RU2822479C2 - Imidazolonylquinoline compounds and therapeutic use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to an atropisomer of an ATM inhibitor of 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(3-fluoro-5-methoxypyridin-4-yl)-7-methoxy-3-methyl-1, 3-dihydroimidazo[4,5-c]quinolin-2-one, specifically to a compound of formula Compound 1, or a pharmaceutically acceptable salt thereof. Also disclosed are Compound 1 in a solid anhydrous form and a pharmaceutical composition for treating cancer, containing Compound 1, a pharmaceutically acceptable salt thereof or a solid anhydrous form.

EFFECT: disclosed compound has better inhibiting properties with respect to ATM, and also has better microsomal clearance and low inhibition of phosphodiesterase (PDE) 2A1, as well as PDE4A1A and PDE4E2, and improved solubility in buffer solutions.

5 cl, 52 dwg, 9 ex

Description

FIELD TO WHICH THE INVENTION RELATES

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[4,5-c]quinolin-2-one, as well as its pharmaceutically acceptable salts and solid forms. These compounds are useful in inhibiting, regulating and/or modulating signal transduction by ATM kinase. The invention also provides compositions that include the specified atropisomers, solid forms, pharmaceutically acceptable salts and deuterated derivatives in accordance with the present invention, as well as methods of using these compositions in the treatment of various disorders associated with ATM kinase, in particular cancer.

PREREQUISITES FOR CREATION OF THE INVENTION

Serine/threonine protein kinase ATM (ataxia telangiectasia mutant kinase) belongs to the PIKK family of kinases with catalytic domains that are homologous to phospho-inositide 3 kinases (PI3 kinase, PI3K). These kinases are involved in many key cellular functions such as cell growth, cell proliferation, migration, differentiation, cell survival and cell adhesion. Specifically, these kinases respond to DNA damage by activating cell cycle arrest and the DNA repair (DDR: DNA damage response) program. ATM is the product of the ATM gene and plays a key role in the repair of double-stranded DNA lesions (DSBs: double-stranded DNA breaks) through homologous recombination and non-homologous continuum joining (NHEJ). This type of double-strand damage is particularly cytotoxic.

One of the main features of tumors in humans is their genomic instability with specific defects in the DNA repair mechanism, which are still unknown in most types of cancer. This instability represents the therapeutic starting point for chemotherapy, which is predominantly administered over time. In addition, there are several syndromes in which the underlying genetic factor is a loss-of-function mutation of a gene that modulates the response to double-stranded DNA damage. This leads to the inclusion of ataxia-telangiectasia, which is caused by a defective ATM gene. The common feature of all these syndromes is that they cause excessive sensitivity to radiation (Lavin & Shiloh (1997) Annu. Rev. Immunol. 15: 177; Rotman & Shiloh (1998) Hum. Mol. Genet. 7: 1555, which are completely incorporated herein by reference). ATM-deficient cells are accordingly sensitive to agents and other measures that cause double-stranded DNA damage, making ATM an attractive target for chemical and radiation sensitization in cancer treatment.

Thus, ATM (ataxia-telangiectasia mutant kinase) is a key regulator of double-strand DNA break repair, which is induced by commonly used radiotherapy and chemotherapy. ATM transmits extensive signals to multiple downstream effectors, including p53. Unrepaired double strand breaks lead to checkpoint activation, cell cycle arrest, and ultimately tumor cell death. Thus, ATM is an attractive point of intervention to inhibit repair of induced double-strand breaks.

The compound Wortmannin was among those initially studied in this context, this compound demonstrated radiation sensitization that could be due to, among other things, ATM inhibition. However, this compound was not suitable for therapeutic use due to in vivo toxicity. Based on the chemical structure of the PI3K inhibitor LY294002, KuDOS Pharmaceuticals identified the ATM inhibitor: KU-55933 (2-morpholino-6-(thianthren-1-yl)-4H-pyran-4-one). Sensitization to ionizing radiation and double-stranded DNA damaging chemotherapeutic agents has been achieved with this compound (Hickson, I., et al. (2004), Cancer Res 64, 9152-9159, which is incorporated herein by reference in its entirety). However, KU-55933 was unsuitable for in vivo use, presumably due to its high lipophilicity. Subsequently, KU-60019 (2-((2S,6R)-2,6-dimethylmorpholino)-N-(5-(6-morpholino-4-oxo-4H-pyran-2-yl)-9H-thioxanthene- 2-yl)acetamide) and KU-559403 (2-(4-methylpiperazin-1-yl)-N-[5-(6-morpholino-4-oxopyran-2-yl)thioxanthen-2-yl]acetamide), and KU-559403 was considered promising enough to enter clinical trials for the treatment of advanced solid tumors.

There are other ATM inhibitors that support the above view and are currently in clinical development, such as AZD0156, AZD1390 and M3541, including clinical trials in combination with radiation therapy.

Although significant progress has been made in the field of ATM inhibitors, there remains a need to provide a compound that has potent ATM kinase inhibition, as well as beneficial selectivity over other kinases, beneficial bioavailability, and/or reduced side effects.

SUMMARY OF THE INVENTION

Providing small molecules that effectively inhibit, regulate and/or modulate ATM kinase signaling is desirable and is an object of the present invention. In addition, it is desirable to provide ATM inhibitors that are selective, i.e. have no or significantly lower activity against other kinases. In addition, it is desirable to provide ATM inhibitors (ATMi) that exhibit beneficial properties against known targets that cause unwanted side effects. Thus, one goal is to provide compounds that have reduced side effects and/or associated toxicity. It is further an object of the present invention to provide an ATM inhibitor with good bioavailability. Another or alternative object of the present invention is to provide an ATM inhibitor with beneficial properties in solid form, such as advantageously low hygroscopicity and/or other physical properties.

At least one objective, as described above, and additional objectives are/are solved using 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[4,5-c]quinolin-2-one (Compound Y), as well as solid forms, pharmaceutically acceptable salts and compositions thereof .

One aspect of the invention provides two compounds that are atropisomers of Compound Y and are represented by the formulas:

and pharmaceutically acceptable salts thereof.

Another aspect of the present invention provides solid forms of Compound 1:

Another aspect relates to certain particularly preferred pharmaceutically acceptable salts of Compound 1, in particular Compound 1 fumarate, Compound 1 edisylate and Compound 1 napsylate, which together may also be referred to hereinafter as "Compounds 1-a".

Another aspect of the present invention provides deuterated Compounds 3, 4 and 5, which are represented by the following formulas:

or their atropoisomers or pharmaceutically acceptable salts.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 displays the annotated ¹ H NMR spectrum of Compound 1.

Figure 2 displays the annotated ¹³ C NMR spectrum of Compound 1.

Figure 3 displays the annotated ¹⁹ F NMR spectrum of Compound 1.

Figure 4 displays the UV-visible spectrum of Compound 1 in methanol.

Figure 5 shows the HPLC chromatogram of Compounds 1 and 2.

Figure 6 shows the preparation of Compound 1.

Figure 7 displays the crystal structure of Compound-1-dibenzoyl-D-tartrate (A) and its X-ray powder diffraction (XPD) (B).

Figure 8 shows the crystal structure of Compound-2-dibenzoyl-L-tartrate.

Figure 9 displays a diagram of the dissolution profile without absorption in biorelevant FaSSIF medium of Compound 1 and its specific salts.

Figure 10 displays the X-ray powder diffraction (XPD) profile of the solid form of Compound 1 fumarate.

Figure 11 displays the X-ray powder diffraction (XPD) profile of the solid form of Compound 1 nasylate.

Figure 12 displays the X-ray powder diffraction (XPD) profile of the solid form of Compound 1 edisylate.

Figure 13 shows the X-ray powder diffraction (XPD) profile of the A2 solid form of Compound 1.

Figure 14 shows the X-ray powder diffraction (XPD) profile of the A1 solid form of Compound 1.

Figure 15 displays the X-ray powder diffraction (XPD) profile of the A3 solid form of Compound 1.

Figure 16 displays the X-ray powder diffraction (XPD) profile of the NF9 solid form of Compound 1.

Figure 17 displays the X-ray powder diffraction (XPD) profile of the solid form of the HI hydrate of Compound 1.

Figure 18 shows the X-ray powder diffraction (XPD) profile of the solid form of H2 hydrate of Compound 1.

Figure 19 displays the X-ray powder diffraction (XPD) profile of the NF19 solid form of Compound 1.

Figure 20 shows the potent inhibition of tumor growth induced by ionizing irradiation (IR) and oral co-administration of Compound 1 (6 x 5 days, 2 Gy; FaDu SCCHN tumor model).

Figure 21 shows the results of an in vivo evaluation of the antitumor activity of Compound 1 and a comparator ATM inhibitor in combination with olaparib in a triple-negative breast cancer xenograft model derived from patient HBX-10.

Figure 22 shows the DSC (Differential Scanning Calorimetry) heating curve of Form A2 of Compound 1.

Figure 23 shows the TGA (thermogravimetric analysis) heating curve of Form A2 of Compound 1.

Figure 24 shows the water absorption isotherm in the EVA (dynamic vapor sorption) method (25°C) of Form A2 of Compound 1.

Figure 25 shows the DSC heating curve of Form A1 of Compound 1.

Figure 26 shows the TGA heating curve of Form A1 of Compound 1.

Figure 27 shows the DSP isotherm of water absorption (25°C) of Form A3 of Compound 1.

Figure 28 displays the DSC heating curve of Form H2 of Compound 1 (hydrate).

Figure 29 shows the TGA heating curve of Form H2 of Compound 1 (hydrate).

Figure 30 shows the DSP isotherm of water absorption (25°C) of Form H2 of Compound 1 (hydrate).

Figure 31 displays the DSC heating curve of Compound 1 fumarate (Form NF6).

Figure 32 shows the TGA heating curve of Compound 1 fumarate (Form NF6).

Figure 33 shows the DSP isotherm of water absorption (25°C) of fumarate Compound 1 (Form NF6).

Figure 34 displays the DSC heating curve of the napsylate of Compound 1 (NF7).

Figure 35 shows the TGA heating curve of the napsylate of Compound 1 (NF7)

Figure 36 displays the DSP isotherm of water absorption (25°C) of the nasylate of Compound 1 (NF7.)

Figure 37 displays the DSC heating curve of the edisylate of Compound 1 (NF8).

Figure 38 shows the TGA heating curve of Compound 1 (NF8) edisylate.

Figure 39 displays the DSP isotherm of water absorption (25°C) of the edisylate of Compound 1 (NF8).

Figure 40 shows X-ray powder diffraction (X-ray powder diffraction) of the methanolate of Compound 1 in solid form S1.

Figure 41 shows the RPD of the mixed hydrate/methanolate of Compound 1 in solid form S2.

Figure 42 shows the RPD of the THF (tetrahydrofuran) solvate of Compound 1 in solid form S3.

Figure 43 shows the RPD of the dioxane solvate of Compound 1 in solid form NF11.

Figure 44 shows the RPD of the chloroform solvate of Compound 1 in solid form NF15.

Figure 45 shows the RPD of acetic acid solvate of Compound 1 in solid form NF16.

Figure 46 shows the RPD of the acetic acid solvate of Compound 1 in solid form NF18.

Figure 47 displays the RPD of the 1,4-dioxane solvate of Compound 1 in solid form NF29.

Figure 48 shows the RPD of the dichloromethane solvate of Compound 1 in solid form NF32.

Figure 49 displays the RPD of the NMP (N-methyl-2-pyrrolidone) solvate of Compound 1 in solid form NF33.

Figure 50 shows the RPD of the acetonitrile solvate of Compound 1 in solid form NF35.

Figure 51 shows the RPD of the 1,4-dioxane solvate of Compound 1 in solid form NF36.

Figure 52 shows the RPD of the dimethylacetamide solvate of Compound 1 in solid form NF37.

DETAILED DESCRIPTION OF THE INVENTION

International patent application WO2016/155844, which is incorporated herein by reference in its entirety, discloses imidazolonyl quinoline compounds that effectively inhibit, regulate and/or modulate ATM kinase signaling. Such connections include Connection Y:

Compound Y is designated Example 4 in WO 2016/155844 and is active in a variety of assays and therapeutic models demonstrating selective inhibition of ATM kinase over PI3Kalpha, PI3Kbeta, PI3Kdelta, PI3Kgamma and mTOR (in enzymatic and cellular assays).

The terms used in this application to define compounds are generally based on IUPAC rules for chemical compounds and, in particular, organic compounds.

It has now surprisingly been discovered that Compound Y exists in the form of two atropisomers that can be isolated and are advantageously stable, and that the corresponding atropisomers exhibit surprising and highly desirable characteristics.

In accordance with 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

• 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 their pharmaceutically acceptable salts.

Compounds 1 and 2 are represented by the following formulas:

where the bold and dotted portions of Compounds 1 and 2 indicate partial rotation of the pyridine ring outside the plane in which the tricyclic ring is located.

One skilled in the art will appreciate that the term "atropoisomer" as used herein refers to a stereoisomer that results from limited rotation about a single bond that creates a chiral axis. In addition, it should be understood that the barrier to rotation around a single bond must be high enough to allow isolation of a single atropisomer. This rotation barrier may result, for example, from steric interactions with other residues of the same molecule, and thereby limits the possibility of rotation around a single bond. Both steric and electronic factors, which can enhance or oppose each other, also play a role.

The use of chiral compounds containing asymmetric carbon atoms is, in principle, well known in drug development. In particular, it is known in the art that racemic mixtures of two chiral compounds typically consist of one more active and one less active enantiomer compared to a racemic mixture. Thus, using only one of the two enantiomers may be beneficial in increasing the overall potency of the compound.

However, the use of atropoisomers, which are stereoisomers that arise only due to hindered rotation around a single bond, is generally considered undesirable. In particular, atropoisomers are generally considered a barrier in drug discovery because the stability of these isomers depends on energy differences resulting from steric deformation or other factors that create a barrier to rotation around a single single bond. Unlike chiral compounds, which are formed by asymmetric carbon atoms, atropisomerism is difficult to predict. Namely, it is usually not possible to easily predict the stability of an atropisomer. In particular, the height of the electrical barrier determines the time of interconversion of the two corresponding atropisomers. Interconversion of a biologically active atropisomer into the corresponding other atropisomer may thus reduce its biological activity and cause off-target or other undesirable effects. Therefore, only stable atropisomers with a sufficiently high energy barrier may be suitable for drug detection.

Surprisingly, it was found that the atropisomers of Compound 1 and Compound 2 do not undergo significant interconversions to the corresponding other atropisomer even after long periods of time exceeding ten years (rotational half-life >10 years was determined using computer modeling) and at temperatures above room temperature. The inversion temperature of atropisomers was estimated to be greater than 100°C in solution. This very good stability was confirmed experimentally. This makes these atropisomers very suitable for pharmaceutical use, production, formulation and provides a sufficient shelf life.

The absolute structure of Compound 1 was determined based on the (2S,3S)-dibenzoyl-D-tartaric acid salt, as well as by X-ray diffraction of solid form A2, which will be described in more detail below. The structure of Compound 1 was confirmed by spectroscopy (NMR, MS, IR and UV), X-ray diffraction, elemental analysis and polarimetry. The ¹ H-, ¹³ C- and ¹⁹ F-NMR spectra of Compound 1 are shown in Figures 1-3, the UV-visible spectrum is shown in Figure 4. The RPD of solid form A2 of Compound 1 is illustrated in Figure 13. Crystal structure and RPD of Compound-1- Dibenzoyl-D-tartrate is shown in Figures 7A and B, the crystal structure of Compound-2-dibenzoyl-L-tartrate is illustrated in Figure 8.

Compounds 1 and 2 are very potent ATM kinase inhibitors. As illustrated by Table 1, Compound 1 has clearly higher ATM inhibition values in all assays compared to Compound Y, which is a mixture of Compounds 1 and 2:

Moreover, these compounds are selective over related kinases, including mTOR (>30,000 nM), DNA-PK and, most notably, ATR (serine/threonine protein kinase). Surprisingly, both Compounds 1 and 2 were less potent ATR kinase inhibitors compared to Compound Y, i.e. more preferable from the point of view of selectivity.

While Compound 2 was no different from Compound Y with respect to ATM inhibition (see Table 1), it had significantly greater selectivity over both ATR (serine/threonine protein kinases) and DNA protein kinase than Compound Y, which is obvious from Table 2 above.

Thus, the compounds of the present invention may be particularly advantageously used to selectively target specific DNA repair mechanisms, in particular homologous recombination, which specifically targets double-stranded DNA damage.

An additional advantage of the selective ATM inhibitors of Compounds 1 and 2 is reduced toxicity, particularly in relation to off-target effects, and thus better tolerability of higher doses of the compound. Thus, the compounds according to the invention open up new possibilities in cancer therapy. For example, Compounds 1 and 2, most preferably Compound 1, can be used in targeted combination therapies, eg, those that include a potent and selective ATM inhibitor and a potent and selective other inhibitor, eg, an ATR inhibitor.

Overall, Compound 1 was found to have the most advantageous overall combination of these properties. Surprisingly, it not only has the best ATM inhibitory properties, but also has the best microsomal clearance values and the lowest inhibition of phosphodiesterase (PDE) 2A1, as well as PDE4A1A and PDE4E2. Phosphodiesterase inhibition itself is associated with a variety of pharmacological effects, and PDE inhibitors are available as drugs to treat a variety of conditions, including depression, multiple sclerosis, and chronic obstructive pulmonary disease, to name a few, but all of which in this case cause off-target effects. effects. PDE4 inhibition is also known to be associated with a risk of nausea and is therefore avoided. Therefore, high IC50 values, ie weak inhibition of these off-targets, are desirable. As can be seen from Table 3 below, Compound 1 has IC50 values for PDE4 inhibition that are approximately 5 times higher than those of Compound Y.

Table 4 illustrates other beneficial properties of Compound 1, including a favorable high bioavailability of approximately 80% and favorable properties against CYP (cytochrome P450) and hERG (cardiac ion channel), indicating that no safety-relevant interactions are expected with cardiac ion channel Kv11.1 hERG.

In addition, it was unexpectedly found that both Compounds 1 and 2 showed significantly improved solubility in biological buffer solutions (see Table 5 below) compared to Compound Y.

The predicted slow clearance from human plasma and the high bioavailability of Compound 1 contribute to the corresponding low dose requirements.

The structures presented for Compound 1 or 2 are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, H, C, N in each case also include heavier isotopes of these atoms. This applies in particular to H, in which case deuterium or tritium can advantageously be used, and to the replacement of carbon by carbon enriched in ¹³ C or ¹⁴ C, which is also within the scope of the present invention. In some preferred embodiments, isotope-enriched atoms are not used, but rather the atoms are used in their natural forms in terms of isotope distribution.

Reference to compounds or salts in accordance with the present invention should be considered to include solvated forms, that is, solvates thereof, which means solvates of both the free form and the salt. Solvates mean adducts of inert solvent molecules with compounds that are formed due to their mutual attraction. Solvates are, for example, mono- or dihydrates or alcoholates. Exemplary solvate options are disclosed in more detail below.

As stated above, the present invention provides two stable atropisomers, Compounds 1 and 2. The preparation of these two atropisomers is typically based on separation and purification methods, as will be described in more detail below. One skilled in the art will appreciate that this may not provide perfectly pure products. However, the present invention provides Compound 1 substantially free of Compound 2 and Compound 2 substantially free of Compound 1. "Substantially free" in this context preferably means that substantially pure Compound 1 may contain a maximum of 20% by weight of Compound 2 or salts thereof, preferably a maximum of 15% by weight, more preferably a maximum of 10% by weight, for example a maximum of 5% by weight, a maximum of 2.5% by weight, a maximum of 1% by weight, a maximum of 0.5% by weight or a maximum of 0.1% by weight of Compound 2 or a salt thereof, the balance up to 100% being Compound 1. In one example, substantially pure Compound 1 may consist of 99% by weight of Compound 1 and 1% by weight of Compound 2. The same applies and to Compound 2, that is, Compound 2 that is substantially free of Compound 1, will preferably mean that pure Compound 2 may contain a maximum of 20% by weight of Compound 1 or a salt thereof, with the preferred ranges disclosed for Compound 1 being are equally applicable (in turn) by analogy. Also, 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 herein below, even without specific mention, unless expressly stated otherwise.

In another embodiment, Compound 1 or 2, respectively, contains no more than about 5.0 HPLC area percent of total organic impurities and, in some embodiments, no more than about 3.0 HPLC area percent of total organic impurities and , in some embodiments, does not contain more than about 1.5 area percent of total HPLC organic impurities of the total area of the HPLC chromatogram. In other embodiments, Compound 1 or 2 contains no more than about 1.0 HPLC percent of any single impurity; no more than about 0.6 percent of the HPLC area of any individual impurity and, in some embodiments, no more than about 0.5 percent of the HPLC area of any individual impurity relative to the total area of the HPLC chromatogram. For example, for an HPLC chromatogram, the method described in EXAMPLE 3.3 can be used to analyze the purity of the corresponding atropisomers. In this case also, 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 hereinafter, unless expressly stated otherwise.

In accordance with another embodiment, the present invention provides a pharmaceutical composition that contains an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof. In an alternative embodiment, the pharmaceutical composition contains an effective amount of Compound 2 or a pharmaceutically acceptable salt thereof. In accordance with another embodiment, the present invention provides a method for preparing such a composition as described herein (eg, a composition that may include an effective amount of Compound 1 or 2). Reference to Compound 1 or 2 should be understood in this context to mean that any amount of the corresponding other atropisomer may be only that amount that is in accordance with the definitions of “substantially free” of the corresponding other atropisomer, “substantially free of impurities” or total the amount of impurities, respectively, that is, will be taken into account in the amount of the said compound, and not be present separately. The same applies to the corresponding salts. As stated above, reference to Compound 1 or 2 or a salt thereof shall equally include any solid form or solvate, such as those further disclosed herein below, unless specifically stated otherwise. In exemplary embodiments, Compound 1 is contained in the pharmaceutical composition in its free, that is, non-salt, form.

In other embodiments, a method of treating cancer is provided using a compound or composition, respectively a pharmaceutical composition or a pharmaceutically acceptable salt of a compound, as described herein. According to another embodiment, 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 the treatment of cancer. In another embodiment, the present invention provides a compound or pharmaceutical composition as described herein for use as a medicine, particularly for the treatment of cancer. Again, reference to a compound or a salt thereof will include any solvate form or solid form of these compounds or salts, such as those disclosed hereinafter, unless explicitly stated otherwise.

Free form and salts

The compounds according to the invention can be used in their free form, that is, as represented in the formulas given above. For example, the free form of Compound 1 has been found to be particularly useful, and illustrative solid forms thereof, including a preferred solid form, will be described in more detail below.

On the other hand, the present invention also includes the use of these compounds in the form of their pharmaceutically acceptable salts, which can be prepared from various organic and inorganic acids by methods known in the art. Acceptable pharmaceutically acceptable salts of the compounds according to the invention can be prepared by conventional methods. The compound of the invention may be converted to its associated acid addition salt using an acid, for example by reacting the compound and an equivalent or excess amount of acid in a suitable solvent such as, for example, THF or acetone, followed by crystallization and cooling of the resulting saturated solution . Alternatively, antisolvent crystallization or evaporation crystallization may be used.

Examples of suitable pharmaceutically acceptable salt of the compound according to the present invention, in particular Compound 1, include HCl salt, sulfate salt, tosylate salt, besylate salt, lactate salt, in particular L-lactate salt.

Preferred salts of Compound 1 may include the fumarate salt, the napsylate salt and the edisylate salt, which together may hereinafter also be referred to as Compound 1a.

Compound 1 napsylate can be prepared from Compound 1 using naphthalenesulfonic acid and either THF or acetone as a solvent. This results in good crystallinity. The preferred ratio of Compound 1: napsylate is approximately 1:1.

The edisylate of Compound 1 can be prepared by using ethanesulfonic acid and cooling crystallization from acetone. The preferred ratio of Compound 1:edisylate is approximately 1:1. In this case, good crystallinity was obtained.

Fumarate Compound 1 can be prepared using antisolvent vapor diffusion in THF using n-pentane as the antisolvent and fumaric acid as the acid. The ratio of Compound 1:fumarate was shown to be 1:0.9. The resulting salt has very good general physical properties and good crystallinity. The fumarate salt is the preferred salt option, due in part to its desirable lack of hygroscopic properties.

Detailed examples of suitable methods for preparing the fumarate, napsilan and edisylate salts of Compound 1 in accordance with the present invention are disclosed in EXAMPLE 5.

These salts of Compound 1 were found to exhibit a higher initial dissolution rate than the parent Compound 1. The non-absorption dissolution profile in FaSSIF buffer solution (pH 6.5) of Compound 1 and its specific salts is shown as an example in Figure 9. Salts with The favorable dissolution characteristics are Compound 1 edisylate, Compound 1 fumarate and Compound 1 napsylate. The starting Compound 1 was used as a mixture of various crystalline and solvated forms.

One skilled in the art will appreciate that the anionic acid moiety and Compound 1 are ionically bound to the form of Compound 1-a. It is believed that Compound 1-a may exist in a variety of physical forms. For example, Compound 1-a may 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 in which a compound or its salts can crystallize.

In some embodiments, Compounds 1-a are crystalline solids 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, or at least about 95% by weight of crystalline Compound 1-a, is present. In other embodiments, at least about 99% by weight crystalline Compound 1-a is present. These percentages refer to the absolute weight of Compound 1-a (100 wt %).

In an illustrative embodiment, the present invention provides a solid form of the fumarate of Compound 1 characterized by an X-ray powder diffraction profile substantially identical to the profile depicted in Figure 10 and/or characterized by one or more peaks in an X-ray powder diffraction profile selected from approximately the following.

The fumarate salt of Compound 1 is anhydrous. Its solid form may also be referred to as fumarate-NF6. The DSC heating curve, TGA heating curve and DSP water absorption isotherm (25°C) of fumarate-NF6 are shown in Figures 31, 32 and 33. The results of dissolution without absorption measurements are presented in the Table below (data regarding dissolution without absorption in FaSSIF at pH 6.5, the method is described in the experimental part):

As used herein, the term “about/about” when used in reference to a degree 2-theta value refers to the specified value of ±0.3 degrees 2-theta (°2θ). In some embodiments, “about/about” refers to ±0.2 degrees 2-theta or ±0.1 degrees 2-theta, most preferably ±0.2 degrees 2-theta.

Any solid form, respectively polymorph, described in this application may be characterized by one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more X-ray diffraction or powder X-ray diffraction (°2θ) peaks. Any solid form or polymorph described herein is preferably characterized by at least six X-ray diffraction peaks (°2θ, preferably ±0.2°2θ). Preferred peaks for solid form or polymorph characterization are indicated in bold and asterisks in the corresponding peak lists.

In a further illustrative embodiment, the present invention provides a solid napsylate form of Compound 1 characterized by an X-ray powder diffraction profile substantially corresponding to that depicted in Figure 11 and/or characterized by one or more X-ray powder diffraction peaks selected from approximately the following.

The thermal properties of the nasylate salt of Compound 1 and its water adsorption properties are shown in Figures 34, 35 and 36. The solid form obtained for the nasylate salt may also be referred to as NF7 nasylate. In addition, the dissolution profile is represented by the following experimental data for dissolution without absorption (FaSSIF, pH 6.5):

In a further illustrative embodiment, the present invention provides a solid edisylate form of Compound 1 characterized by a powder x-ray diffraction profile substantially corresponding to that depicted in Figure 12 and/or characterized by one or more peaks in its powder x-ray diffraction profile selected from approximately the following.

Thermal properties and dissolution data for the edisylate salt of Compound 1 are illustrated in Figures 37, 38 and 39. The solid form of the edisylate salt may also be referred to as NF8. It is an anhydrous/salt form. Data regarding dissolution without absorption (FaSSIF, pH 6.5) is provided in the table below:

In accordance 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 substantially free of Compound 2 or its salts. In additional embodiments, Compound 1-a or another salt of Compound 1 is also provided substantially free of impurities. In another embodiment, Compound 1-a or any other salt of Compound 1 contains no more than about 5.0 HPLC area percent of total organic impurities relative 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 its salts", "free of impurities" and percentage of total area of organic impurities are equally applicable here. The same applies by analogy to any of the salts of any of the compounds and, in particular, the atropisomeric compounds in accordance with the present invention.

In accordance with another embodiment, the present invention includes a pharmaceutical composition that contains an effective amount of Compound 1-a. In accordance with another embodiment, the present invention provides a method for preparing such a composition as described herein (eg, a composition that may include an effective amount of Compound 1-a). Another embodiment provides a method of treating cancer using Compound 1-a or a composition thereof in accordance with the present invention. In accordance with another embodiment, the present invention provides for the use of a composition described herein in the manufacture of a medicament for the treatment of cancer. Compound 1-a may be present in the (pharmaceutical) composition in the same amounts as indicated for Compound 1. With regard to the presence of another atropisomer or a salt thereof in the composition, accordingly, the considerations set out above for Compound 1 are equally applicable by analogy.

Solid forms and solvates

In accordance with another aspect, the present invention provides solid forms of Compound 1 or 2, in particular Compound 1.

Those skilled in the art will appreciate that the compounds of the present invention may exist in a variety of physical forms. For example, they may be in solution, suspension or solid form.

In some preferred embodiments, Compound 1 is in solid form. Solid forms are generally preferred here because they allow the formulation of solid pharmaceutical compositions. When Compound 1 is in solid form, the compound may be amorphous, crystalline, or a mixture thereof. Exemplary solid forms of Compound 1 are described in more detail below.

In accordance with one embodiment, the present invention provides Compound 1 as an amorphous solid. Amorphous solids are well known to those of ordinary skill in the art and are typically prepared by methods such as lyophilization, spray drying, or accelerated precipitation.

In other embodiments, Compound 1 is a crystalline solid. As used herein, the term "polymorph" refers to the different crystal structures in which a compound can crystallize.

In some embodiments, Compound 1 is a crystalline solid 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 some 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 other embodiments, at least about 99% by weight of crystalline Compound 1 is present These percentages refer to the absolute weight of Compound 1 (100 wt %). The same applies, mutatis mutandis, to the acceptable amorphous content in any crystalline form, or polymorph, of all compounds disclosed herein, including those described for salts, anhydrous forms, solvates, and other forms herein.

In accordance with one aspect, the present invention provides a solid form of Compound 1 that is a solid form of anhydrous Compound 1, preferably crystalline anhydrous Compound 1. Five different polymorphic forms of anhydrous Compound 1 are disclosed herein. In the context of the specific solid forms described in this section and including anhydrous forms and solvates, reference to Compound 1 should be understood to refer to the compound itself, that is, its free (non-salt) form.

The first anhydrous crystalline form of Compound 1 is hereinafter referred to as “Form A2” and is a polymorph characterized by an X-ray powder diffraction (XPD) profile substantially identical to that depicted in Figure 13 and has been found to be highly advantageous.

In accordance with one embodiment, Form A2 is characterized by one or more peaks in its X-ray powder diffraction pattern selected from peaks 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 an X-ray powder diffraction pattern selected from peaks 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 an X-ray powder diffraction pattern selected from peaks 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 peaks in its X-ray powder diffraction pattern selected from peaks at about 7.3, about 9.6, about 12.7, about 16.2, about 22.6, and approximately 25.1 degrees 2-theta. Specifically, in embodiments, Form A2 is characterized by six or more or substantially all of the peaks in its X-ray powder diffraction profile selected from peaks 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 2-theta.

In an illustrative embodiment, Form A2 may be characterized by one or more, preferably six and up to substantially all peaks in its X-ray powder diffraction (XRD) profile selected from those presented below:

It will be appreciated that the polymorphic form described above can be characterized, for example, by reference to any of the peaks in its corresponding X-ray diffraction (XRD) profile. As stated previously, bold font and asterisks indicate peaks that may be favored for polymorph characterization.

Form A2 may be characterized by one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more RPD peaks (°2θ ) according to the table above. Any polymorph described herein is advantageously characterized by at least six RD or RPD peaks (°2θ, preferably ±0.2).

Form A2 can be further characterized by having a monoclinic crystal system and a P2 space group of ₁ . Form A2 can be further characterized by one or more of the following parameters of its unit cell, as indicated in the following Table:

Form A2 of Compound 1 has favorable overall properties, which is further supported by its thermal behavior and water uptake as shown by the DSC heating curve (Figure 22), TGA heating curve (Figure 23) and the DSP water uptake isotherm (at 25°C) ( Figure 24). Form A2 absorbs negligible amounts of water (<2%) even up to a relative humidity of 100% and is thus superior to, for example, Form A3.

Another advantage of Form A2 is its favorable dissolution behavior, as shown in the following table, which presents the amounts of Compound 1 in Form A2 dissolved at various times (data regarding dissolution without absorption in FaSSIF at pH 6.5, method described in the experimental section) .

Form A2 can be prepared from Compound 1 by cooling crystallization from alcohols, as one example. Appropriate methods and reaction conditions are described in detail in EXAMPLE 7.

For example, Form A2 crystals having favorable properties can be reproducibly produced by a controlled crystallization process that includes:

a) Preparation of a dispersion of Compound 1, for example a hydrate of Compound 1, such as the H2 hydrate form of Compound 1, in a suitable solvent, for example an alcohol,

b) Heating the dispersion to obtain a solution, preferably a clear solution,

c) Controlled cooling of the solution,

d) Adding Form A2 seed crystals,

e) Controlled cooling of the seed crystal solution, for example at a rate of approximately 0.1°C/min.

Suitable alcohols and crystallization conditions, such as temperature, can be obtained from EXAMPLES 7.1-73, which are applicable beyond the specific embodiment. The present invention also relates to anhydrous crystalline Compound 1 in Form A2, which can be obtained using the above process or essentially in accordance with any of the processes described in EXAMPLES 7.1-7.4.

The other anhydrous crystalline form of Compound 1 is hereinafter referred to as “Form A1” and is a polymorph that exhibits a powder X-ray diffraction pattern substantially consistent with that depicted in Figure 14. Suitable methods for its preparation are described in the EXAMPLE. 7.

Form A1 may be characterized by one or more, preferably six and substantially all peaks in its X-ray powder diffraction profile selected from those listed below:

The thermal properties of Form A1 of Compound 1 were assessed using differential scanning calorimetry (DSC) and

thermogravimetric analysis (TGA), as shown in Figures 25 and 26.

The 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 profile substantially consistent with that shown in Figure 15. A suitable method for its preparation is described below in EXAMPLE 7.

Form A3 may be characterized by one or more, preferably six and substantially all peaks in its X-ray powder diffraction profile selected from those listed below:

As can be seen from Figure 27, Form A3 of Compound 1 exhibits very low water adsorption up to about 70% relative humidity. Form A3 can be further characterized by crystal system and unit cell parameters as listed in Table 7 below. Dissolution data without absorption in FaSSIF at pH 6.5 of Compound 1 in Form A3 is given in the table below:

The 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 profile substantially consistent with that shown in Figure 16. The method of its preparation is described in EXAMPLE 7.

The NF9 form may be characterized by one or more, preferably six and up to all peaks in its X-ray powder diffraction profile selected from those presented below:

In a further embodiment, the present invention provides hydrates of Compound 1, preferably a solid form of the hydrate of Compound 1, preferably a crystalline hydrate of Compound 1. Disclosed herein are two different hydrates, respectively polymorphic forms of the hydrates of Compound 1. As mentioned above, in the context of these particular solid forms, a reference to Compound 1 should be understood as a reference to the compound itself, i.e. its free (non-salt) form.

The first crystalline hydrate form of Compound 1 is hereinafter referred to as “Form H1” and is a polymorph characterized by an X-ray powder diffraction profile substantially consistent with that depicted in Figure 17. Suitable methods for its preparation are described in EXAMPLE 7.

The HI form may be characterized by one or more, preferably six and substantially all, peaks in its X-ray powder diffraction profile selected from those presented below.

The second hydrate crystalline form of Compound 1 is hereinafter referred to as “Form H2” and is a polymorph characterized by an X-ray powder diffraction profile that is substantially consistent with that depicted in Figure 18. Suitable methods for its preparation are described in EXAMPLE 7.

Form H2 may be characterized by one or more, preferably six and substantially all, peaks in its X-ray powder diffraction profile selected from those presented below.

The hydrate of Compound 1 as crystalline Form H2 can be further characterized by having a triclinic crystal system and space group P1. The H2 form can be characterized using one or more of the following unit cell parameters, as listed in Table 7 below.

The thermal and water adsorption properties of Form H2 are illustrated in Figures 28, 29 and 30. Dissolution data without absorption in FaSSIF (pH 6.5) for Form H2 of Compound 1 were determined to be as follows:

The fifth crystalline form of anhydrous Compound 1 is referred to hereinafter as “Form NF19” and is a polymorph that exhibits an x-ray powder diffraction profile that is substantially consistent with that depicted in Figure 19. Suitable methods for its preparation are described in EXAMPLE 7.

The NF19 form may be characterized by one or more, preferably six and up to all, peaks of its X-ray powder diffraction profile selected from those listed below:

The solid forms A1, A3, H1 and H2 may also or alternatively be characterized by the presence of a specific crystal system, space group and/or unit cell parameters selected from a, b, c, α, β, γ and V, as listed below in the Table 7:

The present invention also relates to the following solvate forms, which can be easily obtained by crystallization from suitable solvents, however, they have been found to have significantly less advantage 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 S2), THF solvate of Compound 1 (solid form, designated S3), 1,4-dioxane solvate forms of Compound 1 in multiple solid forms (solid forms, designated as NF11 [from an experiment converting the suspension into anhydrous form at ~26 mg/200 µl in 1,4-dioxane at room temperature], NF29 [from a crystallization experiment at cooling 50-5°C in 1,4-dioxane], NF36 [from an anhydrous suspension experiment at -52 mg/150 µl in 1,4-dioxane at room temperature]), chloroform solvate of Compound 1 (solid form designated NF15), acetic acid solvate of Compound 1 in various solid forms (solid forms designated NF16 [from room temperature crystallization experiment in acetic acid], NF18 [from evaporation crystallization experiment at 50°C in acetic acid]), dichloromethane (DCM) solvate of Compound 1 (solid form , designated as NF32), NMP (N-methyl-2-pyrrolidone) solvate of Compound 1 (solid form, designated as NF33), acetonitrile solvate of Compound 1 (solid form, designated as NF35), dimethylacetamide (DMAA) solvate of Compound 1 (solid form designated NF37). The RPDs of the solid forms of these solvates are shown in Figures 40-52, and the corresponding peaks are listed in the tables below:

In accordance with another embodiment, the present invention provides a pharmaceutical composition that contains an effective amount of Compound 1 in Form A2 and is the preferred solid form. In accordance with another embodiment, the present invention provides a method for preparing such pharmaceutical compositions as described herein, for example, a pharmaceutical composition that includes an effective amount of Compound 1 in Form A2. Another embodiment relates to a method of treating cancer using a pharmaceutical composition containing an effective amount of Compound 1 in Form A2, as 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 the treatment of cancer. In a further embodiment, the present invention provides Form A2 for use as a medicine, preferably for the treatment of cancer. As set forth above, the exemplary embodiments, ranges, purities, etc. disclosed for Compound 1 are equally valid for Form A2.

In accordance with another embodiment, the present invention provides a pharmaceutical composition that includes an effective amount of Compound 1 as any of the solid forms of Compound 1 anhydrous or Compound 1 hydrate as described above. In accordance with another embodiment, the present invention provides a method for preparing such pharmaceutical compositions as described herein, for example, a pharmaceutical composition that includes an effective amount of Compound 1 in one such solid form. Another embodiment provides a method of treating cancer using a pharmaceutical composition containing an effective amount of Compound 1 in one of the solid forms as described herein. In accordance with another embodiment, the present invention provides the use of Compound 1 in solid form, as described herein, in the manufacture of a drug for the treatment of cancer. In a further embodiment, the present invention provides a solid form of Compound 1, as described herein, for use as a drug, preferably for the treatment of cancer. As discussed above, the exemplary embodiments, ranges, purities, etc. disclosed for Compound 1 are equally valid for solid forms.

The present invention also provides a solid form of the anhydrous Compound 1 or the hydrate of Compound 1 as described herein, which is or can be prepared in accordance with the method described in EXAMPLE 7.

Deuterated Embodiments

In accordance with a further aspect, the present invention provides deuterated derivatives of Compound Y. In accordance with one embodiment, the present invention provides:

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

• 1-[3-fluoro-5-(trideuteriomethyl)-4-pyridyl]-7-methoxy-3-methyl-8-[3-methyl-(trideuteriomethyl)pyrazol-4-yl]imidazo[4,5-c ]quinolin-2-one (Compound 4) and

• 8-(1,3-dimethylpyrazol-4-yl)-1-[3-fluoro-5-(trideuteriomethoxy)-4-pyridyl]-7-methoxy-3-methyl-imidazo[4,5-c]quinoline -2-on (Compound 5),

as well as their salts.

Compounds 3, 4 and 5 are represented by the following formulas:

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

or their salts.

Consistent with what has been set forth above with respect to Compounds 1 and 2, the present invention provides Compounds 4-a, 5-a, 6-a, 4-b, 5-b, 6-b substantially free of the corresponding another atropoisomer, including any salt thereof. In additional embodiments, these compounds are also provided substantially free of impurities. In another embodiment, these compounds contain no more than about 5.0 area percent of total organic impurities as determined by HPLC, relative 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 its salts", "free from impurities" and percentage of total area of organic impurities are, by analogy, equally applicable herein with respect to the corresponding others atropoisomers of the corresponding compound.

In accordance with another embodiment, the present invention provides a pharmaceutical composition that contains an effective amount of at least one of compounds 3, 4 or 5, an atropisomer thereof, or a pharmaceutically acceptable salt. In accordance with another embodiment, the present invention provides a method for preparing such pharmaceutical compositions described in this application. Another embodiment provides a method of treating cancer using a pharmaceutical composition described herein. In accordance with another embodiment, the present invention provides the use of a composition described herein in the manufacture of a medicament for the treatment of cancer. The exemplary and preferred embodiments disclosed above for Compound 1 are equally applicable to these compounds.

Receipt

Compounds 1 and 2 according to the present invention can be prepared starting from Compound Y, which was described previously. As disclosed in WO 2016/155884, 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(3-fluoro-5-methoxypyridin-4-yl)-7-methoxy-3-methyl- 1,3-dihydroimidazo[4,5-c]quinolin-2-one (Compound Y) can be prepared according to the following reaction sequence:

Typical reaction conditions for each of these steps a - e are given in EXAMPLE 1, as well as methods for preparing the starting compounds. Other suitable reaction conditions will be apparent to one skilled in the art.

Compounds 1 and 2 can then be prepared by suitable separation methods from Compound Y, illustrative embodiments of which are presented in EXAMPLES 1, 2 and 3.

Atropoisomers can be separated starting with Compound Y using chiral chromatography, including supercritical fluid chromatography (SFC). Examples of suitable methods are described in detail in EXAMPLES 1 and 3.

The corresponding undesired atropisomers can be subjected to racemization, for example, thermal racemization to obtain Compound Y for use as a new starting material, as shown schematically below as one example.

In an alternative embodiment, Compounds 1 and 2 can be prepared starting from Compound Y by crystallization using an optically active acid, for example dibenzoyltartaric acid. The reaction of Compound Y with an optically active acid produces salts of a pair of atropisomers. These salts of Compounds 1 and 2 exhibit different physicochemical properties (eg, solubility, phase distribution) and can be separated using these differences.

As illustrated by the scheme below, in one embodiment, Compound Y reacts with an optically active acid to form a mixture of two salts in the mother liquor, whereby a salt of Compound 2 (L-salt B) first precipitates and is removed by filtration , and the corresponding salt of Compound 1 (L-salt A) is collected only after further concentration of the mother liquor and precipitation. The salt of Compound 1 is then first converted to its free form and isolated, and then reacted with a corresponding other optically active form of the acid to produce the corresponding salt of Compound 1, which in the next step is converted to the free base having high optical purity. In this case, Compound 2 can be racemized to obtain Compound Y as fresh starting material.

A detailed example of the production scheme is provided in EXAMPLE 2 and Figure 6.

The deuterated Compounds 3, 4 and 5 of the present invention can be prepared as described in detail in EXAMPLE 6, and the atropisomers, salts, solvates and solid forms are prepared, respectively, in the same manner as those of Compounds 1 and 2.

Application

Hereafter, any general reference to “compounds of the present invention” shall apply to all embodiments of the compounds of the present invention, including Compound 1 or 2, or pharmaceutically acceptable salts, solvates or solid forms thereof, and may be read as “Compound 1 or 2 , or a pharmaceutically acceptable salt, solvate or solid form thereof." Likewise, any reference to deuterated compounds in accordance with the present invention should include not only Compounds 3, 4 or 5, but also any atropisomer, salt or solid form of any of the above.

The invention also covers the use of these atropisomers, pharmaceutically acceptable solid forms, their solvates and salts of ATM inhibitors, their deuterated forms, as well as atropisomers and pharmaceutically acceptable salts thereof for inhibiting, regulating and/or modulating the ATM kinase signaling cascade and thus provides new tools for research and/or diagnostics. Accordingly, the invention further relates to the use of the compounds of the present invention, including their deuterated forms, for inhibiting ATM kinase. The term "inhibition" refers to any reduction in activity that is based on the action of specific compounds according to the invention, since the latter are able to interact with the target molecule in such a way that recognition, binding and blocking becomes possible. These compounds have a high affinity for ATM kinase. In addition, the compounds are highly selective and thus allow essentially exclusive and direct recognition of ATM kinase. For research and/or diagnostic use, deuterated compounds, e.g. Compounds 3, 4 or 5, or their atropisomers or salts or solid forms, are considered useful, for example for use in assays.

The invention generally covers the use of the compounds of the invention, including deuterated compounds, in the treatment of diseases that are caused, mediated and/or aggravated by ATM kinase activity.

Thus, the present invention broadly relates to the compounds of the invention, including deuterated compounds, for use as a medicine.

Thus, the present invention also provides compounds of the invention, including deuterated compounds, for use in the treatment of any disease that is caused, mediated and/or aggravated by ATM kinase activity. The present invention accordingly also relates to the use of compounds, including deuterated compounds, in accordance with the invention for the preparation of a medicament for the treatment of any disease that is caused, mediated and/or aggravated by ATM kinase activity. In other words, the present invention also discloses a compound of the invention, including a deuterated compound, for use in the treatment of diseases affected by ATM kinase inhibition.

In addition, the compounds or deuterated compounds in accordance with the invention can also be used as reagents for testing kinase-dependent signaling pathways in animal and/or cell culture models or in clinical diseases mentioned in this application. As discussed herein, these signaling pathways are important in various diseases.

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

The invention further describes a method for treating cancer and/or tumors, wherein an effective amount of at least one compound or a pharmaceutically acceptable salt, solvate, deuterated or solid form thereof according to the invention is administered to a subject being treated. Preferred subjects for the purposes of the invention are humans or animals, especially preferably humans.

The cancer/tumor may be selected, in particular, from the group of cancers/tumors of squamous epithelium, bladder, stomach, kidney, head, neck, esophagus, cervix, thyroid, intestine, liver, brain, prostate, genitourinary tract, lymphatic system, larynx, lung, skin, blood and immune system 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 lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma. It should be understood that the sensitization of cancer cells must include cells of the same types of cancers and tumors mentioned above.

The present invention also relates to a medicinal product containing a compound according to the invention and/or a pharmaceutically acceptable salt, solvate, deuterated or solid form thereof.

The invention further relates to a pharmaceutical composition containing 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.

By "drug" and "pharmaceutical composition" is meant any composition that can be used for the treatment of patients who, at least temporarily, have a pathogenetic modification of the general condition or condition of certain parts of the patient's body, preferably due to cancer and/or tumors.

Delivery of the compounds, or pharmaceutical composition, of the present invention into a cell or organism can be accomplished in accordance with the invention by any method that allows the ATM kinase to come into contact with the compounds present in the pharmaceutical composition, thereby inducing a response. The pharmaceutical composition in accordance with the present invention can be administered orally, transdermally, transmucosally, transurethrally, vaginally, rectally, pulmonaryly, enterally and/or parenterally. The type of administration chosen depends, for example, on the indication, dose administered, individual specific parameters, etc. Injections can be intradermal, subcutaneous, intramuscular, or intravenous. Administration can be carried out, for example, using so-called vaccine guns or syringes. It is also possible to provide the substance in the form of an aerosol, which enters the body when inhaled by a patient, preferably a person.

In preferred embodiments, the compounds of the present invention (in any of their forms) are administered orally. Oral administration is beneficial in terms of patient compliance. Thus, the pharmaceutical compositions are preferably solid pharmaceutical compositions for oral administration.

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, is that they are easily formulated as a solid dosage form for oral administration due to their good stability and high bioavailability.

Compositions

The compositions or pharmaceutical compositions in accordance with the present invention can be prepared using conventional solid or liquid excipients corresponding to the desired type of administration, in a suitable dosage and in a manner which is known per se. Thus, pharmaceutically acceptable excipients known to one skilled in the art may generally form part of the pharmaceutical composition according to the invention, wherein the amount of excipient(s) that are combined with the active compound to prepare a unit dose varies depending on the dose and route of administration. Such pharmaceutically acceptable excipients include fillers, stabilizers, complexing agents, antioxidants, solvents, binding agents, lubricants, salts, buffers, preservatives, pH adjusters and the like. Examples of excipients of this type are water, vegetable oils, benzyl alcohols, alkylene glycol, polyethylene glycol, Collifor, glycerol triacetate, gelatin, carbohydrates such as, for example, lactose or starch, hydroxypropyl methylcellulose (HPMC), magnesium stearate, talc and petroleum jelly.

As mentioned above, the present pharmaceutical composition is preferably intended for oral administration. The pharmaceutical composition may generally be in the form of a tablet, film-coated tablet, dragee, lozenge, capsule, pill, powder, granule, syrup, juice, drop, solution, dispersion, suspension, suppository, emulsion, implant, cream, gel, ointment. , paste, lotion, serum, oil, spray, aerosol, adhesive, patch or bandage. Forms for oral administration are preferably tablets, film-coated tablets, dragees, lozenges, capsules, pills, powders, granules, syrups, juices, drops, solutions, dispersions or suspensions.

In addition, parenteral pharmaceutical compositions may be contemplated, such as, for example, suppositories, suspensions, emulsions, implants or solutions, preferably oily or aqueous solutions. For topical use, the compounds of the present invention may be formulated in the usual manner with at least one pharmaceutically acceptable excipient, such as, for example, microcrystalline cellulose, and optionally other excipients, such as, for example, humectants, to obtain compositions that can be applied to the skin, such as, for example, creams, gels, ointments, pastes, powders or emulsions, or to obtain liquid compositions that can be applied to the skin, such as, for example, solutions, suspensions, lotions, serums, oils, sprays or aerosols.

The pharmaceutical composition may also be in the form of an injection solution. To prepare the injection solution, you can use an aqueous medium, such as, for example, distilled water or physiological saline solutions. The pharmaceutical composition can also be presented in the form of a solid composition, for example, in a lyophilized state, and then can be prepared for administration by injection by adding a solubilizing agent, such as, for example, distilled water or a buffer. One skilled in the art will be familiar with the basic principles of preparing lyophilisates.

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

A "therapeutically effective amount" of a compound according to the invention refers to an amount effective at required doses and for periods of time that, when administered to a patient with an ATMi modulated or ATMi dependent condition, preferably cancer, will produce the intended therapeutic effect, effect, e.g. alleviation, amelioration, palliation or elimination of one or more manifestations of a condition, respectively cancer, in a patient or any other clinical outcome during the course of treatment of a patient. A therapeutically effective effect does not necessarily occur with a single dose and can only occur after a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations. Such therapeutically effective amount may vary depending on factors such as the disease state, age, sex and weight of the individual, as well as the ability of the compound of the invention, alone or in combination, to produce the desired response in the individual. A therapeutically effective amount is also an amount such that the therapeutically beneficial effects outweigh any toxic or harmful effects of the compound of the invention.

In one embodiment, the compound of the invention (or salt, solvate, deuterated or solid) is administered at a dose of 5 mg to 1 g per dosage unit, for example 10 to 750 mg per dosage unit, for example 20 up to 500 mg per dosage unit, for example, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325 or 350 mg per unit. The biologically effective dose of Compound 1 is estimated to range from 25 to 350 mg once daily.

Due to their surprisingly potent and/or selective inhibition of ATM kinase, which regulates cellular processes through double-stranded DNA repair, the compounds of the invention can be administered at advantageously low doses and achieve similar or even superior biological efficacy compared to less potent or less potent ones. selective inhibitors. A reduced dose is usually associated with a reduction in medical side effects. In addition, highly selective inhibition is usually also reflected in a reduction in unwanted side effects.

“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For the purposes of this invention, useful or desired clinical results include, but are not limited to, reduction, alleviation of one or more symptoms of the disease being treated, most preferably cancer; reduction in the severity of the disease; delay or slow progression of the disease; improvement, temporary relief or stabilization of a painful condition; or other useful results. It should be appreciated that reference to “treatment” includes prevention as well as alleviation of established symptoms of a condition. Thus, “treating” a condition, disorder, or disease includes: (1) preventing or delaying the onset of clinical symptoms of the condition, disorder, or disease developing in a subject who may be affected by or predisposed to the condition, disorder, or disease, but has not yet experiences or does not demonstrate clinical or subclinical symptoms of a condition, disorder or disease, (2) suppression of a condition, disorder or disease, that is, stopping, reducing or delaying the progression of a disease or its relapse (in the case of maintenance treatment) or at least one of its clinical or subclinical symptom, or (3) alleviation or weakening of the disease, i.e. ensuring the reversal of a condition, disorder or disease or at least one of its clinical or subclinical symptoms. In some embodiments, “treatment” includes (1) and (2).

“Tumor,” as applied to a subject diagnosed or suspected of having cancer, refers to a malignant or potentially malignant neoplasm or tissue mass of any size and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or fluid areas. The different types of solid tumors are named according to the types of cells that form them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Leukemias (blood cancers) do not usually form solid tumors.

“Administering” a compound to a patient (and the grammatical equivalents of this phrase) refers to direct administration, which may be the administration to the patient by a healthcare professional or may be administration by the patient himself, and/or indirect administration, which may be the act of prescribing the drug. For example, a physician who instructs a patient to self-administer a drug or gives a patient a prescription for a drug should be considered to be administering the drug to the patient in the context of the present invention.

All of the above and additional excipients or other components of the drug or pharmaceutical composition are known to the person skilled in the art and can be specially formulated for training in accordance with the invention in routine experiments.

Combination therapy

Drugs and pharmaceutical compositions that contain a compound according to the invention and the use of these compounds for the treatment of kinase-mediated disorders are a very promising approach, in particular for the treatment of cancer. The compounds of the invention may be administered as monotherapy, as set forth above, in combination with other treatments, such as, for example, chemotherapy or radiation therapy. As stated above, reference to a compound will include any salt, solvate, deuterated or solid forms thereof.

The key involvement of ATM in DNA repair processes and evidence that ATM kinase deficiency allows mammalian cells to become more sensitive to radiation provides the potential for the therapeutic use of ATM-specific inhibitors as part of the treatment of cancers, such as solid tumors, through radiation therapy and/or chemotherapy. wherein chemotherapy is predominantly aimed at inducing double-stranded DNA damage. As previously explained, ATM is an attractive intervention for therapy-induced inhibition of DNA double-strand breaks. Thus, the compounds of the present invention in any of their forms are very beneficial in combination with radiation therapy and/or DNA damaging chemotherapy.

Accordingly, the present invention relates to the combination of a compound according to the invention and radiation therapy (RT). Thus, the present invention relates to a compound of 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. In other words, 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 the treatment of cancer and/or tumors in combination with radiation therapy, and thus to a method of treating cancer comprising administering the compound in accordance with the present invention or a pharmaceutically acceptable salt or solid form thereof in combination with radiation therapy. The present invention further provides a compound of the present invention or a pharmaceutically acceptable salt or solid form thereof for use in sensitizing cancer cells to ionizing radiation or radiation therapy (RT).

Compound 1 has been shown to produce significant dose-dependent antitumor responses in vivo when combined with clinically relevant radiation regimens (eg, radiation therapy). EXAMPLE 8 and Figure 20 present the results achieved in detail.

An acceptable administration regimen may include, for example, administering a dose of radiation therapy of 15 Gray (Gy) administered in 5 fractions (3 Gy per fractional day) over a period of weeks (that is, for consecutive 5 days followed by 2 days without) and The compound according to the invention is administered orally on the same days, which can be repeated at least once.

Industrial irradiation methods that are used clinically preferably include photon irradiation (classical, electromagnetic x-ray/gamma ray), proton irradiation, heavy ion irradiation (ionized carbon) and neutron irradiation, without any limitation. These radiotherapies and other suitable methods of radiation therapy in accordance with 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, which are incorporated herein by reference in their entirety. As the most common application, photon irradiation has been technically improved using IMRT (Intensity Modulated Radiation Therapy) and imaging techniques (3D conformal radiation therapy) to plan the treatment and achieve the most precise focusing possible.

According to a further aspect, the present invention relates to a combination of a compound of the present invention and a DNA damaging agent; thus, to a compound according to the present invention for use in the treatment of cancer and/or tumor in combination with a DNA damaging agent, use of a compound according to the invention for the preparation of a medicament for the treatment of cancer in combination with a DNA damaging agent, and a method cancer treatment comprising administering a compound of the present invention and a DNA damaging agent. As generally explained in this application above, the compound will include pharmaceutically acceptable salts, solid forms and solvates, in particular with respect to the composition and method of treatment.

Administration of the DNA damaging agent and the compound of the present invention may be simultaneous or sequential.

As used herein, a DNA damaging agent is an agent that is capable of causing DNA damage in a cell, especially a cancer cell, with exemplary embodiments mentioned below.

As explained previously, ATM kinase is a key regulator of DNA double-strand break (DSB) repair that is induced by commonly used cancer therapeutics such as ionizing radiation (IR) and DNA damaging agents. During DSB events, ATM signals to numerous downstream effectors, including p53. Unrepaired DSBs lead to checkpoint activation, cell cycle arrest, and ultimately tumor cell death.

In one embodiment, the present invention provides a pharmaceutical composition that includes a therapeutic active compound in accordance with the invention and a DNA damaging agent.

A DNA damaging agent suitable for use in a combination (therapy) that includes a pharmaceutical composition or kit is preferably selected from the group consisting of:

• alkylating agents such as altretamine, bendamustine, busulfan, carmustine, chlorambucil, chlormethine, cyclophosphamide, dacarbazine, ifosfamide, improsulfan tosylate, lomustine, melphalan, mitobronitol, mitolactol, nimustine, ranimustine, temozolomide, thiotepa, treosulfan, mechloroethamine, he, apaziquon , fotemustine, glufosfamide, palifosfamide, pipobromane, trofosfamide, uramustine tremosustintepacil, tremosustin theorem, sulfan, mechlorethamine, carboquone, apaziquone, fotemustine, glufosfamide, palifosfamide, pipobromane, trofosfamide, uramustine;

• platinum compounds such as carboplatin, cisplatin, eptaplatin, miriplatin hydrate, oxaliplatin, lobaplatin, nedaplatin, picoplatin, satraplatin;

• topoisomerase inhibitors, for example, irinotecan, SN38, topotecan, camptothecin, rubitecan, belotecan, etoposide, daunorubicin, doxorubicin, aclarubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, amsacrine;

• poly(ADP-ribose) polymerase (PARP) inhibitors, for example, olaparib, niraparib, veliparib;

• ATR (ataxia-telangiectasia and Cas13-related) inhibitors, e.g. M6620 (VX-970: 3-[3-(4-methylaminomethyl-phenyl)-isoxazol-5-yl]-5-[4-(propan-2 -sulfonyl)-phenyl]-pyrazin-2-ylamine), M4344 (VX-803: 2-amino-6-fluoro-pyrazole[1,5-a]pyrimidine-3-carboxylic acid [5'-fluoro-4- (4-oxetan-3-yl-piperazin-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;

• DNA-modifying agents, such as amrubicin, bisantrene, decitabine, mitoxantrone, procarbazine, trabectin, clofarabine, amsacrine, brostalicin, pixantrone, laromustine;

• antitumor antibiotics such as bleomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, levamisole, miltefosine, mitomycin C, romidepsin, streptozocin, valrubicin, zinostatin, zorubicin, daunorubicin, plicamycin, aclarubicin, peplomycin, pyrarubicin

• alpha emitters, such as alpharadine ( ²²³ Ra dichloride, Xofgio), ²¹¹ At, ²¹³ Bi, ²²⁵ Ac, ²²⁷ Th;

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

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

The invention can also be implemented in the form of a kit which contains a compound in accordance with the invention. The kit consists of individual packages of (a) an effective amount of a compound of the present invention and/or a physiological salt, solvate or solid form thereof and (b) an effective amount of an additional active compound. The additional active compound is a DNA damaging agent.

The kit may contain suitable containers such as, for example, boxes or cartons, individual vials, bags or ampoules. The kit may contain, for example, individual 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, such as a DNA damaging agent, in dissolved or lyophilized form. The kit in accordance with the invention may also contain an article that contains written instructions or directs the user to written instructions that explain how to handle the compounds in accordance with the invention.

Thus, it should be noted that the compounds according to the invention can be used individually and/or in combination with other therapeutic measures, such as, for example, surgery, immunotherapy, radiation therapy and/or chemotherapy. The latter refers to targeted therapy using any desired active compounds (chemical or biological, including nME: new molecular entities, NCE: new chemical entities, and NBE: new biological entities) as monotherapy and/or targeted/non-targeted combination therapy.

All documents that are cited herein are intended to be incorporated in their entirety into the disclosure of the present invention by reference.

It goes without saying that the present invention is not limited to the specific compounds, pharmaceutical compositions, uses and methods as described herein, since such things may vary. Moreover, it goes without saying that the terminology used in this application serves solely to describe specific embodiments and is not intended to limit the scope of protection of the invention. As used in the specification hereto, including the appended claims, singular word forms such as, for example, “any” or “that” include a plural equivalent unless the context specifically indicates otherwise. For example, a reference to “compound” includes a single compound or a plurality of connections, which in turn may be identical or different, or a reference to “method” includes equivalent steps and methods that are known to one skilled in the art. A reference to an object as "containing" certain functions should be interpreted as meaning that the object must include those functions, but this does not preclude the presence of other functions as long as they do not render the object inoperable.

experimental part

The compounds according to the invention exhibit beneficial properties as evidenced by various parameters and experimental results. The experimental methods that were used to analyze and characterize the compounds of the invention in all their forms are presented below.

Analyzes

Measuring kinase activity is a method well known to one skilled in the art. Generic assays for determining kinase activity using substrates such as histone (Alessi et al. (1996) FEBS Lett. 399 (3): 333) or myelin basic protein are described in the literature (Campos-Gonzalez & Glenney (1992) JBC 267:14535). Various assay systems are available to identify kinase inhibitors. In the proximity scintillation assay (Sorg et al. (2002) J Biomolecular Screening 7:11) and flash plate assay, radioactive phosphorylation of the protein or peptide substrate is measured using ATP. In the presence of an inhibitory compound, a decrease in the radioactive signal or its absence at all is detected. In addition, homogeneous resonance time-resolved fluorescence energy transfer (HTR-FRET) and fluorescence polarization (FP) technologies are useful as analysis methods (Sills et al. (2002) J Biomolecular Screening 191). Other non-radioactive ELISA methods use specific phospho-antibodies (phospho-Abs). Phospho-ATs bind only the phosphorylated substrate. This binding can be detected by chemiluminescence using a second peroxidase-conjugated anti-sheep antibody.

For the purposes of the present invention, the relevant target properties of the compounds were assessed using the following assays:

ATM Kinase Assay - Determination of ATM Inhibition (IC ₅₀ ATM):

The IC ₅₀ value was determined using an ATM kinase biochemical assay. The assay consists of two steps: an enzymatic reaction and a detection step. First, ATM protein (ataxia-telangiectasia mutant kinase) and the test substance are incubated at various concentrations with the addition of p53 substrate protein and ATP. ATM mediates phosphorylation of p53 at several positions, including amino acid S15. The amount of phosphorylated p53 is determined using specific antibodies and the TR-FRET technique. The ATM enzymatic assay is performed as a 384-well TR-FRET-based assay (HTRFTM, Cisbio Bioassays). In the first step, purified human recombinant ATM protein (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 inhibitor and without test substance as a negative or neutral control. The assay buffer includes 25 mM HEPES pH 8.0, 10 mM Mg(CH ₃ COO) ₂ , 1 mM MnCl ₂ , 0.1% BSA and 0.01% Brij® 35.5 mM dithiothreitol (DTT). Test solution solutions were dispensed into microtiter plates using ECHO 555 (Labcyte). In the second step, purified human recombinant c-myc tagged with p53 (human p53, full length, GenBank ID BC003596, expressed in insect Sf21 cells) and ATP were added, and the reaction mixture was incubated at 22°C for 30-35 minutes. The pharmacologically relevant analytical volume was 5 μl. The final concentration in the assay during reaction mixture incubation was 0.3–0.4 nM ATM, 50–75 nM p53, and 10 μM ATP. The enzymatic reaction was stopped by adding EDTA. The formation of phosphorylated p53 as a result of the ATM-mediated reaction in the presence of ATP was determined using specific antibodies [labeled with europium (Eu) fluorophore as donor and d2 as acceptor (Cisbio Bioassays)] that allow FRET. 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, 0.1006% Tween-20, 0.005% Brij® 35, 0.21 nM anti-phospho-p53(ser15)-Eu antibody and 15 nM anti-cmyc-d2 antibody) were added to the reaction mixture. After incubation, typically 2 hours (1.5 to 15 hours) for signal development, the plates were analyzed in a plate reader (En Vision, PerkinElmer) using the TRF method (and using laser excitation). After excitation of the europium donor at a wavelength of 340 nM, the emitted fluorescent light of both d2 at 665 nM and the Eu donor at 615 nM was 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 measurement results were processed using Genedata Screener software. Determination of IC ₅₀ was carried out, in particular, by fitting a dose/effect curve to the data points using non-linear regression analysis.

IC ₅₀ = half maximum inhibitory concentration

ATP = adenosine triphosphate

TR-FRET = time-resolved fluorescence inductive resonance energy transfer

HTRF® = Homogeneous Time Resolved Fluorescence

HEPES = 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid

Mg(CH ₃ COO) ₂ = magnesium acetate

MnCl ₂ = manganese (II) chloride

BSA = bovine serum albumin

EDTA = ethylenediaminetetraacetate

TRF = time resolved fluorescence

The following abbreviations are used throughout this specification unless otherwise noted.

The method for determining the IC ₅₀ value at an ATP concentration of 1000 μM differs from the above only in the ATP concentration.

Cellular pCHK2 assay:

A fluorescence-based high-abundance assay was used to identify substances that inhibit phosphorylation of protein kinase CHK2 (checkpoint kinase 2) at threonine amino acid position 68 in HCT116 cells.

In vitro cell-based immunofluorescence assay to identify inhibitors of bleomycin-induced phosphorylation of CHK2 (phospho-Thr68) in the human colon carcinoma cell line HCT116:

HCT116 cells were seeded at a defined cell density in 384-well plates in culture medium (high glucose DMEM, 2 mM GlutaMax, 1 mM Na pyruvate, 10% fetal bovine serum (FCS) and incubated overnight at 37°C and 10 % CO _2. The next day, the test substances were added in a certain concentration range (from 1 nM to 30 μM) in combination with 10 μM bleomycin, where the concentration of the DMSO solvent was kept constant at 0.5% after incubation for four hours at 37. C and 10% CO ₂ , cells were fixed (5 min., 4% formaldehyde in PBS), permebialized (10 min., 0.2% Triton X-100 in PBS) and after blocking nonspecific binding sites (10% goat serum, 1% BSA in PBS) were incubated overnight at 4 C with a specific anti-pCHK2 antibody (cell signaling system #2661) pCHK2 (Thr68) was detected using Alexa488-labeled secondary anti-rabbit IgG antibody. Parallel DNA staining. propidium iodide allowed for cell counting. pSNK2 signal was determined using a high concentration imager (Molecular Devices IMX Ultra) and automated image analysis using the device's MetaXpress software. The number of cell nuclei that have a pCHK2 signal above a certain background was determined.

DMEM: Dulbecco's modified Eagle's medium; FBS: fetal bovine serum, PBS: phosphate-buffered saline (abbreviations are used throughout this description unless otherwise noted).

In addition, the effect, in particular the inhibition of other kinases and thus the selectivity of the compound according to the invention can be determined using the following assays:

ATR/ATRIP kinase assay

The IC ₅₀ value was determined using the ATR/ATRIP enzymatic assay. The analysis consisted of two stages: an enzymatic reaction and a detection stage. First, the ATR/ATRIP protein mixture (ataxia telangiectasia and Rad3-related protein/ATR interacting protein), test compound at various concentrations, p53 as substrate protein, and adenosine triphosphate (ATP) were incubated in assay buffer. ATR phosphorylates p53 at Serl5 and other residues. The amount of phosphorylated p53 was then determined using specific antibodies and TR-FRET assay technology.

Details: ATR/ATRIP enzyme assay was performed as TR-FRET- (HTRF™, Cisbio Bioassays) based on a 384-well assay. In the first step, purified 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) was incubated in assay buffer for 15 minutes at 22 °C with the test compound at various concentrations or without the test compound (as a negative control). The assay buffer contained 25 mM HEPES pH 8.0, 10 mM Mg(CH ₃ COO) ₂ , 1 mM MnCl ₂ , 0.1% BSA, 0.01% Brij® 35, and 5 mM dithiothreitol (DTT). Echo 555 (Labcyte) was used to dispense compound solutions. Then, in a second step, purified human recombinant STH-tagged p53 (human p53, full length, GenBank ID: BC003596, expressed in Sf21 insect cells) and ATP were added to the reaction mixture and incubated for 25-35 minutes, typically 25 minutes, at 22°C. The pharmacologically relevant volume was 5 μl. Final concentrations in the assay during reaction incubation were 0.3-0.5 nM, typically 0.3 nM ATR7ATRIP, 50 nM p53, and 0.5 μM ATP. The enzymatic reaction was stopped by adding EDTA. The formation of phosphorylated p53 as a result of the ATM-mediated reaction in the presence of ATP was determined using specific antibodies [labeled with europium (Eu) fluorophore as donor and d2 as acceptor (Cisbio Bioassays)] that allow FRET. For this purpose, 2 μl of a stop solution containing antibodies (12.5 mM HEPES pH 8.0, 125 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)-E antibody and 15 nM anti-cmyc-d2 antibody) were added to the reaction mixture. After signal development for 2 hours, the plates were analyzed on an EnVision microplate reader (PerkinElmer) using the TRF laser excitation method. When the europium donor was excited at a wavelength of 340 nM, the emitted fluorescent light from the d2 acceptor at 665 nM, as well as from the Eu donor at 615 nM, was measured. The amount of phosphorylated p53 is directly proportional to the ratio of the amounts of light emitted, i.e. relative fluorescence units (rfu) at 665 nM and 615 nM. The measurement results were processed using Genedata Screener software. Specifically, IC ₅₀ values were determined in the usual manner by fitting a dose/effect curve to the data points using nonlinear regression analysis.

For abbreviations, see the list above.

Analysis of cellular pCHK1

Chk1 kinase acts downstream of ATR and plays a key role in regulating the DNA damage checkpoint. Activation of Chk1 involves phosphorylation at Ser317 and Ser345 (considered to be the preferred target of ATR phosphorylation/activation) and occurs in response to blocked DNA replication and some forms of genotoxic stress. Phosphorylation at Ser345 serves to translocate Chk1 to the nucleus upon checkpoint activation.

This assay measures the reduction in Chk1 phosphorylation (Ser 345) in HT29 colon adenocarcinoma cells following compound and hydroxyurea treatment (which promotes fork arrest due to dNTP depletion) using an immunocytochemical procedure and high-content imaging.

For HT29 assays, cells were plated in culture medium (high glucose DMEM (no phenol red), 2 mM glutamax, 1 mM pyruvate, 10% FCS, Greiner 384 well plates, black, μclear #781090 (2500 cells/well/30 µl) and incubated for at least 20 hours at 37°C, 10% CO ₂ and 90% relative humidity. Diluted test compound (final content 1 nM - 30 µM) and hydroxyurea (final content 3 mM) were added simultaneously. , and the cells were incubated for 4 hours at 37°C. After fixation/permibialization with 100% MeOH (-20°C, cold) and permibialization with 0.2% Triton X-100, the complete immunocytochemical procedure was performed using specific anti. -pChkl antibody (Cell Signaling, #2348BF) and fluorescently labeled secondary antibody (Alexa Fluor® 488 goat anti-rabbit F(ab')2 fragment, Invitrogen A11070) and parallel nuclear staining for cell counting.

Nuclear localized pChk1 signal was determined on an ImageXpress Ultra high content confocal reader and presented as % positive cells (nuclei).

DNA-RK analysis

The kinase assay was performed as HTRF® using a 384 well assay. At the first stage, the DNA-PK protein complex was incubated with or without the test compound for 15 minutes at 22°C. After the addition of STK substrate 1-biotin (Cisbio), Mg-ATP, DNA and staurosporine, the reaction mixture was incubated for 60-80 minutes (depending on the activity of the DNA-PK protein complex) at 22°C. Echo 555 (Labcyte) was used to distribute compound solutions. The assay buffer consisted of 25 mM HEPES pH 7.4, 11 mM MgCl ₂ , 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 analysis during incubation of the reaction mixture were 50-100 ng/cell of the DNA-RK protein complex (depending on the activity of the DNA-RK protein complex), 1 μM STK substrate 1-biotin, 10 μM Mg-ATP, 80 ng /cell of DNA from calf thymus and 1 µM staurosporine. The enzymatic reaction was stopped by adding EDTA. The formation of the phosphorylated STK substrate l -biotin as a result of the DNA-PK-mediated reaction was determined using a specific anti-phospho-STK antibody (Cisbio) labeled with europium (Eu) as a donor and streptavidin labeled with XL665 (Cisbio) as an acceptor , allowing FRET. For this purpose, 4 μl of a stop solution containing antibodies and streptavidin (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-phopho-STK antibody and 160 nM streptavidin-XL665). After signal development for 1 hour, the plates were 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 emission wavelengths of 665 nM (phosphopeptide sensitive wavelength/XL665 emission) to units at 620 nM (europium reference wavelength). 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, J. G. Schiro, H. Huang, L. E. Burgess, E. A. Kesicki, and J. Halbrook.)

Cellular analysis of phosph. DNA protein kinase

HCT116 cells were cultured in MEM alpha medium with 10% FBS and 2 mM glutamine at 37°C and 10% CO ₂ . Cells were separated from the bottom of the culture vessels using trypsin/EDTA, centrifuged in centrifuge tubes, transferred to fresh medium, and cell density was 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 next day, 10 μM bleomycin (a DNA intercalator and double-strand break inducer) and test substances in fresh culture medium were added to the cells and cultured for a further six hours. Cell lysis was then carried out, and cell lysates were applied to a 96-well ELISA plate (Sigma-Aldrich WH0005591M2: total DNA-PK, Abeam ab 18192 or Epitomics EM09912: phosphoserine 2056 DNA-PK), which was blocked and coated with specific DNA-PK antibodies and incubated at 4°C overnight. 96-well ELISA plates were then treated with detection antibody (Abeam ab79444: total DNA-PK) and streptavidin-horseradish peroxidase conjugate. The development of the enzymatic reaction was carried out using a chemiluminescent reagent, chemiluminescence was measured using Mithras LB940. Phospho-DNA-protein kinase-specific antibody signals were normalized to that of anti-whole protein DNA-protein kinase C antibody. IC ₅₀ values or percentages were determined by reference to the signal level of the bleomycin-treated control group (100% of control). DMSO control was used as a blank experiment.

MEM: minimal nutrient medium; DMSO: dimethyl sulfoxide

PDE2A1 assay

A commercially available assay was used (Cerep, cat. no. 4071, SOP n1C1054) which is designed to evaluate the effect of compounds on human phosphodiesterase-2A1 activity, quantified by measuring the production of 5'AMP from cAMP using recombinant human enzyme expressed in Sf9 cells.

The test compound, standard compound, or water (control) was added to a buffer containing 40 mM Tris/HCl (pH 7.4), 8 mM _MgCl2 and 1.7 mM EGTA/NaOH, 1.8 μM cAMP and 1 μCi [ ³ H]cAMP. The reaction was then initiated by adding enzyme (about 2.5 units) and the mixture was incubated for 20 min. at 22°C. For basal control measurements, the enzyme was excluded from the reaction mixture. After incubation, SPA (Scintillation Proximity Assay) beads were added.

After 30 min. with shaking at 22°C, the amount of [ ³ H]5'AMP was quantified using a scintillation counter (Topcount, Packard). The results were expressed as percentage inhibition of control enzyme activity.

The standard inhibitory control compound was EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), which was tested in each experiment at several concentrations to obtain an inhibition curve from which its IC ₅₀ value was calculated.

References: Maurice D.H., Ke N., Ahmad F., Wang Y., Chung J. and Manganiello V.C. (2014), Advances in targeting cyclic nucleotide phosphodiesterases, Nat. Rev. Drug Discov., Vol. 13 Issue 4: p.290.

PDE4D2 analysis

The commercially available Cerep assay (cat. no. 4077; SOP n1C1045) was used. The assay was designed to evaluate the effect of the test compounds on human phosphodiesterase-4D2 activity, quantified by measuring the production of 5'AMP from cAMP using recombinant human enzyme expressed in Sf9 cells.

The test compound, control compound, or water (control) were 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 was then initiated by adding enzyme (about 1.5 units) and the mixture was incubated for 20 min at 22°C. For basal control measurements, the enzyme was excluded from the reaction mixture. After incubation, SPA (Scintillation Proximity Assay) beads were added.

After 30 min. at a temperature of 22°C with shaking, the amount of [ ³ H]5'AMP was quantified using a scintillation counter (Topcount, Packard). The results were expressed as percentage inhibition of control enzyme activity.

The standard inhibitory control compound was Ro 20-1724, which was tested at several concentrations in each experiment to obtain an inhibition curve from which its IC50 value was calculated.

Bibliographic reference as above.

PDE42A1 analysis

A commercially available Seger assay (cat. no. 4074; SOP n1C1056) was used, which is designed to evaluate the effect of compounds on human phosphodiesterase-4A1 A activity, quantified by measuring the production of 5'AMP from cAMP using recombinant human enzyme expressed in Sf9 cells.

The test compound, standard compound, or water (control) were 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 was then initiated by adding enzyme (about 10 units) and the mixture was incubated for 20 min. at 22°C. For basal control measurements, the enzyme was excluded from the reaction mixture. After incubation, SPA (Scintillation Proximity Assay) beads were added.

After 30 min. at 22°C with shaking [ ³ H]5'AMP was quantified using a scintillation counter (Topcount, Packard). The results were expressed as percentage inhibition of control enzyme activity.

The standard inhibitory control compound was Ro 20-1724, which was tested at several concentrations in each experiment to obtain an inhibition curve from which its IC ₅₀ value was calculated.

Bibliographic reference as above.

Additional parameters considered important were determined as follows:

Method for studying microsomal stability (intrinsic systemic clearance)

A microsomal stability assay (Clint) was used to measure in vitro clearance. The assay involves measuring the rate of disappearance of a compound due to its intrinsic relationship to metabolism (“intrinsic” means that extinction is not influenced by other properties such as permeability, binding, etc., which play a role in quantifying in vivo clearance). Microsomal stability (intrinsic clearance, Clint) and thus metabolic stability is usually expressed in µl/min/mg protein. This can be thought of as the volume of solution that 1 mg of microsomes can clear the compound in one minute.

Devices

A Tecan Genesis workstation (RSP 150/8) was used to carry out microsomal incubation. 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).

UHPLC conditions

Column: Acquity UPLC VEN C18, 2.1 θ 50 mm, 1.7 µM (Waters)

Mobile phases: A = 0.1% formic acid in water; B = acetonitrile

Flow rate: 0.750 ml/min; detection: ESI (electrospray ionization time-of-flight mass spectrometry), MRM (selective reaction monitoring); for injection: 10 µl; column temperature: 50°C

Chemical reagents

• Potassium phosphate buffer: 0.05 M potassium phosphate buffer pH 7.4 containing 1 mM MgCl ₂

• NADPH (reduced nicotinamide adenine dinucleotide phosphate): 22.5 mg NADPH-Ka4 in 1.8 ml potassium phosphate buffer

• Acetonitrile: 50 vol. % acetonitrile (1 volume of acetonitrile, 1 volume of water)

• DMSO: 20 vol. % DMSO in water

• Stock solution of 20 mg/ml human or mouse liver microsomes (protein)/ml in phosphate buffer

• Stock solution of 10 mM compound in 100% DMSO

Microsomal incubation

Dilutions of the test compounds were performed in 2 stages, starting with a 10 mM stock solution of the corresponding compound in 100% DMSO. The first 4 μl of the stock solution was added to 196 μl of 20 vol. % DMSO. In the second step, 10 μL of the first dilution was added to 1590 μL of potassium phosphate buffer to achieve a final concentration of 1.25 μL in the final compound dilution. Thus, the amount of organic solvent in the analysis was minimal (<1%).

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

Incubation was carried out in a 96-well incubation plate. 160 μl per well of the final dilution of the compound was transferred to the incubation plate. Four samples of each compound dilution were analyzed. 20 μl/well of liver microsome solution was added to each well, and then the samples were pre-incubated for 5 minutes at 37°C with agitation at 800 rpm. Two reference compounds (verapamil and dextromethorphan) were used in parallel in each experiment for each species (human or mouse microsomes) to provide system performance for comparison.

160 μl of acetonitrile per well was added to a separate stop plate.

After pre-incubation, i.e. at time t ₁ = 0 minutes, 18 μl of incubated compound solution samples per well (containing acetonitrile) were transferred and added to a stop plate to prevent reaction (0 minutes control samples, 4 samples per compound). In the same way, 18 μl samples of the incubated solution of the reference compound were transferred and added to the well (containing acetonitrile) on the stop plate at time t ₁ = 0 minutes and again after 30 minutes (14), the solubility and chemical stability of the compound were checked.

To initiate the reaction, 26 μl of NADPH (cofactor) solution was added to all wells that included a pre-incubated compound dilution or control solution, except those wells that included a pre-incubated compound dilution were to be used as 30-minute controls, where instead, 26 μl of phosphate buffer was added. Then the incubation was continued at 37°C and stirring at 800 rpm.

After t ₂ = 5 minutes, t ₃ = 10 minutes and t ₄ = 20 minutes of incubation time (i.e. after the start of the reaction), 20 µl samples of the incubated compound solution (4 samples per compound) and the control compound solution were transferred and added to the well stop plate with acetonitrile.

After t ₄ = 30 minutes of incubation time, 20 μl of incubated compound solution samples (4 samples per compound) and 20 μl of 30 minute control samples (containing buffer instead of NADPH) as well as 20 μl of incubated reference compound solution samples were transferred and added to the stop cell. tablet with acetonitrile.

Quenched samples were centrifuged at 4000g for 1 hour at 4°C. 80 μL of supernatant was transferred into 96-well plates for analysis using LC-MS/MS.

Data analysis

The microsomal/metabolic stability of the compound was determined by measuring the change in LC-MS/MS peak area over time. Data were fit to a log-linear model following Michaelis/Menten. The Clint value was calculated from the slope (k) of the linear-log-transformed concentration on the time graph divided by the number of microsomes (0.5 mg/ml): Clint (μl/min/mg protein) = k* 1000/protein concentration. Assay Explorer software was used to automatically calculate the slope k of the decline.

Kv11.1 (hERG) ion channel activity (patch clamp assay)

Method for detection and characterization of test substances that interfere with the Kv11.1 (hERG) channel: Kv111 (hERG, human cardiac specific potassium channel gene) is a potassium channel that plays a central role in the repolarization of ventricular cardiomyocyte cells.

Patch-clamp measurements were performed at room temperature on whole-cell configurations of human embryonic kidney cells. Whole-cell configurations were performed using an automatic patch-clamp device (Patchliner™, Nanion Technologies, Munich). This system is based on glass chips, with which it is possible to carry out automated whole-cell measurements on up to 8 whole cells simultaneously. The glass chip has a hole of a certain size through which the cell is transferred into the Gigaseal by applying reduced pressure and turns into a whole cell configuration. Buffer, cell suspension and test substances were added to the microchannels of the chip using a Teflon-coated pipette.

Cells were clamped to a holding potential of −80 mV. To measure substance-stimulated Kv11.1 channel inhibition, the following voltage protocol was used at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage flux is subtracted using the P4 method. Cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cells were collected, the seal was improved by adding a seal enhancer buffer. Once the whole-cell configuration was achieved, the compaction enhancer buffer was washed away and replaced with extracellular buffer. The measurement was started in extracellular buffer for 1.5 min. DMSO (vehicle control, 0.1% DMSO) was then applied and the control flow was recorded for 3 minutes. The test substance was then added twice at the same concentration, and the potassium flux was measured for 3.5 min. in every case.

If the test substance measured at an initial concentration of 10 µM was less than (-)50% effect (threshold value) (for example, (-)60% effect), then the test substance was added cumulatively to determine the dose-response relationship with increasing concentrations, where each concentration was measured for 5 minutes.

The Kv11.1 ion channel blocker (hERG) quinidine was used as a reference substance. The effects of the test substances and quinidine were standardized to appropriate vehicle controls. The effect on Kv11.1 (hERG) channel activity was assessed by potassium flux at -40 mV. For the calculation, the flow was estimated for the corresponding final route. Kv11.1 channel (hERG) inhibition induced by the test substance was standardized to vehicle control (0.1% DMSO).

During the measurement, an aliquot of the test substance was taken to determine the concentration. The sample was immediately measured by HPLC and the final concentration was determined from the calibration curve.

If the test substance measured at an initial concentration of 10 µM was greater than or equal to (-)50% effect (threshold value) (for example, (-)30% effect, i.e. 30% inhibition at 10 µM ), K _i was calculated according to the following formula: K _i = 1.0E-5 × (100 + % effect) / (-% effect), [M].

The result of measuring the (-)30% effect at a concentration of the test substance of 10 μM gave a K _i value of 23 μM.

Cytochrome P-450 enzymes (CYP)

In the human body, drugs are converted into water-soluble compounds using enzyme systems to facilitate their elimination. These enzyme systems include microsomal cytochrome P-450 enzymes, abbreviated CYP. The assay is designed to determine whether a compound may be a potent inhibitor of certain CYP isoforms. The assay involves the use of recombinant CYP isoforms and their reductase produced by overexpression in baculovirus-infected insect cells. The CYP response occurs through inhibition of the CYP isoform of interest together with the luminometric CYP substrate under conditions of NADPH regeneration. P450-GloTM luminometric substrates are derivatives of beetle luciferin ((4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazolecarboxylic acid or D-luciferin), a firefly luciferase substrate from beetles. P450-GloTM luminometric substrates do not interact directly with luciferase, but are converted by the corresponding CYP isoform to a luciferin product, which luminesces when reacted with luciferin detection reagent (LDR). This allows enzyme activity to be quickly quantified by brightness. The degree of inhibition is measured by determining the IC ₅₀ value (Crespi et al., Methods Enzymol. 357: 276-284, 2002). The following commercially available screening systems/assays were 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 (Luciferin-PPXE) Screening System (Promega Corporation; V9910). Bioavailability

Predicted human bioavailability is derived from measured bioavailability values in the preclinical mouse, rat and dog (beagle) species and calculated for the predicted pharmacologically effective dose in humans (in silico GastroPlus modeling).

X-ray powder diffraction (RPD) method

X-ray powder diffraction (XRD) patterns, such as those presented in Figures 7B and 10-19 (and others), were obtained using the following technique: samples were prepared in a combinatorial 96-well plate (containing an X-ray amorphous foil as the bottom) or between X-ray amorphous films. Measurements were carried out in transmission geometry with Cu-K _α1 radiation on a Stoe StadiP 611 diffractometer. Scanning was carried out simultaneously in the range 0-36° 2θ (step width 0.03° 2 θ 30 seconds per step), or covered 1-65° 2 θ (step width 0.015° 2 θ, 15 seconds per step), respectively.

Single crystal X-ray diffraction:

Single crystal X-ray structure data were obtained using an Agilent SuperNova diffractometer equipped with a CCD detector using Cu-Kα radiation. Measurements were carried out at 200 K (Form H2) or 298 K (Form A1, A2, A3, H1), respectively. Single crystal data were used to determine the crystal system and unit cell parameters.

Differential scanning calorimetry (DSC):

DSC scans were obtained on a Mettler-Toledo differential scanning calorimeter with an autosampler under an inert nitrogen gas atmosphere (50 ml/min). Survey scanning was carried out in 40 μl A1 cuvettes with open lids at a temperature of 25-300°C at a speed of 5°C/min.

Thermogravimetric analysis (TGA):

TGA scans were obtained on a Mettler-Toledo thermogravimetric analyzer with an autosampler under an inert nitrogen gas atmosphere (50 ml/min). Survey scanning was carried out in 100 μl cuvettes without lids at a temperature of 25-300°C at a speed of 5°C/min. Experiments were baseline corrected using a blank with an empty 100 µL A1 cuvette without a cap using the same temperature profile.

Dynamic Vapor Sorption (DVS):

DSP isotherms of water vapor sorption were obtained on a DSP device with a microbalance and an incubator (DSV-Intrinsic, Surface Measurement Systems, SMS). Powder samples were carefully weighed into disposable aluminum cups and placed in the sample area of the chipboard apparatus. For humidification, a total nitrogen flow rate of 200 ml/min was used. (combined dry and wet flows). Water vapor sorption isotherms were obtained at 25°C using the initial desorption segment 1 from 40% RH (relative humidity) to 0% RH (in 10% increments), adsorption segment from 0% RH to 98% RH (in 10% increments) RH and final step 8% RH, respectively), and final desorption segment 2 from 98% RH to 0% RH (with initial step 8% RH and 10% RH, respectively). For all stages of relative humidity, an equilibrium state of dm/dt ≤ 0.0005 wt was used. %/min. with a minimum RH step time of 10 minutes and a maximum RH step time (timeout) of 360 minutes.

Dissolution profiles without absorption of solid forms:

Absorption-free dissolution profiles for solid forms were obtained using the shake flask method with excess solid material in FaSSIF (pH 6.5) with time-resolved sampling to determine dissolved amounts of API.

Approximately 10-20 mg of solid sample was weighed into glass vials. 7 ml of appropriate medium (pre-warmed to 37° C.) was added and the suspension was shaken at 450 rpm. at 37°C. After 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 120 minutes, 24 hours and 48 hours, 1 ml of the suspension was taken and filtered through a 0.2 μM syringe filter. The clear filtrate was subjected to HPLC analysis after suitable dilution to measure the amount of dissolved compound/form (may also be called API).

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 determining pH-dependent solubility. and miniaturized dissolution without absorption:

Dissolved compound/form levels were analyzed by HPLC under the following conditions:

• Column: Chromolite RP-18e 100 - 3 mm

• Solvent A: water/formic acid (999:1; v/v)

• Solvent B: acetonitrile/formic acid (999:1; v/v)

• Injected volume: 5 µl

• Column temperature: 37°C

• HPLC gradient:

EXAMPLES

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, in particular, are to be interpreted so that they are not limited to the specifically illustrated features or combinations of features, but instead, the illustrative features may be freely varied or combined so 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 as such to those skilled in the art. Likewise, modifications to the preparation methods, particularly the reaction conditions, will be apparent to one skilled in the art.

EXAMPLE 1: Preparation of Compounds 1 and 2

Compound Y was prepared according to the procedure disclosed in WO 2016/155844, after separating Compounds 1 and 2 from Compound Y:

a) Synthesis of 6-bromo-N-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-nitro-quinoline-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, then the reaction was quenched by adding ice water (100 ml). The precipitate was filtered, washed with ice water and dried to obtain 1.00 g (94%) of 6-bromo-M-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-nitro-quinoline-4- amine in the form of a yellow solid.

b) Synthesis of 6-bromo-N ⁴ -(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-quinoline-3,4-diamine

6-Bromo-M-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-nitro-quinoline-4-

amine (990 mg, 2.20 mmol) dissolved in methanol (100 ml) was prepared under a protective nitrogen atmosphere. Raney nickel (100 mg, 1.17 mmol) was then added to the solution, and the reaction mixture was stirred for 30 minutes under a hydrogen atmosphere at normal pressure. After introducing nitrogen, the suspension was filtered and the filtrate was dried in vacuum. The filtrate was evaporated to dryness under vacuum. The residue was crystallized from ethyl acetate/petroleum ether to give 0.86 g (99%) of 6-bromo-N4-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-quinoline-3,4-diamine in as a yellow solid.

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) was dissolved in tetrahydrofuran (20 ml) . 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 adding ice water (200 ml). The precipitate was filtered, washed with ice water and dried to obtain 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 light 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

Under a dry protective nitrogen atmosphere, 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) was dissolved in N,N-dimethylformamide (5 ml). 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 for 10 minutes at room temperature. The reaction was then quenched by adding ice water (100 ml). The resulting precipitate was filtered and dried under vacuum to obtain 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 light yellow solid.

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

8-bromo-1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-methyl-imidazo[4,5-c]quinolin-2-one was dissolved in an atmosphere of inert argon gas in a closed equipment (150 mg, 0.33 mmol), 1-3-dimethyl-4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (88.4 mg, 0.40 mmol), Pd (PPh ₃ ) ₄ (76.6 mg, 0.07 mmol) and potassium carbonate (91.6 mg, 0.66 mmol) in 1,4-dioxane (15 ml) and water (5 ml). The reaction mixture was heated to 80°C with stirring for 2 hours. The mixture was then cooled to room temperature and the reaction mixture was reduced to drying in vacuo. The residue was purified by chromatography using silica (ethyl acetate/methanol = 97:3, v/v). The eluate was evaporated to dryness and the resulting crude product was purified by preparative reverse phase HPLC (water/acetonitrile). After reduction of product fractions, 1-(3-fluoro-5-methoxy-4-pyridyl)-7-methoxy-3-methyl-8-(1,3-dimethylpyrazol-4-yl)imidazo[4,5-c]quinoline -2-one (70 mg, 47%) was obtained as a colorless solid.

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

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) which was obtained above was separated by chiral HPLC using SFC (supercritical fluid chromatography) to obtain Compounds 1 and 2. The substance was applied to a chiral Lux Cellulose-2 column and separated at speed flow 5 ml/min. using CO ₂ /2-propanol+0.5% diethylamine (75:25) as solvent and using detection at a wavelength of 240 nM. Reduction of product fractions under reduced pressure gave 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 (25.0 mg, 50%) and 8-(1,3-dimethyl-1H-pyrazol-4 -yl)-1-(SWa)-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1,3-dihydro-imidazo[4,5-c]quinoline- 2-one (22.1 mg, 44%), both as colorless solids.

The starting compounds are easy to prepare, for example as shown below:

EXAMPLE 2: Isolation of Atropisomers and Purification of Compound 1

Compounds 1 and 2 can be separated from Compound Y, as shown in Scheme 1 and Figure 6, and as discussed in detail below. One skilled in the art will appreciate that the process described below is equally applicable to compounds 3-a and 3-b from 3, 4-a and 4-b from 4, and 5-a and 5-b from 5.

Scheme 1: Preparation of chiral salts

Stage 1:

1. Acetone (108 L, 20 volumes), purified water (8.13 L, 1.5 volumes) and Compound Y (5.42 kg, 1.0 eq) were added to a 200 L reactor at 20 ~ 25° WITH.

2. The reactor was charged with dibenzoyl-L-tartaric acid (4.33 kg, 1.0 eq.).

3. Heated to 52-55°C until a clear solution was obtained, stirred for 0.5 hours at 52-55°C.

4. Cooled to 20 ~ 25°C.

5. Filter and wash the precipitate once with acetone (5.4 L, 1 vol.).

6. The precipitate was collected and dried to obtain L-salt B (L-salt of Compound 2) (light yellow solid with 95.9% chiral purity).

7. Concentrate the mother liquor to obtain L-salt A (L-salt of Compound 1).

8. L-salt A and dichloromethane (38 L, 7 volumes) were added to a 100 L reactor and the pH was adjusted to 8-9 with saturated NaHCO ₃ solution.

9. Collect the dichloromethane (DCM) layer, extract saturated NaHCO ₃ with DCM (16.3 L, 3 volumes), combine the DCM layers, wash the DCM layer with H ₂ O (10.8 L, 2 volumes).

10. Concentrate the DCM layer. Then acetone (5.4 L, 1 volume) was added at 20 ~ 25°C.

11. Stir at 20 ~ 25°C for 1 hour.

12. Filter, collect the residue and dry to obtain Compound 1 (3.50 kg, white solid with 73.2% chiral purity, Y: 64.6%).

Stage 2:

1. L-salt B and DCM (16.2 L, 3 volumes) were added to a 50 L flask and the pH was adjusted to 8-9 with saturated NaHCO ₃ solution.

2. The DCM layer was collected, the aqueous DCM layer was extracted (5.4 L, 1 volume), the DCM layers were combined, and the DCM layer was washed with H20 (5.4 L, 1 volume).

3. Replaced DCM with 2-ethoxyethanol (1.8 L, 0.3 vol) twice under vacuum at 40 ~ 50°C.

4. The volume was adjusted with 2-ethoxyethanol to 5.4 l (1 vol.).

5. Heated to 128-130°C to obtain a suspension, stirred for 44 hours at 128-130°C.

6. IPC (49.5:50.5 ratio).

7. Cooled to 15-20°C.

8. Filter and wash the precipitate with methyl tert-butyl ether (2.7 L, 0.5 volume).

9. The precipitate was collected and dried to obtain Compound Y (1.60 kg, off-white solid, overall yield: 29.5%).

Stage 3:

1. Acetone (70 L, 20 volumes) was added to a 100 L reactor at 20 ~ 25 °C, stirred and Compound 1 was added (3.5 kg, 1.0 eq.), water was charged (5.3 L, 1 ,5 vol.).

2. Load dibenzoyl-D-tartaric acid (2.8 kg, 1.0 eq.) at 35 ~ 40°C, heat to 52 ~ 55°C to obtain a clear solution, stir for 0.5 hours at 52 °C ~ 55°C.

3. Cooled to 20 ~ 25°C in an oil bath and stirred for 17 hours at 20 ~ 25°C.

4. Filter and wash the precipitate with acetone (3.5 L, 1 vol.).

5. The precipitate was collected to obtain D-salt A (D-salt of Compound 1) (a light yellow solid with a chiral purity of 98.7%).

6. Concentrate the mother liquor, then add a saturated solution of NaHC03 (15.5 L, 3.5 volumes) and H20 (15.5 L, 3.5 volumes).

7. Stirred for 0.5 hours at 20 ~ 25°C.

8. Filter and wash the precipitate with H ₂ O (3.5 L, 1 vol.).

9. The precipitate was collected on a filter and dried to obtain Compound Y (1.53 kg, white solid at a ratio of 50.3:49.7, yield: 43.7%).

Stage 4:

1. Add D-salt A (D-salt of Compound 1) and acetone (17.5 L, 5 volumes) to a 50-liter reactor, heat to 52-55°C to obtain a suspension solution, stir for 0.5 hour at 52 ~ 55°C.

2. Cooled to 20 ~ 25°C and stirred for 17 hours at 20 ~ 25°C.

3. Filter and wash the precipitate with acetone (3.5 L, 1 vol.).

4. Collect the filter cake and dry it to give D-salt A (3.23 kg, light yellow solid, 99.2% chiral purity).

5. D-salt A was added to a 50 L reactor, then a saturated solution of NaHCO ₃ (16 L, 5 volumes) and H ₂ O (16 L, 5 volumes) was added.

6. Stirred for 0.5 hours at 20 ~ 25°C.

7. Filter and wash the precipitate with H ₂ O (16 L, 5 vol.).

8. The precipitate was collected and dried to obtain Compound 1 (1638 g, white solid with a chiral purity of 99.1% and 99.9% by HPLC, overall yield: 30.2%).

EXAMPLE 3: Chromatographic separation, purification and analysis

3.1 Compounds 1 and 2 can be separated from Compound Y by 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, e.g. on a Chiralpak 1C column (5 mm, 150 x 4.6 mm i.d.), e.g. using isocratic elution with a mobile phase containing: H ₂ O/ ACN 50/50 v/v (ACN: acetonitrile). One skilled in the art will appreciate that the same procedure can be used for compounds 3-a and 3-b, 4-a and 4-b, and 5-a and 5-b.

The chromatogram thus obtained is illustrated in Figure 5 (column and elution as above, flow rate 1.00 ml/min; UV 260 nm; T _C and T _S : 25 ± 5 °C, S _conc 0.20 mg/ ml; injected volume 10 ml)

3.2 As an alternative to the supercritical fluid chromatography conditions mentioned above, preparative supercritical fluid chromatography can be used, including, for example: Chiralpak AS-H column (20 mm × 250 mm, 5 µM); isocratic elution (20:80 ethanol: _CO2 with 0.1% v/v _NH3 ), BPR (suction pressure regulated): approximately 100 bar above atmospheric pressure; column temperature 40°C, flow rate 50 ml/min, injection volume 2500 μl (125 mg) and detector wavelength 265 nM.

3.3 To analyze the purity of the corresponding atropisomers, supercritical fluid chromatography can again be used, for example using the following setup: Chiralpak AS-H column (4.6 mm × 250 mm, 5 µM); isocratic elution (20:80 ethanol: _CO2 with 0.1% v/v _NH3 ), BPR (suction pressure regulated): approximately 125 bar above atmospheric pressure; column temperature 40°C, flow rate 4 mL/min, injection volume 1 μL, and detector wavelength 260 nM.

EXAMPLE 4: Stability of Compounds 1 and 2

Quantum mechanics calculations of rotational barriers

LaPlante et al. (ChemMedChem, 2011, 6(3), 505-513) describe a quantum mechanical workflow for estimating the energy barriers to axial rotation of drug-like molecules. A similar approach was followed: 3D structures of all input molecules were generated using CORINA (Corina version 3.6, Molecular Networks, Germany) and subsequently minimized using Macromodel (version 11.1, Schrodinger, LLC, New York, NY). Based on these input structures, rotational energy barriers were calculated from snapshots with a non-rigid angle between two planes using the program Jaguar (version 9.1 release 14, Schrodinger, LLC, New York, NY) using the B3LYP/6-31G** method with torsional angle 15° increments. The structures were optimized before torsional scanning using the same level of theory. The default parameters were used, except that the maximum number of minimization steps was set to 500 and the stop_rxn label was introduced to avoid artificially breaking links. Representative molecular fragments were used for all calculations. The dihedral view obtained from the minimized molecular mechanics structure was used to determine the initial value for the dihedral scan, for example, for Compound 1 in accordance with the present invention or for "LaPlante reference Compound 1" the values were set to 47.32° and 35.8° , respectively. For each torsion about the QM axial link, the energy values were calculated in 24 steps. The torsion profile was obtained by comparing the torsion angle values with the calculated energy values. The lowest energy barrier for each compound that allows interconversion between both isomers was determined. For reference compounds 1-6, the experimentally determined interconversions and calculated energy barriers are known. Computational energy barriers for reference compounds 1–6 range from 9865 to 31316 kcal/mol. These values are consistent with the experimentally determined interconversion coefficients.

Calculations for Compound 1 and Compound 2 of this invention demonstrated a high predicted rotational barrier of 29,205 kcal/mol, which translates into high stability of the atropisomer with a predicted rotational half-life in the range of years (>10 years). The required temperature to initiate racemization of any enantiomeric atropoisomer is >100°C.

One skilled in the art will appreciate that these values are also exemplary for compounds 3-a and 3-b, 4-a and 4-b, and 5-a and 5-b.

EXAMPLE 5: Pharmaceutically Acceptable Salts of Compound 1

Salts of Compound 1 (Compound 1-a), which are pharmaceutically acceptable, were prepared as presented and discussed in detail below.

Preparation of Fumarate Salt (Form NF6):

Approximately 20 mg 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 was dissolved in 1 ml THF at 50°C (analytical grade) in a 4 ml glass vial with a closed cap, equipped with a magnetic stirrer, and stirred using a magnetic stirrer. At approximately 50°C, 5.7 mg of fumaric acid (-1.1 equiv) was added to the hot solution and cooled to 5°C at a cooling rate of 0.1 K/min. The mixture was repeatedly heated to 50°C (for approximately 30 minutes) and cooled to 5°C (at a rate of 0.1 K/min) with stirring 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 additionally subjected to slow vapor diffusion of the antisolvent n-pentane in a closed vial configuration. Finally, the resulting solid material was separated by centrifugation and carefully dried under a stream of nitrogen.

Obtaining Napsylate Salt (Form NF7) - Option 1:

Approximately 10 mg 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 was dissolved in ~250 µl of acetone at 50°C (analytical grade) in a 4 ml glass vial with a closed cap, equipped with magnetic stirrer, and stirred using a magnetic stirrer. At 50°C, approximately 5.6 mg of 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. The mixture was repeatedly heated to 50°C (for approximately 30 minutes) and cooled to 5°C (at a rate of 0.1 K/min) with stirring before a final equilibration step at 5°C for several hours. Finally, the resulting solid material was separated by centrifugation and carefully dried under a nitrogen purge. Obtaining Napsylate Salt (Form NF7) - Option 2:

Approximately 11.5 mg 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 was dissolved in ~250 µl THF (tetrahydrofuran) at 50°C (analytical grade) in a 4 ml glass vial with a closed lid, equipped with a magnetic stirrer, and stirred using a magnetic stirrer. At 50°C, approximately 6.6 mg of naphthalene-2-sulfonic acid (~1.2 eq) was added to the hot solution and cooled to 5°C at a cooling rate of 0.1 K/min. The mixture was repeatedly heated to 50°C (for approximately 30 minutes) and cooled to 5°C (at a rate of 0.1 K/min) with stirring before a final equilibration step at 5°C for several hours. Finally, the resulting solid material was separated by centrifugation and carefully dried under a nitrogen purge.

Preparation of edisylate salt (Form NF8):

Approximately 12.6 mg 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 was dissolved in ~250 μl of acetone at 50°C (analytical grade) in a 4 ml glass vial with a cap. equipped with a magnetic stirrer and stirred using a magnetic stirrer. At 50°C, approximately 6.1 mg of ethanedisulfonic acid (~1.2 equiv) was added to the hot solution and cooled to 5°C at a cooling rate of 0.1 K/min. The mixture was repeatedly heated to 50°C (for approximately 30 minutes) and cooled to 5°C (at a rate of 0.1 K/min) with stirring before a final equilibration step at 5°C for several hours. Finally, the resulting solid material was separated by centrifugation and carefully dried under a nitrogen purge.

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]quinoline -2-she

Stage 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), CD ₃ OD (0.30 ml, 6.74 mmol), N,N-dimethylformamide (3 ml). The mixture was stirred for 1 hour at 100°C. 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 vacuum. The crude residue was purified using preparative HPLC (SHIMADZU (HPLC-10): Column: Atlantis Prep T3 OBD column, 19 * 250 mm, 10 μM; mobile phase: water (10 mmol/L NH ₄ HCO ₃ ) and acetonitrile (acetonitrile content 34%) within 10 minutes); determination: UV 254 nm) with a yield of 35 mg (38%) 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%. Retention time 1.98 min. (method A). [M+H]+ 452. ¹ H NMR (400 MHz, DMSO-d6) ppm = 8.92 (s, 1H), 8.70 (d, J=9.5 Hz, 2H), 7 .83 (s, 1H), 7.54 (s, 1H), 7.00 (s, 1H), 3.94 (s, 3H), 3.78 (s, 3H), 3.60 (s, ZN), 1.75 (s, 3H).

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

Stage b:

8-bromo-1-(3,5-difluoropyridin-4-yl)-7-methoxy-1H,2H,3H-imidazo[4,5-c]quinolin-2-one (3.00 g) was placed in a round bottom flask , 6.96 mmol, 94%) in N,N-dimethylformamide (50 ml). Sodium hydride (1.39 g, 34.8 mmol, 60%) was added at 0°C for 5 minutes followed by CD ₃ I (3.18 g, 20.9 mmol, 95%). The resulting solution was stirred for 15 minutes at room temperature. 500 ml of ice water was added to the mixture, and the solids were collected by filtration. As a result, 2.95 g (99%) of 8-bromo-1-(3,5-difluoro-4-pyridyl)-7-methoxy-3-trideuteriomethyl)imidazo[4,5-c]quinoline-2- it is in the form of a yellow solid. HPLC/MS (purity) 99%. Retention time 0.93 min. (method B) [M+H]+ 424, 426.

Stage in:

8-bromo-1-(3,5-difluoro-4-pyridyl)-7-methoxy-3-trideuteriomethyl)imidazo-[4,5-c]quinoline-2 was placed in a sealed test tube, cleaned and containing an inert atmosphere of argon. -one (1.80 g, 4.20 mmol, 99%), 1,3-dimethyl-4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.97 g, 8.43 mmol, 95%), Pd(PPh ₃ ) ₄ (540 mg, 0.42 mmol, 90%), potassium carbonate (1.22 g, 8.39 mmol, 95%), dioxane (50 ml) and water (10 ml). The mixture was stirred for 2 hours at 80°C and concentrated under vacuum to dryness. The residue was purified by column chromatography (methanol/ethyl acetate, 13:87) to give 1.35 g (71%) 1-(3,5-difluoro-4-pyridyl)-8-(1,3-dimethylpyrazole -4-yl)-7-methoxy-3-(trideuteriomethyl)imidazo[4,5-c]quinolin-2-one as a yellow solid. HPLC/MS (purity) 97%. Retention time 0.91 min. (method B). [M+N]+ 440.

Stage d:

1-(3,5-difluoro-4-pyridyl)-8-(1,3-dimethylpyrazol-4-yl)-7-methoxy-3-(trideuteriomethyl)imidazo was placed in a sealed test tube, cleaned and containing an inert atmosphere of argon. [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 CD ₃ OD ( 2.76 ml, 2.24 g, 62.2 mmol). The mixture was stirred for 2 hours at 100°C. 100 ml of water was added and the resulting mixture was extracted three times with 200 ml of ethyl acetate. The combined organic layers were washed twice with 100 ml 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 give 1.50 g (66%) 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. The melting point was 266-271°C. HPLC/MS (purity) 97%. Hold time 2.00 min. (method B). [M+H]+ 455. 1H NMR (300 MHz, DMSO-d6) ppm. = 8.87 (s, 1H), 8.65 (d, J=7.5 Hz, 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).

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

Stage d:

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%) in N,N-dimethylformamide (30 ml). Sodium hydride (377 mg, 9.43 mmol, 60%) was added at 0°C in 5 additions, then CD ₃ I (1.44 g, 9.44 mmol, 95%). The resulting mixture was stirred for 8 hours at room temperature. 200 ml of ice water was added and the solution was extracted four times with 200 ml of ethyl acetate. The combined organic layers were washed twice with 100 ml brine. The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness and purified using dichloromethane/methanol (19:1) column chromatography to give 400 mg (47%) 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%. Retention time 0.68 min. (method D). [M+N]+ 440.

Stage e:

1-(3,5-difluoro-4-pyridyl)-7-methoxy-3-methyl-8-[3-methyl-1-(trideuteriomethyl)pyrazole-4- was placed in a sealed test tube, cleaned and containing an inert atmosphere of argon. 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 CD ₃ OD (2.45 ml, 53.9 mmol, 98%). The mixture was stirred for 4 hours at 100°C. 100 ml of water was added and the resulting solution was extracted 5 times with 100 ml of ethyl acetate. The combined organic layers were washed twice with 100 ml brine, dried over anhydrous sodium sulfate, concentrated to dryness and purified using dichloromethane/methanol (10:1) column chromatography to give 300 mg (74%) 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 orange solid. HPLC/MS (purity) 95%. Hold time 0.65 min. (method D). [M+H]+ 455. 1H NMR (300 MHz, DMSO-d6) ppm. = 8.87 (s, 1H), 8.65 (d, J=7.5 Hz, 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*50 mm, 2.2 µm; mobile phase A: water/0.05% TFA, mobile phase B: acetonitrile/0.05% TFA; flow rate: 1.0 ml/min; gradient: from 5% B to 100% B in 2.2 minutes, hold 1.0 minutes; 254 nM.

HPLC method B:

Column: Shim-pack XR-ODS, 3.0*50 mm, 2.2 µmM; mobile phase A: water/0.05% TFA, mobile phase B: acetonitrile/0.05% TFA; flow rate: 1.2 ml/min; gradient: from 5% B to 100% B in 2.0 minutes, hold 0.7 minutes; 254 nm.

HPLC method B:

Column: Poroshell NRN-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: from 10% B to 95% B in 2.1 minutes, hold 0.6 minutes; 254 nm.

HPLC method D:

Column: Ascentis Express CI8, 3.0*50 mm, 2.7 µm; mobile phase A: water/0.05% TFA, mobile phase B: acetonitrile/0.05% TFA; flow rate: 1.5 ml/min; gradient: from 5% B to 100% B in 1.2 minutes, hold 0.5 minutes; 254 nm.

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 alcohols.

7.1 Compound 1 was dissolved in 1-propanol at a concentration of approx. 50 mg/ml at 50°C and stirring. The resulting clear solution was cooled to -20°C at a cooling rate of 0.1°C/min, with a final holding period of at least 1 hour at -20°C. Solid-liquid separation was performed by vacuum suction filtration, and the filtered solid sample was dried under dynamic nitrogen purging overnight.

7.2 Compound 1 was dissolved in isobutyl alcohol at a concentration of approximately 40 mg/ml at 50°C with stirring. The resulting clear solution was cooled to -20°C at a cooling rate of 0.1°C/min, with a final holding period of at least 1 hour at -20°C. Solid-liquid separation was performed by vacuum suction filtration, and the filtered solid sample was dried under dynamic nitrogen purging overnight.

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% (w/w; based on the dry weight of Compound 1). The resulting dispersion was heated to 80°C with stirring until a clear solution was obtained. An initial cooling temperature range of 80 to 70°C was run at a rate of 0.5°C/min. followed by holding time at 70°C. At 70°C, anhydrous form A2 seeds (ground particles <50 μm in size; approximately 4.5% relative to the amount in the reactor) were added. The seed crystals were predispersed in approximately 1 ml of 2-PrOH per 100 mg amount of seed. After adding the seed crystals at 70°C, a holding time of 10 min was used. A cooling temperature range of 70°C to 5°C was started at a rate of 0.1°C/min, followed by a holding time of 3 hours at the final temperature (5°C). Solid-liquid separation was performed by vacuum suction filtration, and the filtered solid sample was dried under dynamic nitrogen purge at 70°C overnight.

In an alternative method, Form A2 was prepared by converting a suspension from another polymorphic form as follows:

7.4 The solid material, specifically another polymorphic form of Compound 1, preferably the Form H2 hydrate (the preparation of which is described below), was dispersed in approximately 5.2 vol.-eq. ethyl acetate and stirred at room temperature for 21 hours. The precipitate was filtered off and dried for at least 72 hours at 60°C under vacuum.

B. Preparation of solid Form A1

Solid Form A1 of Compound 1 was prepared using the following two methods:

7.5 Approximately 50 mg 8-(1,3-dimethyl-1H-pyrazol-4-yl)-1-(3-fluoro-5-methoxy-pyridin-4-yl)-7-methoxy-3-methyl-1, 3-dihydro-imidazo[4,5-c]quinolin-2-one (racemic mixture) was subjected to chiral resolution by supercritical fluid chromatography using a Lux Cellulose-2 column and a solvent mixture of CO ₂ /2-propanol + 0.5% diethanolamine (75:25) at a flow rate of 5 ml/min. The resulting fraction was washed with dichloromethane and concentrated at a bath temperature of 30°C to obtain a solid.

7.6 Approximately 10 mg 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, dissolved in 100 µl dichloromethane (DCM), was evaporated at RT (RT: room temperature, approximately 20-25°C) to obtain powder.

B. Preparation of Solid Form A3

1.1 Approximately 12 mg of solid material 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, representing an alternative morphic form other than A3, most preferably form A2, was dispersed in approximately 40 μl of THF and stirred at room temperature (20-25°C) approximately 4 weeks. The resulting solid was separated by centrifugation and carefully dried under ambient conditions to obtain a powder.

D. Preparation of solid form of NF9:

The NF9 Solid Form of Compound 1 was prepared according to the following two methods:

7.8 Approximately 100 mg 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, dissolved in 2000 μl of dichloromethane, was quickly evaporated in vacuo at room temperature (approximately 20-25°C) to obtain a powder.

7.9 Approximately 20 mg 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, dissolved in 1000 μl of acetone at 50°C, was added to 1 equiv. benzoic acid. The solution was cooled to room temperature (approximately 20-25°C). The solution was then subjected to vapor diffusion crystallization at room temperature (approximately 20-25° C.) using a reservoir of n-pentane, slowly diffusion into the solution by gas phase diffusion. After a few days, a solid crystallized out, which was separated by centrifugation and carefully dried under nitrogen to obtain a powder.

D. Preparation of solid hydrate Form H1

Solid Form H hydrate of Compound 1 was prepared according to the following two methods:

7.10 Approximately 12-13 mg of solid material 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, which is an alternative morphic form other than H1, most preferably form A2, was dispersed in approximately 200 μl of methanol and stirred at room temperature (20-25°C) for 5 days. The resulting solid was separated by centrifugation and carefully dried under ambient conditions to obtain a powder.

7.11 Approximately 12 mg 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 dissolved in 750 μl of 1-propanol was evaporated at RT (approximately 20-25°C) to obtain a powder.

E. Preparation of solid hydrate Form H2

7.12 Approximately 19 g of solid material 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, which is an alternative morphic form other than H2, most preferably Form A2, was dispersed in approximately 80 ml of deionized water and stirred at RT (20-25°C) for approximately 4 days. The resulting solid was collected by vacuum filtration and dried at 50° C. under a stream of nitrogen to obtain a powder.

7.13 Approximately 13-14 mg 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, dissolved in 250 µl THF, was quickly poured into a reservoir with 1500 µl deionized water at room temperature (approximately 20-25°C) with turbulent mixing. The resulting precipitate was separated by centrifugation and carefully dried under ambient conditions to obtain a powder.

G. Preparation of Solid Form of NF19

7.14 Approximately 48 mg 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 dissolved in 1500 μl of methanol was evaporated at 50°C to obtain a powder.

EXAMPLE 8: Combination with radiation therapy

In an in vivo pharmacological study (FaDu SSCHN tumor model), combination treatment with Compound 1 and fractionated irradiation for 6 weeks (6 x 5 days, 2 Gy per fraction) significantly increased the effectiveness of ionizing radiation (IO) in a dose-dependent manner according to the level of involvement targets as shown in Figure 20. More details:

The antitumor efficacy of the ATM inhibitor Compound 1 was assessed in combination with ionizing radiation in NMRI nu/nu mice bearing xenografts of human head and neck squamous cells of the FaDu model.

One of the control groups received vehicle only. Another control group received ionizing radiation (IR) only at 30 fractions of IR according to a schedule of 2 Gy for 5 days/2 days without radiation over a time period of 6 weeks. Two groups were treated with a combination of AI (same as control, AI only) and Compound 1 with an oral dose of 10 mg/kg or 25 mg/kg 30 minutes before each AI fraction.

EXAMPLE 9: Combination with PARP inhibition

The efficacy of Compound 1 in combination with olaparib was demonstrated in the HBCx-10 triple-negative breast cancer patient-derived xenograft model developed in immunodeficient female mice. The results are presented in Figure 21.

Seventy (70) mice with subcutaneously grown HBx-10 tumors (P20.1.4/0) ranging from 62.5 to 196.0 mm3 were treated when their mean and median tumor volumes reached 131.16 and 126.00 ^mm3 , respectively .

The study included different groups of 10 mice each:

- In group 1, the vehicle olaparib was administered at a dose of 10 ml/kg p.o. 1 r/day. x 28 in combination with the carrier methocel, administered at a dose of 10 ml/kg p.o. (3 d. + / 4 d. -) x4;

- In group 2, olaparib was administered at a dose of 50 mg/kg p.o. 1 r/day. x 49;

- In group 3, the compared ATM inhibitor ATMix was administered p.o. (3 d. + / 4 d. -) x 5;

- In group 4, olaparib was administered at a dose of 50 mg/kg p.o. 1 r/day. x 49 in combination with ATMix p.o. (3 d. + / 4 d. -) x 7;

- In group 5, olaparib was administered at a dose of 50 mg/kg i.o. 1 r/day. x 49 in combination with Compound 1 at a dose of 100 mg/kg p.o. (3 d. + / 4 d. -) x 7.

Tumors were measured every two weeks during the treatment period and once a week during the follow-up period.

By. - orally d. - days 1 r/day. - once a day.

Claims (10)

1. A compound represented by the following formula: or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, represented by the following formula: 3. Compound 1 according to claim 1 or 2 in solid anhydrous form, which is characterized by peaks at 7.3, 9.6, 11.1, 12.0, 12.7 and 16.2 degrees 2-theta ± 0 .2 degrees 2-theta in the X-ray powder diffraction profile. 4. Compound 1 in solid anhydrous form in accordance with paragraph 3, which is characterized by having a monoclinic crystal structure and a P2 ₁ space group and/or the following parameters of its unit cell: 5. A pharmaceutical composition for the treatment of cancer, which includes Compound 1, a pharmaceutically acceptable salt or a solid anhydrous form according to any one of paragraphs. 1-4, and a pharmaceutically acceptable excipient.

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