18.9, 21.0, 22.6, and 25.1 degrees two-theta.

[0081] In exemplary embodiments, Form A2 may be characterized by one or more, preferably six, and up to substantially all, of the peaks in its X-ray powder diffraction (XRPD) pattern selected from those for:

[0082] [Table 12]

[0083] It will be appreciated that the above polymorphic forms can be characterized, for example, by reference to any of the peaks in their respective X-ray diffraction (XRD) patterns. As previously noted, bold type and asterisks designate peaks that may be preferred for characterizing the polymorphs.

[0084] Form A2 may be characterized by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the XRPD peaks (°2θ) in the table above. Any polymorph described herein is preferably characterized by at least 6 XRD or XRPD peaks (°2θ, preferably ±0.2).

[0085] Form A2 may optionally be characterized in that it has a monoclinic crystal system and a P21 space group. Form A2 may be further characterized by one or more of the following parameters of its unit cell, as listed in the table below:

[0086] [Table 13]

[0087] Form A2 of Compound 1 has favorable overall properties, as further evidenced by its thermal and water uptake behavior, as illustrated by the DSC heating curve in (Figure 22), the TGA heating curve in (Figure 23), and the DVS water uptake isotherm (at 25°C) in (Figure 24). Form A2, for example, adsorbs very little water (<2%) even up to 100% relative humidity, and is thus superior to Form A3.

[0088] Another advantage of Form A2 is its favorable dissolution behavior, as illustrated in the following table, which shows the amount of Compound 1 in Form A2 dissolved in various time periods (non-sink dissolution data of FaSSIF at pH 6.5, method described in the experimental part):

[0089] [Table 14]

[0090] Form A2 can be obtained from Compound 1, for example, by cooling crystallization from alcohol. Suitable methods and reaction conditions are described in detail in Example 7. By way of illustration, crystals of Form A2 with favorable properties can be reproducibly prepared by a controlled crystallization process that includes: a) preparing a dispersion of Compound 1, e.g., a hydrate of Compound 1, such as hydrate form H2 of Compound 1, in a suitable solvent, e.g., an alcohol; b) heating the dispersion to obtain a solution, preferably a clear solution; c) controlling cooling of said solution; d) seeding with Form A2 crystals; e) Controlled cooling of the solution with the seed crystals, illustratively at a rate of about 0.1° C. / min.

[0091] Crystallization conditions, such as suitable alcohols and temperatures, can be derived from Examples 7.1-7.3, which are applicable across specific embodiments. The present invention further relates to anhydrous, crystalline Compound 1 in Form A2, which can be obtained substantially in accordance with any of the processes above or described in Examples 7.1-7.4.

[0092] An additional anhydrous crystalline form of Compound 1 is hereinafter referred to as "Form A1" and is a polymorph characterized by a powder X-ray diffraction pattern substantially in accordance with that depicted in Figure 14. A suitable method for its preparation is described in Example 7.

[0093] Form A1 may be characterized by one or more, preferably six, and up to substantially all, of the peaks in its X-ray powder diffraction pattern selected from those for:

[0094] [Table 15] The thermal properties of Compound 1 Form A1 were evaluated by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), as illustrated in FIGS.

[0095] A third anhydrous crystalline form of Compound 1 is hereinafter referred to as "Form A3" and is a polymorph characterized by a powder X-ray diffraction pattern substantially in accordance with that depicted in Figure 15. A suitable method for its preparation is described in Example 7.

[0096] Form A3 may be characterized by one or more, preferably six, and up to substantially all, of the peaks in its X-ray powder diffraction pattern selected from those for:

[0097] [Table 16]

[0098] As is evident from Figure 27, Form A3 of Compound 1 exhibits very low water adsorption up to about 70% relative humidity. Form A3 may optionally be further characterized by crystal system and unit cell parameters, as listed in Table 7 below. Non-sink dissolution data for Compound 1 in Form A3 in FaSSIF at pH 6.5 is given in the table below:

[0099] [Table 17]

[0100] A fourth anhydrous crystalline form of Compound 1 is hereinafter referred to as "Form NF9" and is a polymorph characterized by a powder X-ray diffraction pattern substantially in accordance with that depicted in Figure 16. A suitable method for its preparation is described in Example 7.

[0101] Form NF9 may be characterized by one or more, preferably six, and up to substantially all, of the peaks in its X-ray powder diffraction pattern selected from those for:

[0102] [Table 18]

[0103] In a further aspect, the present invention provides a hydrate of Compound 1, preferably a solid form of a hydrate of Compound 1, preferably a crystalline hydrate of Compound 1. Two different hydrates, each a polymorphic form of a hydrate of Compound 1, are described herein. As noted above, in the context of these specific solid forms, reference to Compound 1 will be understood as a reference to the compound itself, i.e., its free (non-salt) form.

[0104] A first crystalline form of the hydrate of Compound 1 is hereinafter referred to as "Form H1" and is a polymorph characterized by a powder X-ray diffraction pattern substantially in accordance with that depicted in Figure 17. A suitable method for its preparation is described in Example 7.

[0105] Form H1 may be characterized by one or more, preferably six, and up to substantially all, of the peaks in its X-ray powder diffraction pattern selected from those for:

[0106] [Table 19]

[0107] A second crystalline form of the hydrate of Compound 1 is hereinafter referred to as "Form H2" and is a polymorph characterized by a powder X-ray diffraction pattern substantially in accordance with that depicted in Figure 18. A suitable method for its preparation is described in Example 7.

[0108] Form H2 may be characterized by one or more, preferably six, and up to substantially all, of the peaks in its X-ray powder diffraction pattern selected from those for:

[0109] [Table 20]

[0110] The hydrate of Compound 1 in crystalline form H2 may optionally be characterized in that it has a triclinic crystal system and a P1 space group. Form H2 may be characterized by one or more of the following parameters of its unit cell, as listed in Table 7 below:

[0111] The thermal and water sorption properties of Form H2 are illustrated by Figures 28, 29, and 30. The non-sink dissolution data in FaSSIF (ph 6.5) for Form H2 of Compound 1 was determined as follows:

[0112] [Table 21]

[0113] A fifth crystalline form of anhydrous Compound 1 is hereinafter referred to as "Form NF19" and is a polymorph characterized by a powder X-ray diffraction pattern substantially in accordance with that depicted in Figure 19. A suitable method for its preparation is described in Example 7.

[0114] Form NF19 may be characterized by one or more, preferably six, and up to substantially all, of the peaks in its X-ray powder diffraction pattern selected from those for:

[0115] [Table 22]

[0116] Alternatively, solid forms A1, A3, H1, and H2 may be characterized by having particular crystal systems, space groups, and / or unit cell parameters selected from a, b, c, a, b, g, and V, as listed under Table 7.

[0117] [Table 23]

[0118] The present invention also relates to the following solvate forms, which can be easily prepared by crystallization from the respective solvents, but have been found to be significantly less advantageous in terms of important properties compared to the above forms: methanolate of Compound 1 (solid form designated as S1), mixed hydrate / methanolate of Compound 1 (solid form designated as S2), THF solvate of Compound 1 (solid form designated as S3), 1,4-dioxane solvate form of Compound 1 in numerous solid forms (solid form designated as NF11 [from a slurry conversion experiment of the anhydrous form at ∼26 mg / 200 μL in 1,4-dioxane at room temperature], NF29 [from a cooling crystallization experiment in 1,4-dioxane at 50-5°C], NF36 [from a slurry conversion experiment of the anhydrous form at ∼52 mg / 150 μL in 1,4-dioxane at room temperature], NF40 [from a cooling crystallization experiment in 1,4-dioxane at 50-5°C], NF50 [from a cooling crystallization experiment in 1,4-dioxane at ∼50 mg / 150 μL], NF60 [from a cooling crystallization experiment in 1,4-dioxane at room temperature at ∼50 mg / 150 μL], NF70 [from a cooling crystallization experiment in 1,4-dioxane at ∼50 mg / 150 μL], NF80 [from a cooling crystallization experiment in 1,4-dioxane at ∼50 mg / 150 μL], NF90 [from a cooling crystallization experiment in 1,4-dioxane at ∼50 mg / 150 μL], NF100 [from a cooling crystallization experiment in 1,4-dioxane at room temperature at ∼50 mg / 150 μL], NF110 [from a cooling crystallization experiment in 1,4-dioxane at ∼ [from a slurry conversion experiment of the aqueous form], a chloroform solvate of Compound 1 (solid form designated as NF15), various solid forms of an acetic acid solvate of Compound 1 (solid form designated as NF16 [from an evaporative crystallization experiment in acetic acid at room temperature], NF18 [from an evaporative crystallization experiment in acetic acid at 50° C.], a dichloromethane (DCM) solvate of Compound 1 (solid form designated as NF32), an NMP (N-methyl-2-pyrrolidone) solvate of Compound 1 (solid form designated as NF33), an acetonitrile solvate of Compound 1 (solid form designated as NF35), and a dimethylacetamide (DMAA) solvate of Compound 1 (solid form designated as NF37). The XRPDs of the solid forms of these solvates are shown in Figures 40-52, and the corresponding peaks are listed in the following table:

[0119] [Table 24]

[0120] According to another embodiment, the present invention provides pharmaceutical compositions comprising an effective amount of Compound 1 in Form A2, which is a preferred solid form. According to another embodiment, the present invention provides methods for preparing such pharmaceutical compositions described herein (e.g., pharmaceutical compositions comprising an effective amount of Compound 1 in Form A2). Yet another embodiment provides methods for treating cancer using pharmaceutical compositions containing an effective amount of Compound 1 in Form A2 described herein. According to another embodiment, the present invention provides the use of Compound 1 in Form A2 in the manufacture of a medicament for treating cancer. In a further embodiment, the present invention provides Form A2 for use as a medicament, preferably for the treatment of cancer. Consistent with what has been set forth above, the exemplary embodiments, ranges, purities, etc. disclosed for Compound 1 are equally valid for Form A2.

[0121] According to another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of Compound 1 in any one of the solid forms of anhydrous Compound 1 or a hydrate of Compound 1, as described above. According to another aspect, the present invention provides a method for preparing such a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising an effective amount of Compound 1 in one of those solid forms). Yet another aspect provides a method for treating cancer using a pharmaceutical composition containing an effective amount of Compound 1 in one of the solid forms described herein. According to another aspect, the present invention provides the use of Compound 1 in the solid forms described herein in the manufacture of a medicament for treating cancer. In a further aspect, the present invention provides a solid form of Compound 1 described herein for use as a medicament, preferably for the treatment of cancer. Consistent with what has been set forth above, the exemplary embodiments, ranges, purities, etc. disclosed for Compound 1 are equally valid for the solid form.

[0122] The present invention relates to a solid form of anhydrous Compound 1 or a hydrate of Compound 1, as described herein, which is obtained or can be obtained according to the method described in Example 7.

[0123] Deuteration mode According to a further aspect, the present invention provides deuterated derivatives of compound Y. According to one embodiment, the present invention provides: 8-(1,3-dimethylpyrazol-4-yl)-1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-(trideuterio-methyl)imidazo[4,5-c]quinolin-2-one (compound 3), and

[0124] 1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-methyl-8-[3-methyl-(trideuterio-methyl)pyrazol-4-yl]imidazo[4,5-c]quinolin-2-one (compound 4),

[0125] 8-(1,3-Dimethylpyrazol-4-yl)-1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-methyl-imidazo[4,5-c]quinolin-2-one (compound 5), as well as salts thereof. Compounds 3, 4, and 5 are represented by the following formula:

[0126] [ka] In other embodiments, the present invention provides atropisomers 3-a, 3-b, 4-a, 4-b, 5-a, and 5-b:

[0127] [ka]

[0128] [ka]

[0129] [ka] Or their salts.

[0130] Consistent with what is set forth above with respect to Compounds 1 and 2, the present invention also provides Compounds 4-a, 5-a, 6-a, 4-b, 5-b, and 6-b, which are substantially free of their respective other atropisomers, including any salts thereof. In a further embodiment, these compounds are provided substantially free of impurities. According to another embodiment, these compounds contain no more than 5.0 area percent HPLC of total organic impurities compared to the total area of ​​the HPLC chromatogram. The exemplary and preferred ranges disclosed above for Compound 1 in relation to "substantially free of Compound 2 or a salt thereof," "free of impurities," and area percent of total organic impurities are equally applicable here with respect to the corresponding other atropisomers of each compound.

[0131] According to another aspect, the present invention provides pharmaceutical compositions comprising an effective amount of at least one of compounds 3, 4, or 5, their atropisomers, or pharmaceutically acceptable salts. According to another aspect, the present invention provides methods for preparing such pharmaceutical compositions as described herein. Yet another aspect provides methods for treating cancer using the pharmaceutical compositions described herein. According to another aspect, the present invention provides the use of the compositions described herein in the manufacture of a medicament for treating cancer. The exemplary and preferred aspects described above for compound 1 are equally applicable to these compounds.

[0132] preparation Compounds 1 and 2 according to the present invention can be prepared starting from compound Y, which has been previously described. As disclosed in WO2016 / 155884, 8-(1,3-dimethyl-1H-pyrazol-4-yl)1-(3-fluoro-5-methoxypyridin-4-yl)7-methoxy-3-methyl-1,3-dihydroimidazoquinolin-[4,5c]2-one (compound Y) can be prepared according to the following reaction sequence:

[0133] [ka]

[0134] Exemplary reaction conditions for each of these steps a-e are provided in Example 1 as are methods for obtaining the starting compounds. Other suitable reaction conditions will be readily apparent to one skilled in the art. Compounds 1 and 2 can then be obtained by suitable methods of separation from compound Y, illustrative embodiments of which are provided in Examples 1, 2, and 3.

[0135] [ka]

[0136] Atropisomers may be separated using chiral chromatography, including supercritical fluid chromatography (SFC), starting from compound Y. Examples of suitable methods are described in detail in Examples 1 and 3.

[0137] Each undesired atropisomer can be subjected to racemization, for example thermal racemization, to produce compound Y as a new starting material, as illustrated schematically below, for example.

[0138] [ka]

[0139] In an alternative embodiment, compounds 1 and 2 can be prepared starting from compound Y by crystallization using an optically active acid, such as dibenzoyltartaric acid. Reaction of compound Y with an optically active acid gives a pair of atropisomeric salts. These salts of compounds 1 and 2 exhibit different physicochemical properties (e.g., solubility, phase distribution) and can be separated by taking advantage of these differences.

[0140] As illustrated by the scheme below, in one embodiment, compound Y is reacted with an optically active acid to produce a mixture of two salts in the mother solution, with the salt of compound 2 (L-salt B) precipitating first and being removed by filtration, and the corresponding salt of compound 1 (L-salt A) being recovered only after further concentration and precipitation of the mother solution.

[0141] The salt of compound 1 is then first converted to its free form, isolated, and then reacted with the corresponding other optically active form of the acid to give the corresponding salt of compound 1, which is converted in a subsequent step to the free base with high optical purity, while compound 2 may be subjected to racemization to give compound Y as a fresh starting material.

[0142] [ka]

[0143] Detailed examples of suitable preparative schemes are provided in Example 2 and Figure 6. Deuterated compounds 3, 4, and 5 according to the invention can be prepared as detailed in Example 6, and atropisomers, salts, solvates, and solid forms can be prepared substantially as those of compounds 1 and 2.

[0144] use Hereinafter, any general reference to "a compound according to the invention" will be meant to apply to all embodiments of the compounds of the invention, including Compound 1 or 2, or a pharmaceutically acceptable salt, solvate, or solid form thereof, and may be read as Compound 1 or 2, or a pharmaceutically acceptable salt, solvate, or solid form thereof. Similarly, any reference to a deuterated compound according to the invention will include not only Compound 3, 4, or 5, but also any atropisomers, salts, or solid forms of the foregoing.

[0145] The present invention also encompasses deuterated ATM inhibitors, their atropisomers, pharmaceutically acceptable solid forms, solvates, and salts, as well as their atropisomers, pharmaceutically acceptable salts, for the inhibition, regulation, and / or modulation of the ATM kinase signaling cascade, thus representing novel tools for research and / or diagnosis. The present invention therefore further relates to the use of compounds according to the present invention, including their deuterated forms, for the inhibition of ATM kinase. The term "inhibition" refers to any reduction in activity due to the action of a specific compound according to the present invention in that the latter is able to interact with the target molecule, allowing recognition, binding, and blocking. The compounds are distinguished from ATM kinase by their high affinity. The compounds are also highly selective, thus allowing for virtually exclusive and direct recognition of ATM kinase. For research and / or diagnostic uses, illustratively for use in assays, deuterated compounds, namely compounds 3, 4, or 5, or their atropisomers, salts, or solid forms, are believed to be useful.

[0146] The present invention generally encompasses the use of compounds according to the invention, including deuterated compounds, in the treatment of diseases caused, mediated and / or propagated by the activity of ATM kinase. The present invention therefore broadly relates to compounds according to the invention, including deuterated compounds, for use as medicaments.

[0147] The present invention therefore also relates to compounds according to the invention, including deuterium compounds, for use in the treatment of any disease caused, mediated, and / or propagated by the activity of ATM kinase. The present invention correspondingly relates to the use of compounds according to the invention, including deuterium compounds, for the preparation of a medicament for the treatment of any disease caused, mediated, and / or propagated by the activity of ATM kinase. In other words, the present invention also discloses compounds according to the invention, including hydrogen compounds, for use in the treatment of diseases affected by the inhibition of ATM kinase.

[0148] In addition, compounds or deuterated compounds according to the present invention can also be used as reagents to study kinase-dependent signaling pathways in animal and / or cell culture models or in clinical diseases described herein, which signaling pathways are associated with various diseases, as described herein.

[0149] The present invention also relates to compounds according to the present invention, including their pharmaceutically acceptable salts, solvates, solid and deuterated forms, for use in the treatment of cancer and / or tumors; and to their use in the preparation of a medicament for the treatment of cancer and / or tumors.

[0150] The present invention further teaches a method for the treatment of cancer or tumors, wherein an effective amount of at least one compound according to the invention, or a pharmaceutically acceptable salt, solvate, deuterated or solid form thereof, is administered to the subject to be treated.Preferred subjects in the sense of the present invention are humans or animals (especially preferably humans).

[0151] The cancer / tumor may be selected from the group of cancers / tumors of squamous cell, bladder, stomach, kidney, head, neck, esophagus, cervix, thyroid, intestine, liver, brain, prostate, genitourinary tract, lymphatic system, larynx, lung, skin, blood and immune system, among others, and / or the cancer may be selected from the group of monocytic leukemia, lung adenocarcinoma, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, colorectal cancer, gastric cancer, breast cancer, ovarian cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. It should be understood that the sensitization of cancer cells encompasses cells of the same cancers and tumors as above. The present invention also relates to medicaments comprising the compounds according to the invention and / or their pharmaceutically acceptable salts, solvates, deuterated or solid forms.

[0152] The present invention furthermore relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound according to the invention, and / or a pharmaceutically acceptable salt, solvate, deuterated or solid form thereof, optionally together with at least one pharmaceutically acceptable excipient.

[0153] "Drug" and "pharmaceutical composition" should be taken to mean any composition that can be used in the treatment of patients who, at least temporarily, exhibit a pathogenic alteration of the overall condition of the patient or the condition of an individual part, preferably as a result of cancer and / or tumor. The delivery of the respective pharmaceutical composition of the compound according to the present invention into a cell or organism can be carried out according to the invention in any way that allows the ATM kinase to come into contact with the compound present in the pharmaceutical composition, as a result of which a response is induced.

[0154] The pharmaceutical compositions of the present invention can be administered orally, transdermally, transmucosally, transurethrally, vaginally, rectally, pulmonary, enterally, and / or parenterally. The type of administration selected depends on the indication (especially the dose administered, individual-specific parameters, etc.). Different types of administration may facilitate localized treatment, minimize side effects, and shorten the active compound dose. Injection may be intradermally, subcutaneously, intramuscularly, or intravenously. Administration can be carried out, for example, using a so-called vaccination gun or with a syringe. It is also possible to provide the substance as an aerosol, which is inhaled by an organism, preferably a human patient.

[0155] In a preferred embodiment, the compounds according to the present invention (in any of their forms) are administered orally. Oral administration is preferred in terms of patient compliance. Therefore, the pharmaceutical composition is preferably an oral solid pharmaceutical composition.

[0156] It is an advantage of the compounds according to the present invention, in particular compounds 1 and 2 and their solid forms, in particular compound 1 and its solid form, respectively, that they readily lend themselves to formulation into oral solid dosage forms due to good stability and high bioavailability.

[0157] composition Each composition, pharmaceutical composition according to the present invention, may be prepared using conventional solid or liquid excipients in suitable dosages and in a manner known per se, corresponding to the desired type of administration. Thus, pharmaceutically acceptable excipients known to those skilled in the art can essentially form part of the pharmaceutical compositions according to the present invention, where the amount of excipient combined with the active compound to prepare a single dose varies depending on the dosage and type of administration. Such pharmaceutically acceptable excipients include fillers, stabilizers, complexing agents, antioxidants, solvents, binders, lubricants, salts, buffers, preservatives, regulators, etc. Examples of this type of excipient are water, vegetable oils, benzyl alcohol, alkylene glycols, polyethylene glycols, corifol, glycerol triacetate, gelatin, carbohydrates such as lactose or starch, hydroxypropylmethylcellulose (HPMC), magnesium stearate, talc, and petrolatum.

[0158] As mentioned above, the pharmaceutical composition of the present invention is preferably for oral administration.The pharmaceutical composition can generally be in the form of a tablet, film tablet, sugar-coated tablet, lozenge, capsule, pill, powder, granule, syrup, juice, drop, solution, dispersion, suspension, suppository, emulsion, implant, cream, gel, ointment, paste, lotion, serum, oil, spray, aerosol, adhesive, plaster, or adhesive bandage.The oral administration form is preferably a tablet, film tablet, sugar-coated tablet, lozenge, capsule, pill, powder, granule, syrup, juice, drop, solution, dispersion, or suspension.

[0159] Furthermore, parenteral pharmaceutical compositions, such as suppositories, suspensions, emulsions, implants, or solutions, are preferably oily or may be aqueous solutions.For topical application, the compounds of the present invention can be formulated in a conventional manner with at least one pharmaceutically acceptable excipient, such as microcrystalline cellulose, and optionally with additional auxiliaries, such as moisturizers, to give compositions that can be applied to the skin, such as creams, gels, ointments, pastes, powders, or emulsions, or to give liquid preparations that can be applied to the skin, such as solutions, suspensions, lotions, serums, oils, sprays, or aerosols.The pharmaceutical composition can also be in the form of an injection solution.For the preparation of an injection solution, aqueous media, such as distilled water or physiological salt solution, can be used.

[0160] The pharmaceutical composition may be provided in the form of a solid composition, for example, lyophilized, and then prepared for administration by injection through the addition of a solubilizing agent, for example, distilled water or a buffer solution, etc. Those skilled in the art are familiar with the basic principles of lyophilizate preparation.

[0161] The amount of a compound according to the present invention in a pharmaceutical composition containing at least one pharmaceutically acceptable excipient can be 0.1 to 100 weight percent. It is important that the pharmaceutical composition contains an effective amount of the compound, optionally together with one or more pharmaceutically acceptable excipients. A simple pharmaceutical composition can be a compound according to the present invention in a solid form, such as a powder, in a hard gelatin capsule. The terms "effective amount" or "effective dose" are used interchangeably herein and refer to an amount of a compound according to the present invention that has a therapeutically relevant effect on a disease or pathological change, preferably cancer and / or tumor, in a cell, tissue, organ, or mammal.

[0162] A "therapeutically effective amount" of a compound according to the present invention refers to an amount effective, at the required dosage and for the required duration, that will have the intended therapeutic effect, e.g., alleviation, amelioration, mitigation, or elimination of one or more manifestations of the respective condition of cancer in the patient, or any other clinical result during treatment of the patient. The therapeutic effect does not necessarily occur with the administration of a single dose, and may occur only after the administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. Such a therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound according to the present invention, alone or in combination, to elicit the desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the compound according to the present invention are outweighed by the therapeutically beneficial effects.

[0163] In an embodiment of the invention, a compound according to the invention (or salt, solvate, deuterated, or solid form) is administered at a dose of 5 mg to 1 g per dosage unit, e.g., between 10 and 750 mg per dosage unit, between 20 and 500 mg per dosage unit, such as 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 mg per dosage unit. It has been estimated that the biologically effective dose for Compound 1 should be in the range of 25 to 350 mg qd.

[0164] Due to their surprisingly strong and / or selective inhibition of ATM kinase, which regulates cellular processes through double-stranded DNA repair, the compounds of the present invention can be advantageously administered at low doses, while they achieve the same or even better biological efficacy compared with less potent or less selective inhibitors.Reduced doses are typically associated with reduced medical side effects.In addition, highly selective inhibition is generally also reflected by reduced undesirable side effects.

[0165] "Treating" or "treatment" of a condition or patient refers to taking steps to obtain a beneficial or desired result, including a clinical result. For purposes of this invention, a beneficial or desired clinical result includes, but is not limited to, alleviation, amelioration of one or more symptoms of the disease being treated, most preferably cancer; reduction in the extent of the disease; delay or slowing of disease progression; improvement, palliation, or stabilization of the disease state; or other beneficial result. It is recognized that reference to "treating" or "treatment" includes prophylaxis as well as the alleviation of established symptoms of the condition. "Treating" or "treatment" of a condition, disorder, or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of a condition, disorder, or condition from developing in a subject afflicted with or predisposed to the condition, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., arresting, reducing, or delaying the progression of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) palliating or attenuating the disease, i.e., causing regression of the condition, disorder, or condition or at least one of its clinical or subclinical symptoms. In some embodiments, "treating" includes (1) and (2).

[0166] "Tumor" refers to a subject diagnosed with or suspected of having one, and cancer refers to a malignant or potentially malignant neoplasm or mass of tissue of any size, including primary and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Various types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcoma, carcinoma, and lymphoma. Leukemia (cancer of the blood) does not generally form solid tumors.

[0167] "Administering" or "administration" (and grammatical equivalents of this phrase) of a compound to a patient refers to direct administration, which may be administration to the patient by a medical professional, or self-administration and / or indirect administration, which may be the act of prescribing a drug. By way of example, a physician who instructs a patient to self-administer a drug or provides a patient with a prescription for a drug, is considered, in the context of the present invention, to be administering the drug to a patient.

[0168] All of the above, and further excipients or other components of drug or pharmaceutical formulations, will be familiar to those skilled in the art and may undergo special formulations for the teachings according to the present invention in routine experimentation.

[0169] Combination therapy Drugs and pharmaceutical compositions comprising compounds according to the present invention and the use of these compounds for the treatment of kinase-mediated disorders are a very promising approach, especially for the treatment of cancer. Compounds according to the present invention may be administered as a sole treatment, but preferably in combination with other treatments, such as chemo- or radiotherapy, as outlined above. As stated above, reference to compounds includes any salts, solvates, deuterated or solid forms thereof.

[0170] The key role of ATM in DNA repair process, and the evidence that ATM kinase deficiency allows mammalian cells to become more radiosensitive, allows the use of ATM-specific inhibitors as part of the treatment of cancer, for example, solid tumors, by radiation therapy and / or chemotherapy, and chemotherapy is preferably aimed at inducing DNA double-strand breaks.As explained above, ATM is an attractive intervention to inhibit the repair of treatment-induced DSB.Therefore, the compounds according to the present invention, in any of their forms, are highly advantageous in combination with radiation therapy and / or DNA-damaging chemotherapy.

[0171] Thus, the present invention relates to the combination of a compound according to the present invention and radiation therapy (RT). Thus, the present invention relates to a compound according to the present invention, or a pharmaceutically acceptable salt or solid form thereof, for use in the treatment of cancer and / or tumors in combination with radiation therapy. Expressed differently, the present invention relates to the use of a compound according to the present invention, or a pharmaceutically acceptable salt or solid form thereof, for the preparation of a medicament for treating cancer and / or tumors in combination with radiation therapy, and thus to a method of treating cancer, which involves administering a compound according to the present invention, or a pharmaceutically acceptable salt or solid form thereof, in combination with radiation therapy. The present invention further relates to a compound according to the present invention, or a pharmaceutically acceptable salt or solid form thereof, for use in increasing the sensitivity of cancer cells to ionizing radiation, radiation therapy (RT), respectively.

[0172] Compound 1 has been shown to lead to significant dose-dependent antitumor responses in vivo in combination with clinically relevant radiation schedules (i.e., radiotherapy). Example 8 and Figure 20 provide details of the results achieved.

[0173] A suitable dosing regimen, by way of example, may involve the administration of 15 Gray (Gy) of RT given in 5 fractions (3 Gy given per fraction per day) over a period of one week (i.e., 5 consecutive days on followed by 2 days off), and oral administration of a compound according to the invention on the same days, which may be repeated at least once.

[0174] Industrial irradiation methods preferably used clinically include, but are not limited to, photon irradiation (classical, electromagnetic X-ray / gamma radiation), proton irradiation, heavy-ion irradiation (ionized carbon), and neutron irradiation. These and other suitable radiation treatments within the meaning of the present invention are known to those skilled in the art, for example, from Herrmann et al. (2006) Klinische Strahlenbiologie [Clinical Radiation Biology], Elsevier Munich, 4th Edition, 67-68; Bhide & Nutting (2010) BMC Medicine 8: 25; Choi & Hung (2010) Current Urology Reports 11(3): 172, the entire contents of which are incorporated herein by reference. In its most frequent application, photon irradiation has been technologically refined by IMRT (intensity-modulated radiation therapy) methods and by the ability to use imaging methods (three-dimensional conformal irradiation) and the most precise focusing method possible in irradiation planning.

[0175] According to further aspects, the present invention relates to the following combinations of compounds according to the invention and DNA-damaging agents: compounds according to the invention for use in the treatment of cancer and / or tumors in combination with a DNA-damaging agent, the use of compounds according to the invention for the preparation of a medicament for treating cancer in combination with a DNA-damaging agent, and methods for treating cancer involving the administration of compounds according to the invention and a DNA-damaging agent. As generally described hereinbefore, compounds may include pharmaceutically acceptable salts, solid forms, and solvates, particularly in relation to compositions and treatments. The administration of the DNA-damaging agent and the compound according to the invention may be simultaneous or sequential.

[0176] As used herein, a DNA-damaging agent is an agent capable of inducing DNA damage in cells, particularly preferably cancer cells, with the following exemplary embodiments:

[0177] As previously described, ATM kinase is a key regulator of DNA double-strand break (DSB) repair, which is induced by widely used cancer therapies, such as ionizing radiation (Ir) and DNA-damaging agents. Upon DSB event, ATM signals to a number of downstream effectors, including p53. Unrepaired DSBs lead to the activation of checkpoint responses, cell cycle arrest, and ultimately tumor cell death. In one aspect of the invention, the invention provides a pharmaceutical composition comprising a therapeutically effective compound according to the invention and a DNA damaging agent. DNA-damaging agents suitable for use in the combination (treatment), include pharmaceutical compositions or kits, are preferably selected from the group comprising:

[0178] Alkylating agents, such as altretamine, bendamustine, busulfan, carmustine, chlorambucil, chloromethine, cyclophosphamide, dacarbazine, ifosfamide, improsulfan tosylate, lomustine, melphalan, mitobronitol, mitolactol, nimustine, ranimustine, temozolomide, thiotepa, treosulfan, mechlorethamine, carboquone, apaziquone, fotemustine, glufosfamide, palifosfamide, pipobroman, trofosfamide, and uramustine:

[0179] Platinum compounds, such as carboplatin, cisplatin, eptaplatin, miriplatin hydrate, oxaliplatin, lobaplatin, nedaplatin, picoplatin, satraplatin, etc.;

[0180] Topoisomerase inhibitors, such as irinotecan, SN38, topotecan, camptothecin, rubitecan, belotecan, etoposide, daunorubicin, doxorubicin, aclarubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, and amsacrine; Poly-(ADP-ribose)-polymerase (PARP) inhibitors, such as olaparib, niraparib, and veliparib;

[0181] ATR (ataxia telangiectasia and Rad3 related) inhibitors, M6620 (VX-970: 3-[3-(4-methylaminomethyl-phenyl)-isoxazol-5-yl]-5-[4-(propane-2-sulfonyl)-phenyl]-pyrazin-2-ylamine), M4344 (VX-803: 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid [5'-fluoro-4-(4-oxetan-3-yl-piperazine-1-carbonyl)-3,4,5,6- tetrahydro-2H-[1,4']bipyridinyl-3'-yl]-amide); AZD-6738 (4-[4-[1-[[S(R)]-s-methylsulfonimidoyl]cyclopropyl]-6-[(3R)-3-methyl-4-morpholinyl]-2-pyrimidinyl]-1H-pyrrolo[2,3-b]pyridine) and 2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine;

[0182] DNA modifying agents, such as amrubicin, bisantrene, decitabine, mitoxantrone, procarbazine, trabectedin, clofarabine, amsacrine, brostallicin, pixantrone, laromustine, etc.

[0183] Anticancer antibiotics, such as bleomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, levamisole, miltefosine, mitomycin C, romidepsin, streptozocin, valrubicin, zinostatin, zorubicin, daunorubicin, plicamycin, aclarubicin, peplomycin, pirarubicin, etc. · Alpha emitters, alpharadine (223Ra dichloride, Xofgio), 211At, 213Bi, 225Ac, 227Th;

[0184] Particular preference is given to etoposide, irinotecan, razoxane, sobuzoxane, topotecan, camptothecin, doxorubicin, amsacrine, PARP inhibitors, and ATR inhibitors.

[0185] The efficacy of Compound 1 in combination with the exemplary PARP inhibitor olaparib was demonstrated in the HBCx-10 patient-derived triple-negative breast cancer xenograft model developed in immunodeficient female mice, the results of which are shown in Figure 21. More details of this experiment are described in Example 9.

[0186] The present invention can also be implemented as a kit containing the compound according to the present invention. The kit consists of separate packs containing (a) an effective amount of the compound according to the present invention and / or its physiological salt, solvate, or solid form, and (b) an effective amount of an additional active compound. The additional active compound is preferably a DNA-damaging agent.

[0187] The kit may contain suitable containers, such as a box or carton, individual bottles, bags, or ampoules. The kit may contain, for example, separate ampoules or vials, each containing an effective amount of a compound according to the invention and / or a pharmaceutically acceptable salt, solvate, or solid form thereof, or an effective amount of an additional active compound, a DNA damaging agent in dissolved or lyophilized form, etc. The kit of the invention may also contain written instructions or an article containing written instructions for the user, which explain how to use the compound of the invention.

[0188] In summary, it should be noted that the compounds according to the present invention can be used individually and / or in combination with other treatment modalities, such as surgical intervention, immunotherapy, radiotherapy, and / or chemotherapy, etc. The latter relates to targeted therapy with any desired (chemical or biological, including nME: new molecular entities, NCE: new chemical entities, and NBE: new biological entities) active compound, as monotherapy and / or on-target / off-target combination therapy. It is intended that all documents cited in the description be incorporated herein by reference in their entirety into the disclosure of the present invention.

[0189] It goes without saying that the present invention is not limited to the specific compounds, pharmaceutical compositions, uses, and methods described herein, as such may vary. It goes without saying, further, that the terminology used herein serves exclusively the purpose of describing particular embodiments and is not intended to limit the scope of protection of the present invention. As used in the specification, including the appended claims, singular word forms, such as "a" or "the," include their plural equivalents unless the context specifically dictates otherwise. For example, a reference to a "compound" includes a single compound or multiple compounds, which may be identical or different in sequence, or a reference to a "method" includes equivalent steps and methods known to those skilled in the art. Reference to a feature as "comprising" certain features will be interpreted as meaning that the feature includes those features, but does not exclude the presence of other features unless they render the feature inoperable.

[0190] experiment The compounds according to the invention exhibit advantageous properties as evidenced by various parameters and experimental results. The experimental methods used for the analysis and characterization of the compounds according to the invention are provided below in all their forms.

[0191] Assay Measurement of kinase activity is a technique well known to those skilled in the art. A general test system for determining kinase activity using substrates such as histones (Alessi et al. (1996) FEBS Lett. 399(3): 333) or basic myelin protein is described in the paper (Campos-Gonzalez & Glenney (1992) JBC 267: 14535). Various assay systems are available for identifying kinase inhibitors. In scintillation proximity assays (Sorg et al. (2002) J Biomolecular Screening 7: 11) and flash plate assays, the radioactive phosphorylation of a protein or peptide as a substrate is measured using ATP. In the presence of an inhibitory compound, a reduced or no radioactive signal is detectable. Furthermore, homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET) and fluorescence polarization (FP) techniques are useful assay methods (Sills et al. (2002) J Biomolecular Screening 191). Another non-radioactive ELISA method uses a specific phospho-antibody (phospho-AB), which binds only phosphorylated substrates. This binding can be detected by chemiluminescence using a second peroxidase-conjugated anti-sheep antibody. For the purposes of the present invention, the on-target properties of relevant compounds were evaluated using the following assays:

[0192] ATM Kinase Assay - ATM Inhibition (IC 50 ATM) determination: I C 50 The values ​​were determined with the aid of a biochemical ATM kinase assay. The assay consists of two steps: Enzymatic reaction and detection step. First, ATM (ataxia telangiectasia mutated) protein and test substances are incubated at various concentrations with the addition of substrate protein p53 and ATP. ATM mediates the phosphorylation of p53 at several positions, including amino acid S15. The amount of phosphorylated p53 is determined with the aid of a specific antibody and TR-FRET technology. The enzymatic ATM assay is performed as a TR-FRET (HTRF™, Cisbio Bioassays)-based 384-well assay. In the first step, purified human recombinant ATM (human ATM, full-length, GenBank ID nM_000051, expressed in a mammalian cell line) is incubated in assay buffer for 15 minutes with various concentrations of ATM inhibitors, without test substances as a negative or neutral control. The assay buffer contained 25 mM HEPES pH 8.0, 10 mM Mg(CH3COO)2, 1 mM MnCl2, 0.1% BSA, 0.01% Brij® 35, and 5 mM dithiothreitol (DTT). The test substance solution was dispensed into microplates using an ECHO555 (Labcyte). In the second step, purified human recombinant c-myc-tagged p53 (human p53, full-length, GenBank ID BC003596, expressed in Sf21 insect cells) and ATP were added, and the reaction mixture was incubated at 22°C for 30-35 minutes. The pharmacologically relevant assay volume was 5 μl. The final concentrations in the assay during the reaction mixture incubation were 0.3-0.4 nM ATM, 50-75 nM p53, and 10 μM ATP. The enzymatic reaction is stopped by the addition of EDTA. The formation of phosphorylated p53 as a result of the ATM-mediated reaction in the presence of ATP is detected via a specific antibody that allows FRET (labeled with fluorophorene europium (Eu) as the donor and d2 as the acceptor).Two microliters of antibody-containing stop solution (12.5 mM HEPES pH 8.0, 12.5 mM EDTA, 30 mM sodium chloride, 300 mM potassium fluoride, 0.1006% Tween-20, 0.005% Brij® 35, 0.21 nM anti-phospho-p53(ser15)-Eu antibody, and 15 nM anti-cmyc-d2 antibody) are added to the reaction mixture. After incubation for signal development, usually 2 hours (between 1.5 and 15 hours), the plate is analyzed in a plate reader (EnVision, PerkinElmer) using TRF mode (and with laser excitation). After excitation of the donor europium at a wavelength of 340 nM, the emitted fluorescence light of both the acceptor d2 at 665 nM and the donor europium at 615 nM is measured. The amount of phosphorylated p53 is directly proportional to the amount of light emitted, i.e., relative fluorescence units (RFU) at 665 nm and 615 nm. The measured data were processed by Genedata Screener software. IC. 50 The determination of is performed, inter alia, by fitting a dose / effect curve to the data points by nonlinear regression analysis. I C 50 = half-maximal inhibitory concentration ATP = adenosine triphosphate TR-FRET = Time-resolved Fluorescence Resonance Energy Transfer HTRF® = Homogeneous Time-Resolved Fluorescence HEPES = 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid Mg(CH3COO)2 = Magnesium acetate MnCl2 = manganese(II) chloride BSA = bovine serum albumin EDTA = ethylenediaminetetraacetate TRF = time-resolved fluorescence Abbreviations apply throughout unless otherwise stated. IC at 1000 μM ATP concentration 50 The assay for determining the value differs from the above assay only in the ATP concentration.

[0193] Cellular pCHK2 assay: To identify substances that inhibit the phosphorylation of the protein kinase CHK2 (checkpoint kinase 2) at amino acid threonine 68, an immunofluorescence-based "high content" analytical assay was used in HCT116 cells.

[0194] In vitro cell-based immunofluorescence assay for identification of inhibitors of bleomycin-induced phosphorylation of CHK2 (phospho-Thr68) in the human colon carcinoma cell line HCT116: HCT116 cells are seeded at a defined cell density in 384-well plates in culture medium (DMEM high glucose, 2 mM GlutaMax, 1 mM sodium pyruvate, 10% FCS) and incubated overnight at 37°C and 10% CO2. On the following days, test substances are added at a defined concentration range (1 nM to 30 nM) in combination with 10 μM bleomycin, and the concentration of the solvent DMSO is kept constant at 0.5%. After 4 hours of incubation at 37°C and 10% CO2, cells are fixed (5 minutes, 4% formaldehyde in PBS), permeabilized (10 minutes, 0.2% Triton X100 in PBS), and after blocking nonspecific binding sites (10% goat serum, 1% BSA in PBS), incubated overnight at 4°C with a specific anti-pCHK2 antibody (Cell Signaling #2661). pCHK2 (Thr68) is determined using an Alexa488-labeled secondary anti-rabbit IgG antibody. Parallel staining of DNA with propidium iodide allows for cell number determination. pCHK2 signal is detected using a high-content imager (Molecular Devices IMX Ultra) and automated image analysis using the instrument's MetaXpress software. The number of cell nuclei with pCHK2 signal above a defined background is determined. DMEM: Dulbecco's modified Eagle's medium; FCS: fetal calf serum; PBS: phosphate-buffered saline (abbreviations apply consistently unless otherwise stated) Furthermore, the effect, especially the inhibition, of other kinases and thus the selectivity of the compounds according to the invention can be determined with the aid of the following assays:

[0195] ATR / ATRIP kinase assay I C 50 The values ​​were determined by an ATR / ATRIP enzyme assay. The assay involves two steps: an enzymatic reaction and a detection step. First, a mixture of ATR / ATRIP proteins (ataxia telangiectasia and Rad3-related protein / ATR-interacting protein), the compound of interest at various concentrations, p53 as a substrate protein, and adenosine triphosphate are incubated in an assay buffer. ATR phosphorylates p53 at Ser15 and other residues. The amount of phosphorylated p53 is then detected using a specific antibody and TR-FRET assay technology.

[0196] In detail: The ATR / ATRIP enzyme assay is performed as a TR-FRET-(HTRF™, Cisbio Bioassays)-based 384-well assay. In the first step, purified human recombinant ATR / ATRIP (human ATR, full-length, GenBank ID: NM_001184.3, and human ATRIP, full-length, GenBank ID AF451323.1, co-expressed in a mammalian cell line) is incubated with various concentrations of test compound or without test compound (as a negative control) in assay buffer for 15 minutes at 22°C. The assay buffer contains 25 mM HEPES pH 8.0, 10 mM Mg(CHCOO), 1 mM MnCl, 0.1% BSA, 0.01% Brij® 35, and 5 mM dithiothreitol (DTT). An Echo555 (Labcyte) is used for compound solution distribution. In the second step, purified human recombinant cmyc-tagged p53 (human p53, full-length, GenBank ID: BC003596 (expressed in Sf21 insect cells)) and ATP are then added, and the reaction mixture is incubated at 22°C for 25-35 minutes, typically 25 minutes. The pharmacologically relevant assay volume is 5 μl. The final assay concentrations during the incubation of the reaction mixture are 0.3-0.5 nM, typically 0.3 nM ATR / ATRIP, 50 nM p53, and 0.5 μM ATP. The enzymatic reaction is stopped by the addition of EDTA. The generation of phosphorylated p53 as a result of the ATR-mediated reaction in the presence of ATP is detected using a specific antibody that allows FRET [labeled with the fluorophore europium (Eu) as the donor and d2 as the acceptor]. For this purpose, 2 μl of antibody-containing stop solution (12.5 mM HEPES pH 8.0, 125 mM EDTA, 30 mM sodium chloride, 300 mM potassium fluoride, 0.006% Tween-20, 0.005% Brij® 35, 0.21 nM anti-phospho-p53(ser15)-Eu antibody, 15 nM anti-cmyc-d2 antibody) is added to the reaction mixture.Following 2 hours of signal development, the plate is analyzed in an Envision (PerkinElmer) microplate reader using TRF mode with laser excitation. Upon excitation of the donor europium at 340 nm, the emitted fluorescence light from the acceptor d2 at 665 nm and from the donor europium at 615 nm is measured. The amount of phosphorylated p53 is directly proportional to the ratio of the amount of light emitted, i.e., the ratio of relative fluorescence units (RFU) at 665 nm and 615 nm. Data are processed using Genedata Screener software. In particular, IC. 50 Values ​​are routinely determined by fitting a dose-response curve to the data points using nonlinear regression analysis. For abbreviations, see list above.

[0197] Assay of pCHK1 cells The Chk1 kinase acts downstream of ATR and plays a key role in DNA damage checkpoint control. Activation of Chk1 involves phosphorylation of Ser317 and Ser345 (considered to be preferential targets for phosphorylation / activation by ATR) and occurs in response to blocked DNA replication and certain forms of genotoxic stress. Ser345 phosphorylation helps localize Chk1 to the nucleus after checkpoint activation.

[0198] This assay uses immunocytochemistry and high-content imaging to measure the reduction in phosphorylation of Chk1 (serine 345) in HT29 colon adenocarcinoma cells following treatment with compounds and hydroxyurea (which promotes fork delay by dNTP depletion).

[0199] For the assay, HT29 cells are plated in culture medium (DMEM high glucose (phenol red-free), 2 mM Glutamax, 1 mM Pyruvate, 10% FCS) (2500 cells / well / 30 μl in Greiner 384-well plates, black, μclear #781090) and incubated at 37°C for at least 20 hours in 10% CO2 and 90% rH2O. Diluted test compounds (1 nM-30 μM final) and hydroxyurea (3 mM final) are added simultaneously, and the cells are incubated at 37°C for 4 hours. After fixation / permeabilization with 100% MeOH (-20°C chilled) and permeabilization with 0.2% TritonX-100, a complete immunocytochemistry procedure is performed using a specific anti-pChk1 antibody (Cell Signaling, #2348BF) and a fluorescently labeled secondary antibody (AlexaFluor® 488 goat anti-rabbit F(ab')2 fragment, Invitrogen A11070) and a parallel nuclear stain for cell counting. Nuclear-localized pChk1 signal was detected on the ImageXpress Ultra high-content reader and reported as % positive cells (nuclei).

[0200] DNA-PK assay The kinase assay was performed as a 384-well HTRF-based assay. In the first step, the DNA-PK protein complex was incubated with or without test compounds for 15 min at 22°C. After addition of STK-substrate l-biotin (Cisbio), Mg-ATP, DNA, and staurosporine, the reaction mixture was incubated at 22°C for 60–80 min (depending on the activity of the DNA-PK protein complex).

[0201] An Echo555 (Labcyte) was used for compound solution distribution. The assay buffer consisted of 25 mM HEPES pH 7.4, 11 mM MgCl2, 80 mM KCl, 0.45 mM EDTA, and 0.5 mM EGTA, and contained 1 mM dithiothreitol (DTT), 0.17% BSA, and 0.01% Tween® 20. The pharmacologically relevant volume was 5 μl. The final concentrations in the assay during reaction mixture incubation were 50–100 ng / well DNA-PK protein complex (depending on the activity of the DNA-PK protein complex), 1 μM STK-substrate l-biotin, 10 μM Mg-ATP, 80 ng / well DNA from calf thymus, and 1 μM staurosporine. The enzymatic reaction was stopped by the addition of EDTA. The generation of phosphorylated STK-substrate l-biotin as a result of the DNA-PK-mediated reaction was detected via a specific anti-phospho-STK antibody (Cisbio) labeled with europium (Eu) as the donor and streptavidin labeled with XL665 (Cisbio) as the acceptor, allowing FRET. To this end, 4 μl of antibody- and streptavidin-containing stop solution (12.5 mM HEPES pH 8.0, 125 mM EDTA, 30 mM sodium chloride, 300 mM potassium fluoride, 0.006% Tween-20, 0.005% Brij® 35, 0.179 nM anti-phospho-STK antibody, 160 nM streptavidin-XL665) was added to the reaction mixture. After 1 h of signal development, the plate was analyzed on a Rubystar or Pherastar microplate reader (BMG Labtech). The amount of phosphorylated substrate was directly proportional to the ratio of fluorescence units (excitation wavelength 337 nm) at an emission wavelength of 665 nm (phosphopeptide sensitive wavelength / emission of XL665) to units at 620 nm (reference wavelength europium). IC 50-Values ​​were calculated using Genedata Screener® software (Molecular Cancer Therapeutics 2003, 1257-1264; DNA-dependent protein kinase inhibitors as drug candidates for the treatment of cancer; A. Kashishian, H. Douangpanya, D. Clark, ST Schlachter, C. Todd Eary, JG Schiro, H. Huang, LE Burgess, EA Kesicki, and J. Halbrook.).

[0202] [Table 25]

[0203] Assay of pDNA-PK cells HCT116 cells were cultured in MEM alpha medium with 10% fetal bovine serum and 2 mM glutamine at 37°C and 10% CO2. Cells were detached from the bottom of the culture vessel using trypsin / EDTA, centrifuged in a centrifuge tube, and transferred to fresh medium. The cell density was then determined. 100,000 cells were seeded in 1 ml of culture medium per well of a 24-well cell culture plate and cultured overnight. The following day, 10 μM bleomycin (a DNA intercalator and DNA double-strand breaker inducer) and test substances in fresh culture medium were added to the cells and cultured for an additional 6 hours. Cell lysis was then performed, and the cell lysates were placed on 96-well ELISA plates (Sigma-Aldrich WH0005591M2: total DNA-PK, Abcam ab18192, or Epitomics EM09912: phospho-serine 2056 DNA-PK), which were blocked, coated with DNA-PK-specific antibodies, and incubated overnight at 4°C. Subsequently, the 96-well ELISA plates were treated with a detection antibody (Abcam ab79444: total DNA-PK) and streptavidin-HRP conjugate. The progress of the enzyme reaction was carried out using a chemiluminescence reagent, and chemiluminescence was measured using a Mithras LB940. The signal from the phospho-DNA-PK-specific antibody was normalized to the signal from the antibody against total protein DNA-PKc. IC 50 Values ​​or percentages were determined by referencing the signal level of the bleomycin-treated vehicle control group (100% of control). A DMSO control was used as a blank. MEM: minimum essential medium; DMSO: dimethyl sulfoxide

[0204] PDE2A1 assay A commercially available assay was used (Cerep, catalog ref. 4071, SOP n°1C1054), which is designed to evaluate the effect of compounds on the activity of human phosphodiesterase-2A1, quantified by measuring the formation of 5'AMP from cAMP using the human recombinant enzyme expressed in Sf9 cells.

[0205] Test compounds, reference compounds, or water (control) are added to a buffer containing 40 mM Tris / HCl (pH 7.4), 8 mM MgCl 2 , and 1.7 mM EGTA / NaOH, 1.8 μM cAMP, and 1 μCi [ 3 H]cAMP. The reaction is then initiated by the addition of enzyme (approximately 2.5 U), and the mixture is incubated at 22° C. for 20 minutes before the control measurement. For the reference measurement of the base, the enzyme is omitted from the reaction mixture. Following incubation, SPA beads are added. After 30 minutes at 22°C under shaking, the amount of [3H]5'AMP is quantified in a scintillation counter (Topcount, Packard). Results are expressed as percent inhibition of control enzyme activity.

[0206] The standard inhibitory reference compound was EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), which was tested at several concentrations in each experiment and its IC 50 An inhibition curve is obtained from which values ​​are calculated.

[0207] References: Maurice DH, Ke H., Ahmad F., Wang Y., Chung J. and Manganiello VC (2014), Advances in targeting cyclic nucleotide phosphodiesterases, Nat. Rev. Drug Discov., Vol. 13 Issue 4: p. 290.

[0208] PDE4D2 assay A commercially available assay from Cerep was used (catalog ref. 4077; SOP n°1C1045). The assay is designed to evaluate the effect of test compounds on the activity of phosphodiesterase-4D2, quantified by measuring the formation of 5'AMP from cAMP using a human recombinant enzyme expressed in Sf9 cells.

[0209] Test compounds, reference compounds, or water (control) are added to a buffer containing 40 mM Tris / HCl (pH 7.4) and 8 mM MgCl 2 , 450 nM cAMP, and 0.0125 μCi [ 3 H]cAMP. The reaction is then initiated by the addition of enzyme (approximately 1.5 U), and the mixture is incubated at 22° C. for 20 minutes before the control measurement. For the basal control measurement, the enzyme is omitted from the reaction mixture. Following incubation, SPA (Scintillation Proximity Assay) beads are added. After 30 minutes at 22°C under shaking, the amount of [3H]5'AMP is quantified in a scintillation counter (Topcount, Packard). Results are expressed as percent inhibition of control enzyme activity. The standard inhibitory reference compound was Ro 20-1724, which was tested at several concentrations in each experiment and its IC 50 An inhibition curve is obtained from which values ​​are calculated. References above.

[0210] PDE42A1 assay A commercially available assay was used (catalog ref. 4074, SOP no. 1C1056), which is designed to evaluate the effect of compounds on the activity of human phosphodiesterase-4A1A, quantified by measuring the formation of 5'AMP from cAMP using the human recombinant enzyme expressed in Sf9 cells.

[0211] Test compounds, reference compounds, or water (control) are added to a buffer containing 40 mM Tris / HCl (pH 7.4) and 8 mM MgCl 2 , 450 nM cAMP, and 0.25 μCi [ 3 H]cAMP. The reaction is then initiated by the addition of enzyme (approximately 10 U) and the mixture is incubated at 22° C. for 20 minutes. For the basal control measurement, the enzyme is omitted from the reaction mixture. Following incubation, SPA beads are added. After 30 minutes at 22°C under shaking, the amount of [3H]5'AMP is quantified in a scintillation counter (Topcount, Packard). Results are expressed as percent inhibition of control enzyme activity. The standard inhibitory reference compound was Ro20-1724, which was tested at several concentrations in each experiment and its IC 50 An inhibition curve is obtained from which values ​​are calculated. References above. Additional parameters that are considered important are determined as follows:

[0212] Test Method Microsomal Stability (Intrinsic Clearance) Microsomal stability assays are used to measure in vitro clearance (Clint). The assay involves measuring the rate of elimination of a compound due to its intrinsic tendency to be metabolized ("intrinsic" means that elimination is not affected by other properties such as permeability, binding, etc., which play a role in quantifying in vivo clearance). Microsomal stability (intrinsic clearance, Clint) and, therefore, metabolic stability, are commonly given as μl / min / mg protein. The volume of solution that 1 mg of microsomes can clear in 1 minute can be visualized.

[0213] measurement A Tecan Genesis workstation (RSP150 / 8) was used to perform the microsomal incubations. The analysis was performed using a Waters ACQUITY UPLC system coupled to an ABSciex API3000 mass spectrometer. Data analysis was performed using Assay Explorer (Symyx). UPLC conditions: Column: Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 μm (Waters) Mobile phase: A = 0.1% formic acid in water; B = acetonitrile

[0214] [Table 26] Flow rate: 0.750 ml / min; Detection: ESI, MRM; Injection: 10 μL; Column temperature: 50°C chemicals Potassium phosphate buffer: 0.05M potassium phosphate buffer pH 7.4 containing 1mM MgCl2 NADPH (nicotinamide adenine dinucleotide phosphate): 22.5 mg NADPH-Na₄ in 1.8 ml potassium phosphate buffer Acetonitrile: 50% acetonitrile (1 volume acetonitrile, 1 volume water) DMSO: 20% by volume DMSO in water 20mg / ml human or mouse liver microsome (protein) / ml stock solution in phosphate buffer 10 mM compound stock solution in 100% DMSO

[0215] Microsome incubation Dilutions of test compounds were made in two steps, starting from a 10 mM stock solution of each compound in 100% DMSO. First, 4 μl of the stock solution was added to 196 μl of 20% (v / v) DMSO. In the second step, 10 μl of the first dilution was added to 1590 μl potassium phosphate buffer to achieve a final concentration of 1.25 μM of the final compound dilution. Thus, the amount of organic solvent in the assay was kept to a minimum (<1%).

[0216] The human or mouse liver microsome (protein) solution used in the assay was prepared by mixing 750 μl stock solution (20 mg / ml) and 2250 μl potassium phosphate buffer to a final concentration of 5 mg / ml.

[0217] Incubations were performed in a 96-deep-well incubation plate. 160 μl per well of the final compound dilution was transferred to the incubation plate. Four samples of each compound dilution were assayed. 20 μl / well liver microsome solution was added to each well, and the samples were then preincubated at 37°C for 5 minutes with 800 rpm agitation. Two reference compounds (verapamil and dextromethorphan) were used in parallel in every experiment and for each type (human or mouse microsomes) to ensure system performance and for comparison. To a separate stop plate, 160 μl acetonitrile was added per well.

[0218] After pre-incubation, i.e., at time t1=0 min, 18 μl samples of the incubated compound solution were transferred and added per well (containing acetonitrile) on the stop plate to block the reaction (0 min control sample, 4 samples per compound). At the same time, 18 μl samples of the incubated reference compound solution were transferred and added per well (containing acetonitrile) on the stop plate at time t1=0 min, and 30 min later (t4), the solubility and chemical stability of the compounds were checked.

[0219] To initiate the reaction, 26 μl NADPH solution (cofactor) was added to all wells containing reference solution except those containing preincubated compound dilutions or compound dilutions to be used as 30-minute control samples (in which 26 μl phosphate buffer was added instead). Incubation was then continued at 37°C and 800 rpm agitation.

[0220] In the final assay solution (i.e., in each well containing compound, microsomes (protein), and NADPH in phosphate buffer), the final protein concentration was 0.5 mg / ml and the compound concentration was 1 mg / ml.

[0221] After incubation times t2 = 5 min, t3 = 10 min, and t4 = 20 min (i.e., after the start of the reaction), 20 μl samples of the incubated compound solutions (four samples per compound) and the reference compound solution were transferred and added per well of acetonitrile on the stop plate.

[0222] After an incubation time of t4 = 30 min, 20 μl samples of the incubated compound solution (4 samples per compound) and 20 μl samples of the 30 min control sample (containing buffer instead of NADPH), as well as a 20 μl sample of the incubated reference compound solution, were transferred and added per well of acetonitrile on the stop plate.

[0223] The quenched samples were centrifuged at 4000 g for 1 hour at 4° C. 80 μl of the supernatant was transferred to a 96-well plate for analysis by LCM / MS.

[0224] Data analysis The microsomal / metabolic stability of each compound was determined by measuring the change in LCM / MS peak area over time. Data were fit according to a log-linear model, along with the Michaelis / Menten model. Clint values ​​were calculated from the slope (k) of the linear log-transformed concentration per time plot divided by the microsomal amount (0.5 mg / ml): Clint (μl / min / mg protein) = k * 1000 / protein concentration. Assay Explorer software was used to automatically calculate the slope of the decline, k.

[0225] Kv11.1 (hERG) ion channel activity (patch clamp assay) Methods for detection and characterization of test substances that block Kv11.1 (hERG) channels: Kv11.1 (hERG, human delayed rectifier potassium ion channel gene) is a potassium channel in ventricular cardiomyocytes that plays a central role for cell repolarization.

[0226] Patch-clamp measurements were performed at room temperature in the whole-cell configuration on human embryonic kidney cells (HEK293) stably transfected with the hERG gene.

[0227] Whole-cell configuration was performed using an automated patch clamp device (Patchliner™, Nanion Technologies, Munich). This is a glass chip-based system that allows automated whole-cell measurements of up to eight cells simultaneously. The glass chip has a defined-sized hole through which cells are transferred to the gigaseal by application of vacuum, resulting in whole-cell configuration. Buffers, cell suspensions, and test substances were added to the microchannels of the chip using Teflon-coated pipettes.

[0228] Cells were clamped at a holding potential of -80 mV. For measurements of substance-enhanced inhibition of Kv11.1 channels, the following voltage protocol was applied at 10-second intervals: 51 ms / -80 mV, 500 ms / +40 mV, 500 ms / -40 mV, and 200 ms / -80 mV. Leak currents were subtracted by the P4 method. Cells were resuspended in extracellular buffer (EC) and applied to the chip. After cell collection, the seal was improved by adding seal enhancer buffer. Upon reaching whole-cell configuration, the seal enhancer buffer was washed off and replaced with extracellular buffer. Measurements were initiated at EC for 1.5 minutes. DMSO (vehicle control, 0.1% DMSO) was then applied, and control currents were recorded for 3 minutes. Test substances were then added in duplicate at the same concentrations, and potassium currents were measured in each case for 3.5 min.

[0229] If the measurement result of the test substance at the starting concentration of 10 μM was less than (-)50% effect (threshold) (e.g. (-)60% effect), the test substance was added cumulatively in increasing concentrations, each concentration measured for 5 min, to determine the dose / effect relationship.

[0230] The reference substance used was the Kv11.1 (hERG) ion channel blocker quinidine. The effects of the test substance and quinidine were normalized to the relevant vehicle control. The effect on Kv11.1 (hERG) channel activity was evaluated from the potassium current at -40 mV. For calculation, the current was evaluated for each final trace. The inhibition of the Kv11.1 (hERG) channel induced by the test substance was normalized to the vehicle control (0.1% DMSO). During the measurement, an aliquot of the test substance was selected for concentration determination. The sample was immediately measured by HPLC, and the final concentration was determined from the calibration curve.

[0231] If the measured result of the test substance at the starting concentration of 10 μM is greater than or equal to the (-)50% effect (threshold) (e.g., (-)30% effect, i.e., 30% inhibition of 10 μM), the K i is calculated according to the following formula: i = 1.0E-5 × (100+% effect) / (-% effect), [M]. The (-)30% effect measured at a test substance concentration of 10 μM resulted in a K i Give.

[0232] Cytochrome P-450 enzymes (CYP) In the human organism, drug substances are converted into water-soluble compounds by enzyme systems to facilitate their excretion. These enzyme systems include microsomal cytochrome P-450 enzymes, or CYPs for short. The assay was designed to identify whether compounds may be potent inhibitors of defined CYP isoforms. This involves the use of recombinant CYP isoforms and their reductases obtained by overexpression in baculovirus-infected insect cells. CYP reactions are carried out through the inhibition of luminescent CYP isoforms, which are tested with CYP substrates under NADPH-regenerating conditions. The luminescent P450-Glo™ substrate is a derivative of beetle luciferin ((4s)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazolecarboxylic acid or D-luciferin), a substrate for firefly luciferase from the beetle. The luminescent P450-Glo™ substrate does not react directly with luciferase, but is converted by the respective CYP isoform to a luciferin product that emits light upon reaction with the luciferin detection reagent (LDR). This allows enzyme activity to be rapidly quantified through photometry. The extent of inhibition is measured using the IC 50The value is measured by determining the γ-Lysine β-Lysine (Lysine β-Lysine) value (Crespi et al., Methods Enzymol. 357:276-284, 2002). The following commercially available screening systems / assays are used: P450-Glo™ CYP1A2 Screening System (Promega Corporation; V9770); P450-Glo™ CYP2C8 Assay (Promega Corporation; V8782); P450-Glo™ CYP2C9 Screening System (Promega Corporation; V9790); P450-Glo™ CYP2C19 Screening System (Promega Corporation; V9880); P450-Glo™ CYP2D6 Screening System (Promega Corporation; V9880); P450-Glo™ CYP3A4 Screening System (Luciferin-PPXE) (Promega Corporation; V9910). Bioavailability

[0233] The predicted bioavailability in humans is derived from the measured bioavailability in preclinical models: mice, rats, and dogs (beagles) calculated for the predicted pharmacologically effective dose in humans (in silico GastroPlus simulations).

[0234] Powder X-ray Diffraction (PXRD) Method: X-ray powder diffraction patterns (XRPDs), such as those depicted in Figures 7B and 10 through 19 (and others), were obtained using the following methodology: Samples were prepared in combination 96-well plates (containing X-ray amorphous foils as the base) or between X-ray amorphous films. Measurements were performed on a Stoe StadiP 611 diffractometer using a Cu-K α1The radiography was performed in transmission geometry. Scans simultaneously covered either 0 to 36° 2θ (step width of 0.03° 2θ, 30 seconds per step) or 1 to 65° 2θ (step width of 0.015° 2θ, 15 seconds per step), respectively.

[0235] Single crystal X-ray diffraction: Single crystal X-ray structural data were obtained using an Agilent SuperNova diffractometer equipped with a CCD detector using CuKα radiation. Measurements were performed at 200 K (form H2) or 298 K (forms A1, A2, A3, H1), respectively. The single crystal data were used to determine the crystal system and unit cell parameters.

[0236] Differential scanning calorimetry (DSC): DSC scans were acquired on a Mettler-Toledo heat flow differential scanning calorimeter with an autosampler using a nitrogen inert gas atmosphere (50 ml / min). Scans were performed in open Al 40 μL pans from 25 to 300 °C at 5 °C / min.

[0237] Thermogravimetric analysis (TGA): TGA scans were acquired on a Mettler-Toledo Thermogravimetric Analyser with an autosampler using a nitrogen inert gas atmosphere (50 ml / min). Overview scans were performed in an uncovered Al 100 μL pan from 25 to 300 °C at 5 °C / min. The experiment was baseline corrected by a blank run from an empty uncovered Al 100 μL pan using the same temperature profile.

[0238] Dynamic Vapor Sorption (DVS): DCS water vapor sorption isotherms were obtained using a DVS instrument (DVS-Intrinsic, Surface Measurement Systems, SMS) with a microbalance and incubator. Powder samples were accurately weighed into disposable aluminum pans and placed on the sample position of the DVS instrument. A total nitrogen flow rate of 200 mL / min (combined dry and wet flows) was used for humidification. Water vapor sorption isotherms were obtained at 25 °C using an initial desorption segment 1 from 40% RH (relative humidity) to 0% RH (with a 10% RH step), an adsorption segment from 0% RH to 98% RH (with a 10% RH step and a final 8% RH step, respectively), and a final desorption segment 2 from 98% RH to 0% RH (with an initial 8% RH step and a 10% RH step, respectively). For all Rh steps, equilibrium conditions dm / dt≦0.0005 wt % / min were used, with a minimum Rh step time of 10 min and a maximum Rh step time (timeout) of 360 min.

[0239] Non-sink dissolution profile of solid forms: Non-sink dissolution profiles for solid forms were obtained using a shake-flask method involving excess solid material in FaSSIF medium (pH 6.5), with time-resolved sampling for determination of the amount of dissolved API. Approximately 10–20 mg of solid sample was weighed into a glass vial. 7 ml of the respective medium (preheated to 37°C) was added, and the suspension was shaken at 450 rpm at 37°C. After 5, 10, 15, 30, 60, 120 minutes, 24 hours, and 48 hours, 1 ml of suspension was withdrawn and filtered through a 0.2 μm syringe filter. The clear filtrate was analyzed by HPLC after appropriate dilution to determine the amount of dissolved compound / form (also referred to as API).

[0240] FaSSIF: 3 mM sodium taurocholate; 0.75 mM lecithin; 105.9 mM sodium chloride; 28.4 mM monobasic sodium phosphate and 8.7 mM sodium hydroxide, pH 6.5 HPLC method for pH-dependent solubility and miniaturized non-sink dissolution: The levels of dissolved compounds / forms were analyzed by HPLC according to the following conditions: Chromolith RP-18e column 100~3mm Solvent A: Water / formic acid (999:1; v / v) Solvent B: acetonitrile / formic acid (999:1; v / v) Injection volume: 5μL Column temperature: 37℃ HPLC-Gradient:

[0241] [Table 27] example

[0242] The following examples illustrate the invention described above; however, they are not intended to limit the scope of the invention in any way. The examples should be interpreted in a manner that they are not limited to the specifically exemplified features or combinations of features, but instead, the exemplified features can be freely modified or combined as long as the object of the invention is achieved. The beneficial effects of the compounds, combinations, and compositions of the present invention can also be determined by other analytical methods and experimental setups known to those skilled in the relevant art. Similarly, modifications to the preparation methods, especially in reaction conditions, will be readily apparent to those skilled in the art.

[0243] Example 1: Preparation of Compounds 1 and 2 Compound Y is prepared according to the procedure disclosed in WO2016 / 155844, followed by separation of compounds 1 and 2 from compound Y: [ka]

[0244] a. Synthesis of 6-bromo-N-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-nitro-quinolin-4-amine A solution of 3-fluoro-5-methoxypyridin-4-amine (447 mg, 3.02 mmol) dissolved in N,N-dimethylformamide (5 ml) was prepared under a dry nitrogen atmosphere. Sodium hydride (504 mg, 12.6 mmol, 60%) was then added to the solution, and stirring was continued for 5 minutes at room temperature. 6-Bromo-4-chloro-7-methoxy-3-nitro-quinoline (800 mg, 2.52 mmol) was then added to the reaction mixture, followed by stirring for 15 minutes at room temperature, and then the reaction was quenched by the addition of ice water (100 ml). The precipitate was filtered off, washed with ice water, and dried to give 1.00 g (94%) of 6-bromo-N-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-nitro-quinolin-4-amine as a yellow solid.

[0245] b. Synthesis of 6-bromo-N4-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-quinoline-3,4-diamine 6-Bromo-N-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-nitro-quinolin-4-amine (990 mg, 2.20 mmol) dissolved in methanol (100 ml) was provided under a protective nitrogen atmosphere. Raney-Ni (100 mg, 1.17 mmol) was then added to the solution, and the reaction mixture was stirred under a hydrogen atmosphere at atmospheric pressure for 30 minutes. After introducing nitrogen, the suspension was filtered, and the filtrate was dried under reduced pressure. The filtrate was evaporated to dryness under reduced pressure. The residue was crystallized from a mixture of ethyl acetate / petroleum ether to yield 0.86 g (99%) of 6-bromo-N4-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-quinoline-3,4-diamine as a yellow solid.

[0246] c. Synthesis of 8-bromo-1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3H-imidazo[4,5-c]quinolin-2-one A solution of 6-bromo-N4-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-quinoline-3,4-diamine (0.85 g, 2.20 mmol) dissolved in tetrahydrofuran (20 ml) was then added. 1,1'-carbonyldiimidazole (1.84 g, 11.3 mmol) and Huenig's base (1.46 g, 11.3 mmol) were then added. The reaction mixture was heated to 40°C and stirred for 16 hours. The reaction was then quenched by the addition of ice water (200 ml). The precipitate was filtered off, washed with ice water, and dried to give 0.87 g (94%) of 8-bromo-1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3H-imidazo[4,5-c]quinolin-2-one as a pale yellow solid. d. Synthesis of 8-bromo-1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-methyl-imidazo[4,5-c]quinolin-2-one

[0247] 8-Bromo-1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3H-imidazo[4,5-c]quinolin-2-one (0.86 g, 1.94 mmol) dissolved in N,N-dimethylformamide (5 ml) was provided under a dry, protective nitrogen atmosphere. Sodium hydride (388 mg, 9.71 mmol, 60%) and methyl iodide (2.76 g, 19.4 mmol) were then added. The reaction mixture was stirred at room temperature for 10 minutes. The reaction was then quenched by the addition of ice water (100 ml). The resulting precipitate was filtered and dried under reduced pressure to give 0.70 g (80%) of 8-bromo-1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-methyl-imidazo[4,5-c]quinolin-2-one as a pale yellow solid.

[0248] e. Synthesis of 1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-methyl-8-(1,3-dimethylpyrazol-4-yl)imidazo[4,5-c]quinolin-2-one

[0249] In a sealed apparatus under an argon inert gas atmosphere, 8-bromo-1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-methyl-imidazo[4,5-c]quinolin-2-one (150 mg, 0.33 mmol), 1-3-dimethyl-4-(tetramethyl-1,3,2-dioxoborolan-2-yl)-1H-pyrazole (88.4 mg, 0.40 mmol), Pd(PPh3)4 (76.6 mg, 0.07 mmol), and potassium carbonate (91.6 mg, 0.66 mmol) were provided in 1,4-dioxane (15 mL) and water (5 mL). The reaction mixture was heated to 80°C with stirring for 2 hours. After cooling to room temperature, the reaction mixture was compressed to dryness under reduced pressure. The residue was purified by chromatography on silica (ethyl acetate / methanol = 97:3, by volume). The eluate was pressed to dryness, and the resulting crude product was purified by manual or preparative RP-HPLC (water / acetonitrile). After pressing the product fractions, 1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-methyl-8-(1,3-dimethylpyrazol-4-yl)imidazo[4,5-c]quinolin-2-one (70 mg, 47%) was obtained as a colorless solid.

[0250] f. Separation of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Ra)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydro-imidazo[4,5-c]quinolin-2-one and 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydro-imidazo[4,5-c]quinolin-2-one

[0251] The 1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-methyl-8-(1,3-methylpyrazol-4-yl)imidazo[4,5-c]quinolin-2-one (50.0 mg, 0.11 mmol) obtained above was separated via chiral HPLC using SFC to give compounds 1 and 2. The materials were applied to a chiral column, Lux Cellulose-2, and separated using CO₂ / 2-propanol + 0.5% diethylamine (75:25) as the solvent at a flow rate of 5 ml / min and detection at a wavelength of 240 nm. Compression of the product fractions under reduced pressure afforded 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Ra)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one (25.0 mg, 50%) and 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydro-imidazo[4,5-c]quinolin-2-one) (22.1 mg, 44%), both as colorless solids.

[0252] As an example, the starting compounds are readily available: [ka]

[0253] Example 2: Isolation of atropisomers and purification of compound 1 Compounds 1 and 2 can be isolated from compound Y as shown in Scheme 1 and in Figure 6, and as discussed in detail below. Those skilled in the art will recognize that the process below is equally applicable to compounds 3-a and 3-b from compound 3, 4-a and 4-b from compound 4, and 5-a and 5-b from compound 5.

[0254] [ka]

[0255] Step 1: Acetone (108 L, 20 vol), purified water (8.13 L, 1.5 vol), and compound Y (5.42 kg, 1.0 equivalent) were added to a 1,200 L reactor at 20-25°C. 2. Charged with dibenzoyl-L-tartaric acid (4.33 kg, 1.0 equiv.). 3. Heat to 52-55°C to give a clear solution, which was stirred at 52-55°C for 0.5 hours. 4. Cool to 20-25°C. 5. Filter and wash the filter cake with acetone (5.4 L, 1 vol). 6. The cake was collected and dried to give L-salt B (L-salt of compound 2) (a pale yellow solid with 95.9% chiral purity). 7. The mother solution was concentrated to give L-salt A (L-salt of compound 1). 8. L-Salt A and DCM (38 L, 7 vol.) were added to a 100 L reactor and the pH was adjusted to 8-9 with saturated NaHCO3 solution.

[0256] 9. The dichloromethane (DCM) layer was collected and extracted with saturated NaHCO3 along with DCM (16.3 L, 3 vol.), the DCM layers were combined, and the DCM layer was washed with H2O (10.8 L, 2 vol). 10. The DCM layer was concentrated. Acetone (5.4 L, 1 vol) was then added at 20-25°C. 11. Stirred at 20 to 25°C for 1 hour. 12. Filter, collect the cake and dry to give Compound 1 (3.50 kg, white solid with chiral purity of 73.2%, Y: 64.6%).

[0257] Step 2: 1. L-Salt B and DCM (16.2 L, 3 vol.) were added to a 50 L flask and the pH was adjusted to 8-9 with saturated NaHCO3 solution. 2. Collect the DCM layer, extract the aqueous layer with DCM (5.4 L, 1 vol), combine the DCM layers and wash the DCM layer with H2O (5.4 L, 1 vol). 3. DCM was exchanged with 2-ethoxyethanol (1.8 L, 0.3 vol) twice under reduced pressure at 40-50°C. The volume was adjusted to 4.5.4 L (1 vol) with 2-ethoxyethanol. 5. The mixture was heated to 128-130°C to give a slurry, which was stirred at 128-130°C for 44 hours. 6.IPC (ratio 49.5:50.5). 7. Cool to 15-20°C. 8. Filter and wash the cake with methyl-tertiary butyl ether (2.7 L, 0.5 vol). 9. The cake was collected and dried to give compound Y (1.60 kg, off-white solid, overall yield: 29.5%).

[0258] Step 3: 1. At 20-25°C, acetone (70 L, 20 vol) was added to a 100 L reactor with stirring, and then compound 1 (3.5 kg, 1.0 equiv.) was added and water (5.3 L, 1.5 vol.) was charged.

[0259] Charge with dibenzoyl-D-tartaric acid (2.8 Kg, 1.0 eq) at 2.35-40°C, heat to 52-55°C to give a clear solution, and stir at 52-55°C for 0.5 hours. 3. The mixture was cooled to 20 to 25°C in an oil bath and stirred at 20 to 25°C for 17 hours. 4. Filter and wash the cake with acetone (3.5 L, 1 vol). 5. The cake was collected to give D-salt A (D-salt of compound 1) (a pale yellow solid with 98.7% chiral purity). 6. The mother solution was concentrated, then saturated NaHO3 solution (15.5 L, 3.5 vol) and H2O (15.5 L, 3.5 vol) were added. 7. The mixture was stirred at 20 to 25°C for 0.5 hours. 8. Filter and wash the cake with H2O (3.5 L, 1 vol). 9. The filter cake was collected and dried to give compound Y (1.53 kg, white solid with a ratio of 50.3:49.7, Y: 43.7%).

[0260] Step 4: 1. D-salt A (D-salt of Compound 1) and acetone (17.5 L, 5 vol.) were added to a 50 L reactor and warmed to 52-55°C to give a slurry solution, which was then stirred at 52-55°C for 0.5 hours. 2. The mixture was cooled to 20 to 25°C and stirred at 20 to 25°C for 17 hours. 3. Filter and wash the cake with acetone (3.5 L, 1 vol). 4. The filter cake was collected and dried to give D-salt A (3.23 kg, pale yellow solid with 99.2% chiral purity). 5. D-Salt A was added to a 50 L reactor and saturated NaHCO3 solution (16 L, 5 vol) and H2O (16 L, 5 vol) were added. 6. The mixture was stirred at 20 to 25°C for 0.5 hours. 7. Filter and wash the cake with H2O (16 L, 5 vol.). 8. The cake was collected and dried to give compound 1 (1638 g, white solid with 99.1% chiral purity and 99.9% by HPLC, overall yield: 30.2%).

[0261] Example 3: Chromatographic separation, purification, and analysis 3.1 Compounds 1 and 2 can be isolated from compound Y using chromatography on a chiral stationary phase (see, for example, Chiral Liquid Chromatography; W. J. Lough, Ed., Chapman and Hall, New York, (1989); Okamoto, "Optical resolution of dihydropyridine enantiomers by high-performance liquid chromatography using phenylcarbamates of polysaccharides as a chiral stationary phase", J. of Chromatogr. 513:375-378, (1990)). Compounds 1 and 2 can be isolated by chromatography on a chiral stationary phase, for example, a Chiralpak IC column (5 mm, 150 × 4.6 mm I.D.), using isocratic elution with a mobile phase containing, for example, HO / ACN 50 / 50 v / v (ACN: acetonitrile; v: volume). Those skilled in the art will recognize that the same procedure can be applied for compounds 3-a and 3-b, 4-a and 4-b, and 5-a and 5-b.

[0262] The chromatogram thus obtained is illustrated in Figure 5 (column and elution as above: flow 1.00 ml / min; UV @ 260 nm; Tc and TS: 25 ± 5°C (Sconc 0.20 mg / ml); injected volume 10 ml).

[0263] 3.2 As an alternative to the SFC conditions described above, preparative supercritical fluid chromatography may be used, illustratively involving: Chiralpak AS-H (20mm x 250mm, 5μm) column; Isocratic elution (20:80 ethanol:CO with 0.1% v / v NH), BPR (back pressure reg.): approximately 100 bar above atmospheric pressure; column temperature of 40°C, flow rate of 50 ml / min, injection volume of 2500 μl (125 mg), and detector wavelength of 265 nm.

[0264] 3.3 For analysis of the purity of each atropisomer, SFC may be applied, again illustratively using the following setup: Chiralpak AS-H (4.6 mm × 250 mm, 5 μm) column; isocratic elution (20:80 ethanol:CO with 0.1% v / v NH), BPR (back pressure reg.): approximately 125 bar above atmospheric pressure; column temperature of 40°C, flow rate of 4 ml / min, injection volume of 1 μl, and detector wavelength of 260 nm.

[0265] Example 4 Stability of compounds 1 and 2 Quantum mechanical calculation of rotational barriers LaPlante et al. (ChemMedChem, 2011, 6(3), 505-513) describe a quantum mechanical workflow for estimating the energy barrier to axial rotation of drug-like molecules. A similar approach was applied:

[0266] 3D structures of all input molecules were generated using CORINA (Corina version 3.6, Molecular Networks, Germany) and subsequently minimized by Macromodel (version 11.1, Schroedinger, LLC, New York, NY). Based on these input structures, rotational energy barriers were calculated from relaxed dihedral angle scans using the program Jaguar (version 9.1 release 14, Schroedinger, LLC, New York, NY) using the B3LYP / 6-31G** method with a 15° torsion angle increment. The structures were optimized prior to torsional scans using the same level of theory. Default parameters were used, except that the maximum number of minimization steps was set to 500, and the stop_rxn flag was introduced to avoid artificial bond breakage. Representative molecular fragments were used for all calculations. The dihedral angles obtained from the molecular mechanics-minimized structure were used to determine the initial values ​​for the dihedral angle scan, for example, for compound 1 of the present invention or for "LaPlante Reference Compound 1," and the values ​​were set to 47.32° and 35.8°, respectively. For each torsion, values ​​were calculated in 24 steps in terms of the axial bond QM energy. Torsion profiles were obtained by plotting the torsion angle values ​​against the calculated energy.

[0267] The lowest energy barrier for each compound that allows interconversion between both isomers was determined. Experimentally determined interconversion rates and inferred energy barriers for reference compounds 1-6 are known. Computational energy barriers for reference compounds 1-6 range from 9.865 to 31.316 kcal / mol. These values ​​were fitted to the experimentally determined interconversion rates. Calculations for compound 1 of the present invention and compound 2 of the present invention revealed a high predicted rotational barrier of 29.205 kcal / mol, resulting in conversion to a highly stable atropisomer with a predicted rotational half-life in the range of several years (>10 years). The required temperature for the occurrence of racemization of either enantiomeric atropisomer is >100°C. Those skilled in the art will recognize that these values ​​are also exemplary for compounds 3-a and 3-b, 4-a and 4-b, and 5-a and 5-b.

[0268] Example 5: Pharmaceutically acceptable salts of Compound 1 The salt of Compound 1 (Compound 1-a) is pharmaceutically acceptable and was prepared as shown and discussed in detail below. Preparation of fumarate salt (form NF6): Approximately 20 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one was dissolved in 1 ml THF (PA grade) in a 4 ml glass vial with a sealed cap equipped with a magnetic stirrer and dissolved in 1 ml THF (PA grade) at 50 °C by using a magnetic stirrer. At 50 °C, approximately 5.7 mg fumaric acid (~1.1 equiv.) was added to the hot solution, which was then cooled to 5 °C at a cooling rate of 0.1 K / min. The mixture was repeatedly heated to 50 °C (within approximately 30 min) and cooled to 5 °C under stirring (at 0.1 K / min) before a final equilibration step at 5 °C for several hours. To increase the yield of salt formation in the cooled solution, the mixture was further exposed to slow anti-solvent vapor diffusion of n-pentane in a sealed vial configuration. The final solid component was separated by centrifugation and gently dried under a nitrogen purge.

[0269] Preparation of napsylate-salt (form NF7) - Variant 1: Approximately 10 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one was dissolved in ∼250 μl acetone (PA grade) in a 4 ml glass vial with a sealed cap equipped with a magnetic stir bar and by using a magnetic stirrer at 50 °C. At 50 °C, approximately 5.6 mg naphthalene-2-sulfonic acid (∼1.2 equiv.) was added to the hot solution and cooled to 5 °C at a cooling rate of 0.1 K / min.

[0270] The mixture was repeatedly heated to 50° C. (within about 30 min) and cooled (at 0.1 K / min) under stirring to 5° C. before a final equilibration step at 5° C. for several hours. The final solid component was separated by centrifugation and gently dried under a nitrogen purge.

[0271] Preparation of napsylate-salt (form NF7) - Variant 2: Approximately 11.5 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one was dissolved in ∼250 μl THF (PA grade) in a 4 ml glass vial with a sealed cap equipped with a magnetic stir bar and by using a magnetic stirrer at 50° C. At 50° C., approximately 6.6 mg naphthalene-2-sulfonic acid (∼1.2 equiv.) was added to the hot solution, which was then cooled to 5° C. at a cooling rate of 0.1 K / min.

[0272] The mixture was repeatedly heated to 50° C. (within about 30 min) and cooled (at 0.1 K / min) under stirring to 5° C. before a final equilibration step at 5° C. for several hours. The final solid component was separated by centrifugation and gently dried under a nitrogen purge.

[0273] Preparation of the edisylate-salt (form NF8): Approximately 12.6 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one was dissolved in ∼250 μl acetone (PA grade) at 50 °C in a 4 ml glass vial with a sealed cap equipped with a magnetic stir bar and by using a magnetic stirrer. At 50 °C, approximately 6.1 mg ethanedisulfonic acid (∼1.2 equiv.) was added to the hot solution, which was then cooled to 5 °C at a cooling rate of 0.1 K / min. The mixture was repeatedly heated to 50 °C (within ∼30 min) and cooled to 5 °C under stirring (at 0.1 K / min) before a final equilibration step at 5 °C for several hours. The final solid component was separated by centrifugation and gently dried under a nitrogen purge.

[0274] [Table 28]

[0275] Example 6: Preparation of compounds 3, 4, and 5 Synthesis of 8-(1,3-dimethylpyrazol-4-yl)-1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-methyl-imidazo[4,5-c]quinolin-2-one

[0276] [ka]

[0277] Step A: The following was placed in a sealed tube: 1-(3,5-difluoropyridin-4-yl)-8-(1,3-dimethyl-1H-pyrazol-4-yl)-7-methoxy-3-methyl-1H,2H,3H-imidazo[4,5-c]quinolin-2-one (90.0 mg, 0.20 mmol, 95%), potassium carbonate (85.3 mg, 0.62 mmol), CD3OD (0.30 mL, 6.74 mmol), and N,N-dimethylformamide (3 mL). The mixture was stirred at 100 °C for 1 hour. 10 mL of water was added, and the resulting solution was extracted three times with ethyl acetate (10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The crude residue was purified by preparative HPLC (Shimadzu (HPLC-10): Column: Atlantis PrepT3 OBD column, 19*250 mm, 10 μm; Mobile phase: Water (10 mmol / L NH4HCO3) and acetonitrile (hold at 34% acetonitrile for 10 min); Detector: UV 254 nm) to yield 35 mg (38%) of 8-(1,3-dimethylpyrazol-4-yl)-1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-methyl-imidazo[4,5-c]quinolin-2-one as a white solid. Melting point: 260-262 °C. HPLC / MS (purity): 97%. Rt: 1.98 min (Method A). [M+H]: 452. 1H NMR (400MHz, DMSO-d6)ppm=8.92(s, 1H), 8.70(d, J=9.5Hz, 2H), 7.83(s, 1H), 7 .54(s, 1H), 7.00(s, 1H), 3.94(s, 3H), 3.78(s, 3H), 3.60(s, 3H), 1.75(s, 3H).

[0278] Synthesis of 8-(1,3-dimethylpyrazol-4-yl)-1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-(trideuteriomethyl)imidazo[4,5-c]quinolin-2-one

[0279] [ka]

[0280] Step B: In a round-bottom flask, 8-bromo-1-(3,5-difluoropyridin-4-yl)-7-methoxy-1H,2H,3H-imidazo[4,5-c]quinolin-2-one (3.00 g, 6.96 mmol, 94%) was placed in N,N-dimethylformamide (50 ml). Sodium hydride (1.39 g, 34.8 mmol, 60%) was added within 5 minutes at 0°C, followed by the addition of CD3I (3.18 g, 20.9 mmol, 95%). The resulting solution was stirred at room temperature for 15 minutes. 500 ml of ice water was added to the mixture, and the solid was collected by filtration. This resulted in 2.95 g (99%) of 8-bromo-1-(3,5-difluoro-4-pyridyl)-7-methoxy-3-trideuteriomethyl)imidazo[4,5-c]quinolin-2-one as a yellow solid. HPLC / MS (purity) 99%. Rt0.93 min (method B). [M+H]+424, 426. Step C:

[0281] 8-Bromo-1-(3,5-difluoro-4-pyridyl)-7-methoxy-3-trideuteriomethyl)imidazo-[4,5-c]quinolin-2-one (1.80 g, 4.20 mmol, 99%), 1,3-dimethyl-4-(tetramethyl-1,3,2-dioxoborolan-2-yl)-1H-pyrazole (1.97 g, 8.43 mmol, 95%), Pd(PPh3)4 (540 mg, 0.42 mmol, 90%), potassium carbonate (1.22 g, 8.39 mmol, 95%), dioxane (50 mL), and water (10 mL) were placed in a sealed tube purged with and maintained under an inert atmosphere of argon. The mixture was stirred at 80 °C for 2 hours and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (methanol / ethyl acetate, 13:87) to yield 1.35 g (71%) of 1-(3,5-difluoro-4-pyridyl)-8-(1,3-dimethylpyrazol-4-yl)-7-methoxy-3-(trideuteriomethyl)imidazo[4,5-c]quinolin-2-one as a yellow solid. HPLC / MS (purity) 97%. Rt 0.91 min (Method C). [M+H] 440.

[0282] Step D: 1-(3,5-Difluoro-4-pyridyl)-8-(1,3-dimethylpyrazol-4-yl)-7-methoxy-3-(trideuteriomethyl)imidazo[4,5-c]quinolin-2-one (1.35 g, 2.96 mmol) (N,N-dimethylformamide (30 mL), potassium carbonate (818 mg, 5.92 mmol), and CD3OD (2.76 mL, 2.24 g, 62.2 mmol)) was placed in a sealed tube purged and maintained with an inert atmosphere of argon. The mixture was stirred at 100°C for 2 hours. 100 mL of water was added, and the resulting mixture was extracted three times with 200 mL of ethyl acetate. The combined organic layer was washed twice with 100 mL of brine. The organic layer was dried over anhydrous sodium sulfate, concentrated to dryness, and the crude product was crystallized from methanol / acetonitrile (1:25) to yield 1.50 g (66%) of 8-(1,3-dimethylpyrazol-4-yl)-1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-(trideuteriomethyl)imidazo[4,5-c]quinolin-2-one as an off-white solid. Melting point 266-271 °C. HPLC / MS (purity) 97%. Rt 2.00 min (Method C). [M+H]+455.1H NMR (300MHz, DMSO-d6)ppm=8.87(s, 1H), 8.65(d, =7.5Hz, 2H), 7.78(s, 1H), 7.49(s, 1H), 6.95(s, 1H), 3.89(s, 3H), 3.28(s, 3H), 1.71(s, 3H).

[0283] Synthesis of 1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-methyl-8-[3-methyl-(trideuteriomethyl)pyrazol-4-yl]imidazo[4,5-c]quinolin-2-one

[0284] [ka]

[0285] Step E: In a round-bottom flask, 1-(3,5-difluoropyridin-4-yl)-7-methoxy-3-methyl-8-(3-methyl-1H-pyrazol-4-yl)-1H,2H,3H-imidazo[4,5-c]quinolin-2-one (890 mg, 1.89 mmol, 90%) was placed in N,N-dimethylformamide (30 mL). Sodium hydride (377 mg, 9.43 mmol, 60%) was added at 50°C, followed by CD3I (1.44 g, 9.44 mmol, 95%). The resulting mixture was stirred at room temperature for 8 hours. 200 mL of ice water was added, and the solution was extracted four times with 200 mL of ethyl acetate. The combined organic layer was washed twice with 100 mL of brine. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and purified by column chromatography with dichloromethane / methanol (19:1) to yield 400 mg (47%) of 1-(3,5-difluoro-4-pyridyl)-7-methoxy-3-methyl-8-[3-methyl-1-(trideuteriomethyl)pyrazol-4-yl]imidazo[4,5-c]quinolin-2-one as a yellow solid. HPLC / MS (purity) 98%. Rt 0.68 min (Method D). [M+H]+ 440.

[0286] Step F: 1-(3,5-Difluoro-4-pyridyl)-7-methoxy-3-methyl-8-[3-methyl-1-(trideuteriomethyl)pyrazol-4-yl]imidazo[4,5-c]quinolin-2-one (380 mg, 0.84 mmol, 98%), N-methyl-2-pyrrolidone (20 mL), potassium carbonate (368 mg, 2.53 mmol, 95%), and CD3OD (2.45 mL, 53.9 mmol, 98%) were placed in a sealed tube purged and maintained with an inert atmosphere of argon. The mixture was stirred at 100°C for 4 hours. 100 mL of water was added, and the resulting solution was extracted five times with 100 mL of ethyl acetate. The combined organic layers were washed twice with 100 ml of brine, dried over anhydrous sodium sulfate, concentrated to dryness, and purified by column chromatography with dichloromethane / methanol (10:1) to yield 300 mg (74%) of 1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-methyl-8-[3-methyl-1-(trideuteriomethyl)pyrazol-4-yl]imidazo[4,5-c]quinolin-2-one as an orange solid. HPLC / MS (purity) 95%. Rt0.65min (Method D) [M+H]+455.1H NMR (300MHz, DMSO-d6) ppm=8.87(s, 1H), 8.65(d, J=7.5Hz, 2H), 7.78(s, 1H), 7.49(s, 1H), 6.95(s, 1H), 3.89(s, 3H), 3.55(s, 3H) 1.71(s, 3H). HPLC method A: Column Shim-pack XR-ODS, 3.0*50mm, 2.2μm; Mobile phase A: water / 0.05% TFA, Mobile phase B: acetonitrile / 0.05% TFA; Flow rate: 1.0mL / min; Gradient: 5%B to 100%B in 2.2min, hold 1.0min; 254nm.

[0287] HPLC method B: Column: Shim-pack XR-ODS, 3.0*50mm, 2.2μm; Mobile phase A: water / 0.05% TFA, Mobile phase B: acetonitrile / 0.05% TFA; Flow rate: 1.2mL / min; Gradient: 5%B to 100%B in 2.0min, hold 0.7min; 254nm.

[0288] HPLC method C: Column: Poroshell HPH-C18, 3.0*50 mm, 2.7 μm; Mobile phase A: water / 5 mM NH4HCO3, Mobile phase B: acetonitrile; Flow rate: 1.3 mL / min; Gradient: 10% B to 95% B in 2.1 min, hold 0.6 min; 254 nm.

[0289] HPLC method D: Column: Ascentis Express C18, 3.0*50mm, 2.7μm; Mobile phase A: water / 0.05% TFA, Mobile phase B: acetonitrile / 0.05% TFA; Flow rate: 1.5ml / min; Gradient: 5%B to 100%B in 1.2min, hold for 0.5min; 254nm.

[0290] Example 7: Solid forms and solvates of Compound 1 A. Preparation of Solid Form A2 Solid Form A2 of Compound 1 was prepared by various cooling crystallization processes from alcohol: 7.1 Compound 1 was dissolved in 1-propanol at a concentration of approximately 50 mg / mL at 50°C under stirring. The resulting clear solution was cooled to -20°C at a cooling rate of 0.1°C / min, with a final hold period of at least 1 hour at -20°C. Solid-liquid separation was accomplished by filtration under vacuum, and the filtered solid sample was dried overnight under a dynamic nitrogen purge.

[0291] 7.2 Compound 1 was dissolved in iso-butyl alcohol at a concentration of approximately 40 mg / mL under stirring at 50° C. The resulting clear solution was cooled to −20° C. at a cooling rate of 0.1° C. / min with a final hold period of at least 1 hour at −20° C. Solid-liquid separation was accomplished by filtration under vacuum suction, and the filtered solid sample was dried overnight under a dynamic nitrogen purge.

[0292] 7.3 Compound 1 hydrate form H2 (the preparation of which is described below) was dispersed in 2-PrOH at a concentration level of 12% (m / m; relative to the dry mass of Compound 1). The resulting dispersion was heated to 80°C under stirring to obtain a clear solution. An initial cooling ramp from 80 to 70°C was performed at a rate of 0.5°C / min, with a subsequent hold time at -70°C. At 70°C, seed crystals of anhydrous form A2 (ground particles <50 μm; approximately 4.5% relative to the reactor volume) were added. The seed crystals were pre-dispersed in approximately 1 ml of 2-PrOH per 100 mg seed weight. A 10-minute hold time was applied after seeding at 70°C. A cooling ramp from 70°C to 5°C was performed at 0.1°C / min, followed by a 3-hour hold at the final end temperature (5°C). Solid-liquid separation was accomplished by filtration under vacuum suction, and the filtered solid sample was dried at 70° C. overnight under a dynamic nitrogen purge. In an alternative method, Form A2 was prepared from various polymorphic forms by slurry conversion as follows:

[0293] 7.4 The solid components, in particular the various polymorphic forms of compound 1, most preferably the hydrate form H2 (preparation described below), were dispersed in approximately 5.2 vol-equivalents of ethyl acetate and stirred for 21 hours at RT. The precipitate was filtered off by suction and dried under vacuum at 60° C. for at least 72 hours.

[0294] B. Preparation of Solid Form A1 Solid Form A1 of Compound 1 was prepared by the following two methods: Approximately 50 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one (racemic mixture) was subjected to SFC chiral separation using a Lux Cellulose-2 column and a CO2 / 2-propanol + 0.5% DEA (75:25) mixture at a flow rate of 5 mL / min. The resulting fractions were rinsed with dichloromethane and concentrated at a bath temperature of 30 °C to give a solid.

[0295] 7.6 Approximately 10 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one dissolved in 100 μL of dichloromethane (DCM) was evaporated at RT (RT: room temperature, 20-25°C) to obtain a powder.

[0296] C. Preparation of Solid Form A3 Approximately 12 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one solid material, representing an alternative polymorphic form other than 7.7 A3, most preferably representing Form A2, was dispersed in approximately 40 μL THF and stirred at RT (20-25° C.) for approximately 4 weeks. The resulting solid was isolated by centrifugation and gently dried at ambient conditions to obtain a powder.

[0297] D. Preparation of solid form NF9: The solid form NF9 of Compound 1 was prepared by the following two methods: 7.8 Approximately 100 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one dissolved in 2000 L of dichloromethane was rapidly flash evaporated at room temperature (approximately 20-25°C) to give a powder.

[0298] 7.9 Approximately 20 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one dissolved in 1000 μL of acetone at 50°C was added to 1 equivalent of benzoic acid. The solution was cooled to RT (approximately 20-25°C). The solution was subjected to vapor diffusion crystallization at RT (approximately 20-25°C) using a reservoir of n-pentane, which slowly diffused into the solution via vapor phase diffusion. After several days, a solid crystallized, which was isolated by centrifugation and gently dried under a nitrogen purge to obtain a powder.

[0299] E. Preparation of Solid Hydrate Form H1 Solid Form H of the hydrate of Compound 1 was obtained by the following two methods: Approximately 12-13 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one solid form, representing an alternative polymorphic form other than 7.10 H1, most preferably Form A2, was dispersed in approximately 200 μL of methanol and stirred at RT (20-25° C.) for approximately 5 weeks. The resulting solid was isolated by centrifugation and gently dried at ambient conditions to obtain a powder.

[0300] 7.11 Approximately 12 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one dissolved in 750 μL of 1-propanol was evaporated at RT (approximately 20-25°C) to obtain a powder.

[0301] F. Preparation of Solid Hydrate Form H2 Approximately 19 g of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one solid form, representing an alternate polymorphic form other than 7.12 H2, most preferably Form A2, was dispersed in approximately 80 mL DI water and stirred at RT (20-25° C.) for approximately 4 days. The resulting solid was isolated by vacuum filtration and dried at 50° C. under a nitrogen purge to obtain a powder.

[0302] 7.13 Approximately 13–14 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one dissolved in 250 μL THF was rapidly poured into a reservoir of 1500 μL DI water at RT (approximately 20–25°C) under vigorous stirring. The resulting precipitate was isolated by centrifugation and gently dried at ambient conditions to yield a powder.

[0303] G. Preparation of Solid Form NF19 7.14 Approximately 48 mg of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(Sa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydroimidazo[4,5-c]quinolin-2-one dissolved in 1500 μL of 1-propanol was evaporated at 50° C. to give a powder.

[0304] Example 8: Combination with radiation therapy As shown in Figure 20, in an in vivo pharmacological study (FaDu SSCHN tumor model), combined treatment with Compound 1 and 6-week fractionated radiation (6 x 5 days, 2 Gy per fraction) strongly enhanced the efficacy of IR in a dose-dependent manner, along with the level of target engagement.

[0305] The antitumor efficacy of the ATM inhibitor Compound 1 was evaluated in combination with irradiation (IR) in NMRI nu / nu mice bearing xenografts of the human squamous cell head and neck model, FaDu.

[0306] One control group was treated with vehicle alone. The other control group was treated with IR alone in 30 fractions of 2 Gy on a 5-day on / 2-day off schedule over a 6-week period. Two groups were treated with IR (as in the IR-only control) in combination with Compound 1 at an oral dose of 10 mg / kg or 25 mg / kg, 30 minutes before each IR fraction.

[0307] Example 9: Combination with PARP inhibition The efficacy of Compound 1 in combination with olaparib was demonstrated in an HBCx-10 patient-derived triple-negative breast cancer xenograft model developed in immunodeficient female mice, the results of which are shown in FIG.

[0308] Subcutaneously grown 62.5 and 196.0 mm 3 Seventy mice bearing HBCx-10 tumors (P20.1.4 / 0) between 1999 and 2000 were randomly selected for analysis, with mean and median tumor volumes of 131.16 and 126.00 mm, respectively. 3 Treatment was assigned if the patient reached 100%. The study included various groups of 10 mice each: - In Group 1, vehicle olaparib was administered at 10 ml / kg oral once daily x 28 in combination with 10 ml / kg oral vehicle methocel. (3 days on / 4 days off) x 4; In group 2, olaparib was administered at 50 mg / kg orally once daily x 49; - In group 3, the comparative ATM inhibitor ATMix was given orally alone (3 days on / 4 days off) × 5; In group 4, olaparib was administered at 50 mg / kg orally once daily x 49 in combination with ATMix orally (3 days on / 4 days off) x 7;

[0309] In Group 5, olaparib was administered at 50 mg / kg orally once daily in combination with Compound 1 at 100 mg / kg orally (3 days on / 4 days off) x 7. Tumors were measured every other week during the treatment period and weekly during the follow-up period. po (orally) - d (once a day)

Claims

1. The following formula 【Chemistry 1】 or a pharmaceutically acceptable salt thereof.

2. The following formula 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

3. 10. The pharmaceutically acceptable salt of Compound 1 of claim 1 selected from Compound 1 fumarate, Compound 1 napsylate, and Compound 1 edisylate.

4. The following formula 【Transformation 3】 Compound 1 is a solid anhydrous crystalline compound represented by the formula:

5. 5. The solid anhydrous crystal of Compound 1 of claim 4, characterized by one or more peaks selected from peaks at 7.3, 9.6, 11.1, 12.0, 12.7, and 16.2 degrees 2-theta ± 0.2 degrees 2-theta in a powder X-ray diffraction pattern.

6. The monoclinic crystal system and the P21 space group and / or the following parameters of its unit cell: Table 1 6. A solid anhydrous crystal of Compound 1 according to claim 4 or 5, characterized in that it has the formula:

7. below: 【Chemistry 4】 and pharmaceutically acceptable salts thereof.

8. A pharmaceutical composition comprising the compound of any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, or a solid anhydrous crystal thereof, and a pharmaceutically acceptable excipient.

9. 10. The pharmaceutical composition of claim 8, for use in a method for treating cancer and / or tumors in a patient, comprising administering said pharmaceutical composition to the patient.

10. 10. The pharmaceutical composition of claim 9, wherein the method comprises administering the pharmaceutical composition in combination with radiation therapy.

11. 10. The pharmaceutical composition of claim 9, wherein the method comprises administering the pharmaceutical composition in combination with a DNA-damaging agent.

12. the tumor is selected from the group of squamous, bladder, stomach, kidney, head, neck, esophagus, cervix, thyroid, intestine, bone, liver, brain, prostate, genitourinary tract, lymphatic system, larynx, lung, skin, blood, and immune system diseases; and / or 12. The pharmaceutical composition according to any one of claims 9 to 11, wherein the cancer is selected from the group consisting of monocytic leukemia, lung adenocarcinoma, small cell lung carcinoma, pancreatic cancer, glioblastoma, intestinal carcinoma, breast cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma.

13. 10. Use of a compound according to any one of claims 1 to 7, a pharmaceutically acceptable salt thereof, or a solid anhydrous crystal thereof in the manufacture of a medicament for the treatment of cancer and / or tumors.

14. A compound according to any one of claims 1 to 4 and 7, or a pharmaceutically acceptable salt thereof, for the treatment of cancer and / or tumors, optionally in combination with radiotherapy and / or DNA-damaging agents.

15. A solid anhydrous crystal according to claim 5 or 6 for the treatment of cancer and / or tumors, optionally in combination with radiation therapy and / or DNA-damaging agents.

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