Pharmaceutical salts of arylpyrrole derivatives

Abstract

The present invention relates to (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)-ethyl]benzamide (+)-L-tartrate, processes for its preparation, pharmaceutical compositions containing it, and its use in the treatment of diseases such as cancer.

Description

[Background technology]

[0001] The present invention relates to pharmaceutical salts of atropisomers of triarylpyrrole compounds, methods for their preparation, pharmaceutical compositions containing them, and their use in the treatment of diseases such as cancer.

[0002] The protein expressed by the normal KRAS gene performs essential functions in normal tissue signaling. Mutation of the KRAS gene by single amino acid substitutions, particularly single nucleotide substitutions, causes activating mutations, which are essential steps in the development of many cancers. The resulting mutant proteins are implicated in a variety of malignancies, including lung adenocarcinoma, mucinous adenoma, pancreatic ductal carcinoma, and colorectal cancer. Like other members of the Ras family, the KRAS protein is a GTPase and is involved in many signaling pathways.

[0003] KRAS acts as a molecular on / off switch. When turned on, it recruits and activates proteins necessary for signal propagation from growth factors and other receptors, such as c-Raf and PI-3 kinase. In its active state, normal KRAS binds GTP and has the intrinsic enzymatic activity of cleaving the terminal phosphate of nucleotides and converting it to GDP. Once GTP is converted to GDP, KRAS is turned off. The conversion rate is typically slow but can be dramatically accelerated by accessory proteins of the GTPase-activating protein (GAP) class, such as RasGAP. KRAS can then bind to proteins of the guanine nucleotide exchange factor (GEF) class, such as SOS1, which forces the release of the bound nucleotide. Subsequently, KRAS binds to GTP present in the cytosol, releasing the GEF from ras-GTP. In mutant KRAS, its GTPase activity is directly eliminated, rendering KRAS constitutively active. Mutant KRAS is often characterized by mutations at codons 12, 13, 61, or a mixture thereof.

[0004] The viability of cancer cells harboring mutant KRAS is known to depend on polo-like kinase 1 (PLK1), and silencing PLK1 has been shown to cause death in cells containing mutant KRAS (see Luo et al., Cell. 2009 May 29;137(5):835-848). Therefore, compounds that inhibit PLK1 should be useful for treating cancers arising from KRAS mutations. However, current kinase inhibitors designed to bind to the conserved ATP-binding domain of PLK1 may be too nonselective compared to other kinases to access this mechanism of action (see, e.g., Elsayed et al., Future Med. Chem. (2019) 11(12), 1383-1386).

[0005] PLK1 is a 603-amino acid serine / threonine kinase with a molecular weight of 66 kDa, and is a key regulator of the cell cycle. In particular, PLK1 is important in mitosis, where it is involved in the formation and transformation of the mitotic spindle during the M phase of the cell cycle and the activation of CDK / cyclin complexes.

[0006] All polo-like kinases contain an N-terminal serine / threonine kinase catalytic domain and a C-terminal region containing one or two polo boxes (Lowery et al., Oncogene, (2005), 24, 248-259). In the case of polo-like kinases 1, 2, and 3, the entire C-terminal region, including both polo boxes, functions as a single modular phosphoserine / threonine-binding domain known as the polo-box domain (PBD). In the absence of bound substrate, the PBD inhibits the basal activity of the kinase domain. Phosphorylation-dependent binding of the PBD to its ligand releases the kinase domain and simultaneously localizes the polo-like kinase to specific intracellular structures.

[0007] Because PLKL1 localizes to intracellular anchoring sites via its Polo-box domain, it has been shown that the action of PLK1 can be inhibited by small molecules that disrupt its intracellular localization by inhibiting the function of the PBD (Reindl et al., Chemistry & Biology, 15, 459-466, May 2008).

[0008] The tumor protein p53 functions as a tumor suppressor, inhibiting apoptosis, genomic stability, and angiogenesis. It is known that tumors with both p53 deficiency and high PLK1 expression may be particularly sensitive to PLK1 inhibitors (Yim et al., Mutat Res Rev Mutat Res, (2014). 761, 31-39).

[0009] Thus, evidence in the literature also suggests that small molecules that bind to the PBD and inhibit its function should be effective inhibitors of the PLK1 kinase and, therefore, should also be useful in treating cancers resulting from KRAS and / or p53 mutations. Notably, because the PBD domain is only present in PLK, inhibitors designed against this domain may have greater selectivity than previous ATP-competitive inhibitors, potentially enabling a greater ability to target KRAS mutant and p53-deficient cancers.

[0010] Identifying and developing therapeutic drugs for primary brain cancer has proven particularly challenging. Targeted cancer therapies, particularly those using protein kinase inhibitors, have become a major focus for pharmaceutical and biotechnology companies (Nature Reviews Clinical Oncology 2016, 13, 209-227). However, while over 30 kinase inhibitors have been approved for use in oncology, none of these are intended for the treatment of primary brain cancer. Particularly problematic is that most approved kinase inhibitor oncology drugs lack the quality of drug substance required to achieve the brain exposure required for their use in the treatment of brain cancer (JMC 2016, 59(22), 10030-10066).

[0011] The alkylating agent temozolomide (Temodar®, Temodal®) is currently the first-line treatment for the brain tumor glioblastoma multiforme, frequently used in combination with radiation therapy. However, drug resistance is a major problem in the management of glioblastoma, thus limiting the usefulness of temozolomide. Therefore, at present, malignant glioblastoma remains incurable.

[0012] Polo-like kinase 1 (PLK1) is overexpressed in various tumor types, including glioblastoma multiforme (Translational Oncology 2017, 10, 22-32). Furthermore, recent studies have shown that PLK1 drives checkpoint adaptation and temozolomide resistance in glioblastoma multiforme (Oncotarget 2017, 8, 15827-15837).

[0013] Ependymoma is a brain and spinal cord tumor, and current standard treatment is limited to surgery and radiation. PLK1 is involved in ependymoma, and PLK1 inhibitors are active against ependymoma cell lines [Gilbertson et al., Cancer Cell (2011) 20, 384-399].

[0014] PLK1 has also been investigated as a target for diffuse pontine glioma (DIPG), a highly malignant and invasive pediatric brain tumor [Amani et al. BMC Cancer (2016) 16,647 and Cancer Biology and Therapy (2018) 19,12,1078-1087].

[0015] More specifically, inhibition of PLK1 has been shown to enhance the efficacy of temozolomide in IDH1-mutant gliomas [Oncotarget,(2017)8,9,15827-15837] and inhibit tumor growth in an MMR-deficient, temozolomide-resistant glioblastoma xenograft model [Mol Cancer Ther;17(12)December 2018].

[0016] In the above cases, current inhibitors lack sufficient brain exposure.

[0017] Compounds that inhibit PLKL1, but do not induce drug resistance and show good brain exposure are expected to be useful in the treatment of glioblastoma multiforme and other brain cancers.

[0018] PLK4 is a member of the polo-like kinase family of serine / threonine kinases that plays an important role in centrosome duplication and acts as a central regulator of centriole duplication (Bettencourt-Dias, Curr Biol. 2005 15(24);2199-207). PLK4-dependent changes in the centrosome can lead to asymmetric chromosome segregation during mitosis, which can cause chromosome missegregation and cell death after mitotic defects.

[0019] PLK4 is aberrantly expressed in human cancers and is involved in tumorigenesis and metastasis, highlighting it as a promising target for cancer therapy (Zhao, J Canc Res Clin Oncol., 2019).

[0020] PLK4 is overexpressed in many cancers, including rhabdoid tumors, medulloblastomas, and other embryonal tumors of the brain (Pediatr Blood Cancer. 2017), as well as breast cancer, lung cancer, melanoma, gastric cancer, colorectal cancer, pancreatic cancer, and ovarian cancer. Elevated or overactivated PLK4 is associated with decreased survival in cancer patients, including ovarian, breast, and lung cancer (Zhao, J Canc Res Clin Oncol. 2019).

[0021] PLK4 inhibition has been investigated for the treatment of glioblastoma multiforme, demonstrating that PLK4 plays a key role in regulating temozolomide chemosensitivity. The combination of temozolomide and PLK4 inhibition in glioblastoma PDX models has been shown to enhance antitumor efficacy compared to temozolomide alone (Cancer Letters, Vol. 443, 2019, 91-107).

[0022] PLK4 has been reported to cooperate with p53 inactivation in cancer development, and cancers with PLK4 overexpression and p53 deficiency are predicted to be more likely to form tumors (Sercin, 2016; Nat Cell Biol 18:100-110). Therefore, compounds that inhibit PLK4 activity are expected to be useful for the treatment of p53-mutated cancers.

[0023] Inhibition of PLK4 results in antitumor activity in lung cancer, and activity is seen in cancers harboring wild-type and mutant KRAS (Kawakami, PNAS 2018, 115(8)1913-18). Therefore, compounds that inhibit PLK4 activity are expected to be useful in treating KRAS mutant cancers.

[0024] Current PLK4 inhibitors act at the kinase active site and are suboptimal for brain penetration (Int. J. Mol. Sci. 2019, 20, 2112). Therefore, compounds that inhibit the PBD of PLK4 but also exhibit favorable brain exposure are expected to be useful for the treatment of glioblastoma multiforme and other brain cancers.

[0025] Our earlier international patent application WO2018 / 197714 discloses compounds of the following formula: [ka] wherein ring X is a benzene or pyridine ring, ring Y is a benzene, pyridine, thiophene, or furan ring, and Ar 1 is an optionally substituted benzene, pyridine, thiophene, or furan ring, and R 1 ~R 4 , R 6 , R 7are hydrogen or various substituents). The compounds are described as having anticancer activity and good brain exposure after oral administration, making them good candidates for the treatment of brain cancer. The compounds are active against glioblastoma cell lines and are believed to act as inhibitors of the polo-box domain of PLK1 kinase. The compounds are also disclosed to be active against mutant RAS cancer cell lines (such as HCT116) and should also be useful in the treatment of cancers resulting from KRAS mutations. Summary of the Invention

[0026] It has now been discovered that the compound of Example 33 of WO 2018 / 197714, i.e., 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide, and its analogs, form atropisomers, which are stereoisomers resulting from hindered rotation about a single bond axis, where the energy barrier to rotation is high enough to allow for the isolation of individual rotamers; see LaPlante et al., J. Med. Chem., 54:7005-7022 (2011).

[0027] Atropisomers can be classified into three categories based on the amount of energy required to rotate the chiral axis and racemize and the length of time required for racemization to occur: Class 1 atropisomers have a rotational barrier about the chiral axis of less than 84 kJ / mol (20 kcal / mol) and racemize at room temperature in times measured within minutes; Class 2 atropisomers have a rotational barrier of 84-117 kJ / mol (20-28 kcal / mol) and racemize at room temperature in times measured from hours to months; and Class 3 atropisomers have a rotational barrier of greater than 117 kJ / mol (28 kcal / mol) and racemize at room temperature in times measured in years.

[0028] The stereochemistry of atropisomers can be assigned using the Cahn-Ingold-Prelog R and S system, exemplified by (S)-6,6′-dinitrobiphenyl-2,2′-dicarboxylic acid shown in FIG.

[0029] In this system, the nearest substituents on either side of the aryl-aryl bond are assigned priority in the order a, b, and c. If the substituents a, b, and c are in a counterclockwise configuration, the atropisomer is the S isomer. In the corresponding R isomer, the substituents a, b, and c are in a clockwise configuration.

[0030] Atropisomers of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide and its analogs have been found to be sufficiently stable to be isolated and characterized, and do not racemize to any significant extent when heated to temperatures up to 80°C for 10 days. Therefore, the atropisomers can be classified as Class 3 atropisomers. Atropisomerism is believed to arise because steric interactions between the 2-trifluoromethyl substituent and the aromatic rings attached to the 2- and 5-positions of the pyrrole ring prevent rotation around the bond between the 2-trifluoromethyl-substituted ring and the nitrogen atom of the pyrrole ring.

[0031] The two individual atropisomers of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide have been found to have significantly different biological properties. Thus, typically, one atropisomer of a pair is significantly more active against a particular cancer target than the other atropisomer of the pair. The atropisomer with better biological activity against the intended biological target has been shown by single-crystal X-ray analysis to have the R configuration, i.e., chemical structure (1). [ka]

[0032] It has further been found that there is an approximately 1:1 molar ratio between acid and base, and that the (+)-L-tartrate salt of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is advantageous over the free base form of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide and other salts of the compound.

[0033] (+)-L-Tartaric acid is particularly advantageous in that it is a highly crystalline, stable solid that takes up only surface moisture (less than 1% at 90% RH) with improved water solubility over the free base. These properties make it particularly suitable for pharmaceutical development.

[0034] Thus, in a first aspect (Aspect 1.1), the present invention provides (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)-ethyl]benzamide (+)-L-tartrate, wherein the molar ratio between acid and base is about 1:1.

[0035] In a further aspect, the present invention provides: 1.2 (+)-L-tartrate salt of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide having formula (2): [ka]

[0036] 1.3 (+)-L-tartrate salt of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide, in which there is an approximately 1:1 molar ratio between acid and base and 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is in the form of a single atropisomer.

[0037] 1.4 The (+)-L-tartrate salt according to embodiment 1.3, wherein the single atropisomer is the atropisomer of formula (1).

[0038] 1.5 The (+)-L-tartrate salt according to embodiment 1.3, wherein the single atropisomer is the R atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide.

[0039] 1.6 The (+)-L-tartrate salt according to embodiment 1.3, wherein the single atropisomer is characterized by any one or more of the following parameters: (i) X-ray crystallography data substantially as described in Example 3 herein; (ii) a retention time of about 20 minutes (e.g., about 20.5 minutes) as determined by Chiral HPLC Method 1 herein; and (iii) a specific rotation of about −11.76° as measured using the method described in Example 2 herein.

[0040] 1.7 The (+)-L-tartrate salt according to embodiment 1.3, wherein the single atropisomer is atropisomer A-2 as described in the examples herein.

[0041] 1.8 The (+)-L-tartrate salt according to embodiment 1.3, wherein the (+)-L-tartrate salt is as described in the examples herein.

[0042] 1.9 The (+)-L-Tartrate Salt according to any one of embodiments 1.1 to 1.8, which is in a crystalline form.

[0043] 1.10 The (+)-L-tartrate salt according to embodiment 1.9, which is anhydrous.

[0044] 1.11 The (+)-L-tartrate salt according to embodiment 1.10, which is the anhydrate identified herein as Pattern B.

[0045] 1.12 The (+)-L-tartrate salt according to embodiment 1.9, which is a solvate.

[0046] 1.13 The (+)-L-tartrate salt according to embodiment 1.12, which is a solvate identified herein as Pattern A.

[0047] 1.14 A composition of matter comprising the (+)-L-tartrate salt according to any one of Aspects 1.3 to 1.13, wherein either (a) a single atropisomer is the only atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide present in the composition, or (b) less than 10% by molar amount of any other atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is present relative to the single atropisomer.

[0048] 1.15 A composition of matter according to Aspect 1.14, wherein either (a) a single atropisomer is the only atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide present in the composition, or (b) less than 5% by molar amount of any other atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is present relative to said single atropisomer.

[0049] 1.16 A composition of matter according to Aspect 1.14, wherein either (a) a single atropisomer is the only atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide present in the composition, or (b) less than 2% by molar amount of any other atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is present relative to the single atropisomer.

[0050] 1.17 A composition of matter according to embodiment 1.14, wherein either (a) a single atropisomer is the only atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide present in the composition, or (b) less than 1.5% by molar amount of any other atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is present relative to said single atropisomer.

[0051] 1.18 A composition of matter according to embodiment 1.14, wherein either (a) a single atropisomer is the only atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide present in the composition, or (b) less than 1% by molar amount of any other atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is present relative to said single atropisomer.

[0052] 1.19 The composition of matter of embodiment 1.14, wherein either (a) a single atropisomer is the only atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide present in the composition, or (b) less than 0.1% by molar amount of any other atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is present relative to the single atropisomer.

[0053] definition (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)-ethyl]benzamide (+)-L-tartrate, the compound of Formula (2), and compositions of matter defined in any one of Embodiments 1.1-1.19 may be collectively or generically referred to as "the tartrate salt of the invention." Accordingly, reference to "the tartrate salt of the invention" may be interpreted as a reference to any one of Embodiments 1.1-1.19, unless the context indicates otherwise.

[0054] Isotopes A composition of matter, compound, or salt defined in any one of aspects 1.1 to 1.19 may contain one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of that element. For example, a reference to hydrogen includes within its scope all isotopes of that element. 1 H, 2 H(D), and 3 Similarly, references to carbon and oxygen include 12 C. 13 C, and 14 C, and 16 O and 18 Contains O.

[0055] The isotope may be radioactive or non-radioactive. In one aspect of the present invention, the composition of matter or atropisomer does not contain a radioactive isotope. Such compounds are preferred for therapeutic use. However, in another aspect, the composition of matter or atropisomer may contain one or more radioactive isotopes. Compounds containing such radioactive isotopes may be useful in diagnostic situations.

[0056] Solvates and anhydrides The compositions of matter, compounds or salts defined in any one of embodiments 1.1 to 1.19 may form solvates and anhydrates.

[0057] Particular solvates are those formed by incorporating molecules of a non-toxic, pharmaceutically acceptable solvent (hereinafter referred to as the solvating solvent) into the solid-state structure (e.g., crystalline structure) of the composition of matter or atropisomer of the present invention. Solvates can be prepared by recrystallizing the composition of matter or atropisomer of the present invention with a solvent or mixture of solvents containing the solvating solvent. Whether a solvate has formed in any given instance can be determined by analyzing the composition of matter or crystals of the atropisomer using well-known standard techniques, such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC), and X-ray powder diffraction (XRPD).

[0058] The solvates may be stoichiometric or non-stoichiometric solvates.

[0059] Examples of solvates are those formed with any one of one or more solvents selected from water, alcohols (e.g., methanol, ethanol, and isopropyl alcohol), alkyl alkanoate esters (e.g., ethyl acetate and isopropyl acetate), ethers (especially cyclic ethers such as 1-4-dioxane and tetrahydrofuran), and monocyclic lactams (e.g., N-methylpyrrolidone).

[0060] For a more detailed discussion of solvates and methods used to prepare and characterize them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc. of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

[0061] In addition to forming solvates, the compositions of matter, compounds, or salts defined in any one of embodiments 1.1-1.19 may be provided in anhydrous form. As used herein, the term "anhydrous" refers to a solid particulate form that does not contain water (and preferably does not contain other solvents) within its three-dimensional structure (e.g., crystalline form), although salt or compound particles may have water molecules attached to their outer surface.

[0062] Methods for preparing the compounds of the present invention The (+)-L-tartrate salt of the present invention can be prepared from the atropisomer of formula (1) by reacting it with tartaric acid in a solvent or mixture of solvents, and then isolating the tartrate salt from the solvent or mixture of solvents.

[0063] In one embodiment, the atropisomer of Formula (1) can be dissolved or suspended in one solvent to form a first mixture, (+)-L-tartaric acid can be dissolved or suspended in the same or another solvent to form a second mixture, and the first and second mixtures can then be combined and allowed to stand (e.g., with stirring) for a period of time to allow salt formation to occur, followed by isolation of the (+)-L-tartrate salt.

[0064] When the first and second mixtures are combined, it is preferred that the molar amounts of the atropisomer of formula (1) and (+)-L-tartaric acid are approximately equal; i.e., there is preferably a 1:1 molar ratio between the atropisomer of formula (1) and (+)-L-tartaric acid.

[0065] The (+)-L-tartrate salt can be isolated from the combined mixture by filtration (if a precipitate forms) or by evaporation of the solvent.

[0066] Thus, when two or more solvents are present in the combined mixture, different solvents can be selected to act as co-solvents or anti-solvents.

[0067] The solvent or mixture of solvents can be selected to at least partially keep the (+)-L-tartrate salt in solution when heated, but to cause the salt to deposit as a precipitate when the solvent or mixture of solvents is cooled.

[0068] The solvent used to form the first mixture (the mixture containing the atropisomer of Formula (1)) can be selected from, for example, aliphatic ketones, aliphatic esters of aliphatic acids, non-aromatic cyclic ethers, and aliphatic alcohols.

[0069] A preferred example of an aliphatic ketone is acetone.

[0070] Examples of aliphatic esters of aliphatic acids include C acetic acid 2-4 Alkyl esters are included, a particular example being isopropyl acetate.

[0071] Examples of non-aromatic cyclic ethers include dioxane, 2-methyltetrahydrofuran, and tetrahydrofuran, with a particular example being 2-methyltetrahydrofuran.

[0072] Examples of fatty alcohols are C 2-4 Aliphatic alcohols, more specifically C such as isopropyl alcohol and butanol 3-4 It is an alkanol.

[0073] The solvent used to form the second mixture (the mixture containing (+)-L-tartaric acid) can be selected from, for example, water, non-aromatic cyclic ethers, and aliphatic alcohols.

[0074] A particular example of an aliphatic alcohol solvent for the second mixture is ethanol.

[0075] A specific example of a non-aromatic cyclic ether solvent for the second mixture is tetrahydrofuran (THF).

[0076] Another specific example of a solvent for use in forming the second mixture is water.

[0077] The atropisomeric (+)-L-tartrate salt of formula (1) can exist in several crystalline forms, specifically Pattern A (which is a solvate) and Pattern B (which is an anhydrate). Details of the characterization of the different crystalline forms are provided elsewhere herein. The different crystalline forms can be prepared by varying the solvent and heating conditions used to form the salt.

[0078] In one process for making the (+)-L-tartrate salt of an atropisomer of Formula (1) having Pattern A, a solution of the atropisomer in acetone is mixed with a solution of (+)-L-tartaric acid in ethanol at a temperature ranging from 20°C to 30°C (e.g., about 25°C), the resulting mixture is stirred or otherwise agitated for a time sufficient for salt formation to occur (e.g., 12 to 24 hours), and the salt is then isolated by filtration.

[0079] In another process for making the (+)-L-tartrate salt of the atropisomer of Formula (1) having Pattern A, a solution of the atropisomer in isopropyl alcohol is mixed with a solution of (+)-L-tartaric acid in ethanol at a temperature ranging from 35°C to 45°C (e.g., about 40°C), the resulting mixture is cooled to a temperature ranging from 20°C to 30°C (e.g., about 25°C) over about 1 to 3 hours, and the salt is then isolated by filtration.

[0080] In another process for making the (+)-L-tartrate salt of an atropisomer of Formula (1) having Pattern A, a solution of the atropisomer in 2-methyltetrahydrofuran is mixed with a solution of (+)-L-tartaric acid in ethanol at a temperature ranging from 20° C. to 30° C. (e.g., about 25° C.), the resulting mixture is stirred or otherwise agitated for a time sufficient for salt formation to occur (e.g., 12 to 24 hours), and the salt is then isolated by filtration.

[0081] In one process for making the (+)-L-tartrate salt of the atropisomer of Formula (1) having Pattern B, a solution of the atropisomer in isopropyl acetate at a temperature ranging from 35°C to 45°C (e.g., about 40°C) is mixed with a solution of (+)-L-tartaric acid in ethanol, the resulting mixture is cooled to a temperature ranging from 20°C to 30°C (e.g., about 25°C) over about 1 to 3 hours, and the salt is then isolated by filtration.

[0082] Another process for making the (+)-L-tartrate salt of the atropisomer of Formula (1) having Pattern B involves mixing (either in portions or in a single charge) a solution of the atropisomer in isopropyl acetate at a temperature in the range of 35°C to 45°C (e.g., about 40°C) with a solution of (+)-L-tartaric acid in THF, adding one or more seed crystals of salt Pattern B to obtain a precipitate, cooling the mixture to a temperature in the range of 20°C to 30°C (e.g., about 25°C), and stirring or agitating for a sufficient time (e.g., 12 to 24 hours, especially about 20 hours) to allow the precipitate to mature to a state where it can be isolated by filtration.

[0083] In another process for the (+)-L-tartrate salt of the atropisomer of Formula (1) having Pattern B, a solution of the atropisomer in butanol at an elevated temperature in the range of 70°C to 85°C (e.g., about 80°C) is mixed (either in portions or in a single charge) with a solution of (+)-L-tartaric acid in water, and the resulting mixture is cooled to an intermediate temperature in the range of 65°C to 70°C, followed by the addition of one or more seed crystals of the Pattern B salt, and the mixture is cooled to a low temperature in the range of 3 to 10°C over a period of 8 to 15 hours, after which the resulting mixture is stirred or agitated at or near low temperature for an additional 2 to 8 hours (e.g., about 6 hours), and the Pattern B salt formed is then filtered off.

[0084] The atropisomer of formula (1) is (a) separating a mixture of atropisomers (e.g., a racemic mixture) of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)-ethyl]benzamide; or (b) Atropisomers of formula (3): [ka] with an amine of formula H2N-(CH2)-N(CH3)2 under amide forming conditions It can be prepared by

[0085] Compounds of formula (1), (2) and (3) can be prepared by the route shown in Scheme 1 below. [ka]

[0086] The starting materials for the synthetic route shown in Scheme 1 are 4-cyano-acetophenone (4) and 4-chlorophenacyl bromide (5), both of which are commercially available.

[0087] In step 1, 4-cyanoacetophenone (4) and 4-chlorophenacyl bromide (5) are reacted together to give 4-[4-(4-chlorophenyl)-4-oxo-butanoyl]benzonitrile (6). The reaction is typically carried out in a suitable solvent, such as a mixture of a non-polar (e.g., hydrocarbon) solvent such as benzene or toluene with a tertiary alcohol (e.g., t-butanol), in the presence of a tertiary amine such as triethylamine, and in the presence of a zinc(II) salt (e.g., zinc chloride). The reaction can be carried out at or near room temperature for, for example, 12 to 60 hours.

[0088] In step 2, 4-[4-(4-chlorophenyl)-4-oxo-butanoyl]benzonitrile (6) is reacted with 2-trifluoromethylaniline to give 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzonitrile (7). The reaction is typically carried out in a suitable high-boiling solvent (e.g., dioxane) in the presence of an acid catalyst such as p-toluenesulfonic acid at elevated temperature (e.g., 130-170°C) and / or microwave irradiation. The reaction can be carried out for 1 to 12 hours, for example, 1 to 6 hours.

[0089] In step 3, 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzonitrile (7) is subjected to alkaline hydrolysis to give 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (8). The hydrolysis reaction is typically carried out in the presence of an alkali metal hydroxide, such as sodium hydroxide (typically in excess), in an aqueous solvent which may contain an alcohol, such as methanol, generally with heating, for example, at a temperature in the range of 60-80°C or for a period of up to about 20 hours or more. Once hydrolysis is complete, the acid (8) is typically isolated by cooling and acidifying the reaction mixture.

[0090] Following step 3, one of two possible routes to atropisomer 1 can be followed. In one variation consisting of steps 4b, 5b, and 6, 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid 8 is reacted with N,N-dimethylethylenediamine under amide-forming conditions to give a racemic mixture of atropisomers of 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)-N-(2-(dimethylamino)ethyl)benzamide 9, which is then resolved into the individual atropisomers by chiral separation to give atropisomer 1.

[0091] In another variation, racemic 6,4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (8) is subjected to chiral separation to give atropisomer (3), which is then reacted with N,N-dimethylethylenediamine under amide-forming conditions to give atropisomer (1).

[0092] Carboxylic acids (3) and (8) are reacted with N,N-dimethylethylenediamine in the presence of an amide coupling reagent under amide-forming conditions. Examples of such amide coupling reagents include 1,3-dicyclohexylcarbodiimide (DCC) (Sheehan et al., J. Amer. Chem. Soc. 1955, 77 ,1067) and 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (also referred to herein as EDC or EDCI) (Sheehan et al, J. Org. Chem., 1961, 26, 2525), which are typically substituted with 1-hydroxy-7-azabenzotriazole (HOAt) (LA Carpino, J. Amer. Chem. Soc., 1993, 115, 4397) or 1-hydroxybenzotriazole (HOBt) (Konig et al., Chem. Ber., 103, 708, 2024-2034), in combination with uronium-based coupling reagents such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and propanephosphonic anhydride (T3P) (see A. Garcia, Synlett, 2007, No. 8, pp. 1328-1329). Specific amide coupling reagents for use in process steps 5a and 5b are HATU and T3P.

[0093] The amide coupling reaction is typically carried out in a non-aqueous, polar, aprotic solvent such as tetrahydrofuran or dimethylformamide or mixtures thereof at or near room temperature (e.g., 18-30°C) in the presence of a non-interfering base, for example, a tertiary amine such as triethylamine or N,N-diisopropylethylamine.

[0094] Chiral separation of the mixture of atropisomers of carboxylic acid (8) and amide (9) can be achieved using a variety of techniques. For example, chiral chromatography can be used to separate the individual atropisomers. The retention times of the atropisomers in the chiral chromatographic procedure provide a means to distinguish and characterize the individual atropisomers, whose NMR and MS properties are typically identical.

[0095] Chiral chromatography columns that can be used to separate individual atropisomers include immobilized chiral stationary phases (CSFs), which can be based on, for example, functionalized amylose or cellulose. Examples of such CSFs are amylose and cellulose functionalized with chloro- and / or methyl-substituted phenylcarbamates. A specific example of a chiral column that can be used to isolate individual atropisomers of the present invention is the "Chiralpak IG" column available from Daicel Corporation.

[0096] Mobile phases that can typically be used with the chiral columns described above include (A) a liquid alkane, such as n-heptane, containing a small amount (e.g., up to 1% (v / v) and more usually about 0.1% (v / v)) of an alkylamine base, such as diethylamine; and (B) alcohols and mixtures thereof, such as a mixture of isopropyl alcohol and methanol (e.g., 70:30 IPA:MeOH). For example, the mobile phase can include a mixture of A:B in a ratio ranging from 80:20 to 95:5, e.g., from about 85:15 to about 90:10. The mobile phase can be used in an isocratic or gradient elution mode; however, in one embodiment of the present invention, an isocratic elution mode is used.

[0097] The atropisomers of the present invention can also be resolved by chiral HPLC under supercritical fluid chromatography (SFC) conditions, in which the mobile phase often comprises a supercritical fluid such as carbon dioxide, together with a co-solvent such as an alcohol or a mixture of alcohols, e.g., methanol, ethanol, and isopropanol.

[0098] The Chiralpak IG column described above can be used in SFC chromatography procedures using carbon dioxide / methanol / isopropanol mixtures as the mobile phase.

[0099] Other chiral column / cosolvent combinations for use in SFC include: Lux Cellulose 4 (MeOH, EtOH); Lux cellulose 2(MeOH); Lux Amylose 1 (MeOH, EtOH); and YMC Amylose-SA (MeOH, EtOH).

[0100] The Lux family of chiral columns is available from Phenomenex, Inc.

[0101] YMC Amylose-SA columns are available from YMC America, Inc.

[0102] The chiral chromatographic methods described above can be used to separate the atropisomer (1) specifically from the racemic mixture (9).

[0103] As an alternative to chiral chromatography, a chiral acid (e.g., (+)-10-camphorsulfonic acid) can be reacted with the racemic mixture (9) to form chiral salts as a mixture of diastereoisomers, which can be separated by crystallization to give the salts of atropisomers (1), which can then be converted to the free bases of atropisomers (1).

[0104] The racemic mixture of atropisomers of carboxylic acid (8) can be resolved by forming a salt with a chiral amine, such as (S)-1-(4-methoxyphenyl)-ethylamine, to form a mixture of diastereoisomers that can be separated, for example, by crystallization with the aid of seed crystals of the salt of the atropisomer (1) with the chiral amine.

[0105] Certain aspects of the above-described processes represent further embodiments of the present invention (embodiments 2.1 to 2.5). Accordingly, the present invention provides:

[0106] 2.1 A process for preparing a compound of formula (1) comprising reacting a compound of formula (3) with N,N-dimethylethylenediamine under amide-forming conditions.

[0107] 2.2 The method of embodiment 2.1, wherein the amide-forming conditions comprise the presence of an amide coupling reagent, eg, an amide coupling agent described herein.

[0108] 2.3 The method of embodiment 2.2, wherein the amide coupling reagent is propanephosphonic anhydride (T3P).

[0109] 2.4 A process for the preparation of compounds of formula (3), comprising chiral separation of compounds of formula (3) from a mixture of atropisomers of formula (8), for example, by chiral chromatography or salt formation with a chiral base and resolution of the resulting chiral salt.

[0110] 2.5 Atropisomeric compounds having formula (3) or salts thereof (for example metal salts such as alkali metal salts or alkaline earth metal salts, or salts with ammonia or organic amines).

[0111] Biological Properties and Therapeutic Uses Evidence presented in the examples below demonstrates that the atropisomers of formula (1) defined herein and their tartrate salts are inhibitors of the Polo-box domains of PLK1 and PLK4 kinases, but not the catalytic domains of PLK1 and PLK4 kinases. Because the PBD domain is present only in PLK, the atropisomers of formula (1) and their tartrate salts should exhibit much higher selectivity (and therefore fewer undesirable side effects due to off-target kinase inhibition) than compounds that are ATP-competitive kinase inhibitors. Results from the study described in Example 7F below, in which the atropisomers of formula (1) were tested against a panel of 97 kinases and showed negligible activity against other kinases, support the high selectivity of the atropisomers of formula (1) for PLK1-PBD and PLK4-PBD over other structurally and functionally similar kinases.

[0112] An additional advantage of inhibiting the PBD domain rather than the catalytic domain is that this may be less prone to inducing drug resistance compared to PLK1 inhibitors that inhibit the catalytic domain.

[0113] The activity of the atropisomer of formula (1) and its tartrate salt as inhibitors of the PBD domain of PLK1 kinase can be demonstrated using the fluorescence polarization (FP) assay described in Narvaez et al., Cell Chemical Biology, 24, 1017-1028, 2017 (see pages 1018 and 1026 (method details)).

[0114] It is believed that the atropisomers of formula (1) and their tartrate salts may be effective in exploiting weaknesses in cellular pathways as a result of constitutively activating mutant KRAS, and therefore may be useful in the treatment of diseases and conditions mediated by modulation of KRAS.

[0115] Mutations in KRAS, caused by single nucleotide substitutions, are associated with various forms of cancer, with KRAS mutations occurring in a high proportion of leukemia, colon cancer, pancreatic cancer, and lung cancer.

[0116] Furthermore, it is believed that the atropisomer of formula (1) and its tartrate salt may be useful in treating cancers characterized by p53 deficiency or mutations in the TP53 gene. PLK1 is believed to inhibit p53 in cancer cells. Therefore, treatment with a PLK1 inhibitor should activate p53 in tumor cells and induce apoptosis.

[0117] The activity of the atropisomers of formula (1) against KRAS mutant and p53-deficient cancers is believed to occur, at least in part, through inhibition of the C-terminal polo-box domain (PBD) of the PLK1 kinase, as described above. KRAS is known to be dependent on its interaction with PLK1.

[0118] Atropisomers of formula (1) induce mitotic arrest with unassembled chromosomes, a property believed to result from the PLK1-PBD and PLK4-PBD inhibitory activity of the atropisomers (see Example 7C below).

[0119] The atropisomer induces mitotic arrest with a multipolar spindle phenotype and causes centriole amplification, a well-described phenotype of PLK4 inhibition (Lei 2018, Cell Death & Disease 9, 1066; Kawakami, PNAS 2018, 115(8)1913-18). These phenotypes are thought to result from the PLK4-PBD inhibitory activity of the atropisomer of formula (1).

[0120] A primary screen for anticancer activity utilizing a cancer cell line (U87MG, human brain (glioblastoma astrocytoma)) is described below in Example 7A. The data obtained showed that the R-atropisomer of formula (1) (atropisomer A-2) had an IC of 4.6 μM against the U87MG cell line. 50 is much more active than the corresponding S-atropisomer (A-1) (IC of 0.22 μM) 50 ) has been demonstrated.

[0121] Two atropisomers (A-2 and A-1) were also tested against a panel of 48 cancer cell lines, and the results are shown below in Example 7B. In all cell lines tested, atropisomer (A-2) of formula (1) was in most cases at least 10 times more active than atropisomer (A-1).

[0122] The data in Example 7B demonstrate that atropisomer (A-2) of formula (1) is active against a wide range of different cancer cell lines, ranging from solid tumors such as pancreatic cancer, colon and colorectal cancer, lung cancer, brain and nerve cancer, and hematological cancers such as lymphoma and leukemia.

[0123] The atropisomers of formula (1) have good oral bioavailability (see Example 7G below) and good brain exposure when administered orally (Example 7G). Thus, the compositions of matter or atropisomers of the present invention should be useful in the treatment of brain cancers such as glioma and glioblastoma.

[0124] Based on the evidence to date, it is believed that atropisomers of formula (1) are useful in the treatment of a wide range of cancers (and their benign counterparts), including those described in the following embodiments.

[0125] Thus, in further aspects (Aspects 3.1 to 3.25), the present invention provides:

[0126] 3.1. A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use as a PLK1-PBD.

[0127] 3.2 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use as a PLK4-PBD inhibitor.

[0128] 3.3 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use as a PLK1-PBD and PLK4-PBD inhibitor.

[0129] 3.4 The cancer is a tumor of epithelial origin (various types of adenomas and carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other cancers), e.g., cancer of the bladder, urinary tract, breast, gastrointestinal tract (including esophagus, stomach (gastric portion), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g., cancer of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., thyroid follicular carcinoma), adrenal gland, prostate, skin and adnexal organs (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematologic malignancies (i.e., leukemia, lymphoma) and premalignant hematologic and borderline malignant disorders, including hematologic malignancies and lymphatic system-related conditions (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [ NK-cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), as well as hematological malignancies and related conditions of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndromes, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia); tumors of mesenchymal origin such as soft tissue tumors Sarcomas of the tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma and glioblastoma, meningioma, ependymoma, pineal tumor, and schwannoma); endocrine tumors (e.g., pituitary tumor, adrenal tumor, pancreatic islet cell tumor, parathyroid tumor, carcinoid tumor, medullary thyroid carcinoma); eye and accessory tumors (e.g., retinoblastoma);A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy), selected from germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinomas); and pediatric and fetal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor); or congenital or other syndromes in which the patient is susceptible to malignancy (e.g., xeroderma pigmentosum).

[0130] 3.4A A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy), wherein the cancer is selected from pancreatic cancer, colon and colorectal cancer, lung cancer, brain and nerve cancer, and blood cancer (e.g., lymphoma and leukemia).

[0131] 3.5 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., anti-cancer agent or therapy), wherein the cancer is selected from pancreatic cancer, colon and colorectal cancer, lung cancer, brain and nerve cancer, and hematological cancers such as lymphoma and leukemia.

[0132] 3.6 The tartrate salt or composition of matter of any one of aspects 1.1-1.19 for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy), wherein the cancer is selected from glioma and glioblastoma (e.g., glioblastoma multiforme, ependymoma, diffuse pontine glioma, IDH1-mutated glioma).

[0133] 3.7 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy), wherein the cancer is selected from rhabdoid tumor, medulloblastoma and other embryonal tumors of the brain; breast cancer, lung cancer, melanoma, gastric cancer, colorectal cancer, pancreatic cancer, and ovarian cancer.

[0134] 3.7 The tartrate salt or composition of matter according to any one of aspects 1.1-1.19, for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy), wherein the cancer is selected from: (a) a tumor of epithelial origin selected from breast cancer, gastrointestinal cancer, exocrine pancreatic cancer, lung cancer, and prostate cancer; (b) hematological malignancies selected from B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], Burkitt lymphoma, mantle cell lymphoma, multiple myeloma, acute myeloid leukemia [AML], chronic myeloid leukemia [CML], and myelodysplastic syndrome; (c) a tumor of mesenchymal origin selected from osteosarcoma and rhabdomyosarcoma; (d) a tumor of the central or peripheral nervous system selected from glioma, glioblastoma, and ependymoma; and (e) Pediatric and embryonal tumors selected from medulloblastoma and neuroblastoma.

[0135] 3.8 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy), wherein the cancer involves PLK1 (e.g., PLK1 is overexpressed).

[0136] 3.9 The tartrate salt or composition of matter for use according to embodiment 3.8, wherein the cancer is as defined in any one of embodiments 3.4 to 3.7.

[0137] 3.10 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy), wherein the cancer involves PLK4 (e.g., PLK4 is overexpressed).

[0138] 3.11 The tartrate salt or composition of matter for use according to embodiment 3.10, wherein the cancer is as defined in any one of embodiments 3.4 to 3.7.

[0139] 3.12 The tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the treatment of cancer, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy), wherein the cancer is characterized by p53 deficiency or a mutation in the TP53 gene.

[0140] 3.13 The tartrate salt or composition of matter for use according to embodiment 3.12, wherein the cancer is as defined in any one of embodiments 3.4 to 3.7.

[0141] 3.14 The tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the treatment of cancer, wherein the cancer is characterized by the presence of a mutated form of KRAS.

[0142] 3.15 The tartrate salt or composition of matter for use according to aspect 3.14, wherein the mutant form of KRAS has a mutation in an amino acid in the protein selected from glycine 12, glycine 13, glutamine 61, and combinations thereof.

[0143] 3.16 The tartrate salt or composition of matter for use according to embodiment 3.14 or 3.15, wherein the cancer is as defined in any one of embodiments 3.4 to 3.7.

[0144] 3.17 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in medicine or therapy, optionally in combination with another therapeutic agent or treatment (e.g., anti-cancer agent or therapy).

[0145] 3.18 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in the prevention or treatment of disease states and conditions characterized by aberrant expression of KRAS protein, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy).

[0146] 3.19 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use as an anti-cancer agent.

[0147] 3.20 A method of treating a subject (e.g., a mammalian subject such as a human) suffering from a cancer as defined in any one of embodiments 3.4-3.16, comprising administering to the subject a therapeutically effective amount of a tartrate salt, or composition of matter, as defined in any one of embodiments 1.1-1.19, optionally in combination with another therapeutic agent or treatment (e.g., an anti-cancer agent or therapy).

[0148] 3.21 Use of a tartrate salt or composition of matter according to any one of embodiments 1.1 to 1.19 for the manufacture of a medicament for the use defined in any one of embodiments 3.1 to 3.9.

[0149] 3.22 A method of inhibiting PLK1-PBD, comprising contacting PLK1-PBD with an effective inhibitory amount of a tartrate salt, or composition of matter, according to any one of embodiments 1.1-1.19.

[0150] 3.23 A method of inhibiting PLK4-PBD, comprising contacting PLK4-PBD with an effective inhibitory amount of a tartrate salt, or composition of matter, according to any one of embodiments 1.1-1.19.

[0151] 3.24 A method for inhibiting PLK1-PBD and PLK4-PBD, comprising contacting PLK1-PBD and PLK4-PBD with an effective inhibitory amount of a tartrate salt or composition of matter according to any one of embodiments 1.1-1.19.

[0152] 3.25 The method of any one of embodiments 3.22 to 3.24, wherein an effective inhibitory amount of a tartrate salt, or composition of matter, of any one of embodiments 1.1 to 1.19 is contacted with PLK1-PBD and / or PLK4-PBD in vivo, e.g., in a mammalian subject, such as a human subject.

[0153] Prior to administration of a tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19, the patient may be screened to determine whether the cancer the patient has or may have is a cancer characterized by elevated levels of PLK1 and / or PLK4 kinase and therefore susceptible to treatment with a compound having activity against PLK1 and / or PLK4 kinase.

[0154] For example, biological samples collected from patients can be analyzed to determine whether the cancer the patient is suffering from or may be suffering from is a cancer characterized by genetic abnormalities or abnormal protein expression that result in upregulation of PLK1 and / or PLK4 kinases. The term upregulation includes increased expression or overexpression, including gene amplification (i.e., multiple gene copies), increased expression due to transcriptional effects, and hyperactivity and activation, including activation due to mutations. Thus, patients may be subjected to diagnostic tests to detect markers characteristic of upregulation of PLK1 and / or PLK4 kinases. The term diagnosis includes screening. Markers include genetic markers, such as measuring DNA composition to identify PLK1 mutations. The term marker also includes markers characteristic of upregulation of PLK1 and / or PLK4, including enzyme activity, enzyme level, enzyme state (e.g., phosphorylated or non-phosphorylated), and mRNA levels of the aforementioned proteins.

[0155] Tumors with upregulation of PLK1 and / or PLK4 kinases may be particularly sensitive to PLK1 inhibitors.Tumors can be preferentially screened for upregulation of PLK1 and / or PLK4.Therefore, patients can be subjected to diagnostic tests to detect markers characteristic of upregulation of PLK1 and / or PLK4.Typically, diagnostic tests are performed on biological samples selected from tumor biopsy samples, blood samples (tumor cell separation and enrichment), stool biopsies, karyotype analysis, pleural effusion, and ascites.

[0156] Methods for identifying and analyzing mutations and upregulating proteins are known to those skilled in the art. Screening methods include, but are not limited to, standard methods such as reverse transcriptase polymerase chain reaction (RT-PCR) or in situ hybridization.

[0157] RT-PCR screening evaluates the level of mRNA in tumors by creating a cDNA copy of mRNA and then amplifying the cDNA by PCR. PCR amplification methods, primer selection, and amplification conditions are known to those skilled in the art. Nucleic acid manipulation and PCR are performed by standard methods, for example, as described in Ausubel, FM et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc., or Innis, MA et al., eds. PCR Protocols: a guide to methods and applications, 1990, Academic Press, San Diego. Reactions and manipulations involving nucleic acid technology are described in Sambrook et al., 2001, 3 rdand "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1999. Alternatively, commercially available kits for RT-PCR (e.g., Roche Molecular Biochemicals) or the methodologies described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659; 5,272,057; 5,882,864; and 6,218,529 may be used, which are incorporated herein by reference.

[0158] An example of an in situ hybridization technique for assessing mRNA expression is fluorescence in situ hybridization (FISH) (see Angerer, 1987 Meth. Enzymol., 152:649).

[0159] Generally, in situ hybridization involves the following major steps: (1) fixation of the tissue to be analyzed; (2) prehybridization treatment of the sample to increase the accessibility of the target nucleic acid and reduce nonspecific binding; (3) hybridization of a nucleic acid mixture to nucleic acids within a biological structure or tissue; (4) posthybridization washes to remove unbound nucleic acid fragments; and (5) detection of the hybridized nucleic acid fragments. Probes used in such applications are typically labeled, for example, with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long, e.g., from about 50, 100, or 200 nucleotides to about 1,000 nucleotides or longer, to allow specific hybridization with the target nucleic acid under harsh conditions. Standard methods for performing FISH are described in Ausubel, F M et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc. and Fluorescence In Situ Hybridization: Technical Overview by John M S Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, 2nd ed.; ISBN: 1-59259-760-2; March 2004, pp. 077-088; Series: Methods in Molecular Medicine.

[0160] Alternatively, the protein products expressed from the mRNA can be assayed by immunohistochemistry of tumor samples, solid-phase immunoassays using microtiter plates, Western blotting, two-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry, and other methods known in the art for detecting specific proteins. Detection methods include the use of site-specific antibodies. Those skilled in the art will recognize that all such well-known techniques for detecting upregulation of PLK1 and / or PLK4 kinases are applicable to the present case.

[0161] Alternatively, or in addition, prior to administration of a tartrate salt or composition of matter described in any one of aspects 1.1-1.19, the patient may be screened to determine whether the cancer the patient has or may have is a cancer characterized by mutant KRAS and therefore susceptible to treatment with a compound that has activity against cancer cells harboring mutant KRAS.

[0162] For example, biological samples collected from patients can be analyzed to determine whether the cancer that the patient is suffering from or may be suffering from is characterized by the presence of mutant KRAS.Thus, for example, the patient can be subjected to a diagnostic test to detect mutations in codons 12, 13, 61, or a mixture thereof, of KRAS protein.Commercially available diagnostic tests for mutant KRAS include the cobas® KRAS Mutation Test from Roche Molecular Systems, Inc. and the therascreen KRAS RGQ PCR Kit from Qiagen Manchester, Ltd.

[0163] Tumors with mutant KRAS may be particularly sensitive to PLK1 and / or PLK4 inhibitors. Methods for identifying and analyzing mutations and upregulating proteins are known to those skilled in the art. Screening methods include, but are not limited to, standard methods such as reverse transcriptase polymerase chain reaction (RT-PCR) or in situ hybridization, as described above.

[0164] Thus, in further aspects (Aspects 3.26 to 3.34), the present invention provides:

[0165] 3.26 The tartrate salt or composition of matter of any one of aspects 1.1 to 1.19 for use in treating cancer in a subject (e.g., a human subject) screened and determined to be afflicted with a cancer characterized by elevated levels of PLK1 kinase (e.g., PLK1 overexpression).

[0166] 3.27 The tartrate salt or composition of matter of any one of aspects 1.1-1.19 for use in treating cancer in a subject (e.g., a human subject) screened and determined to have a cancer characterized by elevated levels of PLK4 kinase (e.g., PLK4 overexpression).

[0167] 3.28 The tartrate salt or composition of matter of any one of aspects 1.1 to 1.19 for use in treating cancer in a subject (e.g., a human subject) screened and determined to be afflicted with a cancer characterized by elevated levels of PLK1 kinase and PLK4 kinase (e.g., PLK1 and PLK4 overexpression).

[0168] 3.29 A tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 for use in treating cancer in a subject (e.g., a human subject) who has been screened and determined to have, or be at risk for, a disease or condition susceptible to treatment with a compound having activity against KRAS.

[0169] 3.30 The tartrate salt or composition of matter of any one of aspects 1.1-1.19 for use in treating a subject (e.g., a human subject) who has been screened and determined to be afflicted with a cancer characterized by mutant KRAS and susceptible to treatment with a compound having activity against cancer cells harboring mutant KRAS.

[0170] 3.31 The tartrate salt or composition of matter for use according to any one of aspects 3.26 to 3.30, wherein the cancer is a cancer as defined in any one of aspects 3.4 to 3.16.

[0171] 3.32 Use of a tartrate salt or composition of matter according to any one of embodiments 1.1 to 1.19 for the manufacture of a medicament for the use defined in any one of embodiments 3.26 to 3.31.

[0172] 3.33 A method of diagnosing and treating a disease state or condition characterized by the presence of a mutant form of KRAS (e.g., cancer, e.g., a cancer defined in any one of embodiments 3.4-3.16), comprising: (i) screening a subject (e.g., a human subject) to determine whether the disease or condition from which the subject is suffering or may be suffering is one that is susceptible to treatment with a compound that has activity against KRAS; and (ii) if the subject is shown to have such a susceptible disease or condition, then administering to the subject a therapeutically effective amount of a tartrate salt, or composition of matter, as described in any one of embodiments 1.1-1.19.

[0173] 3.34 A method of treating a disease state or condition characterized by the presence of a mutant form of KRAS (e.g., cancer, e.g., a cancer defined in any one of aspects 3.4-3.16), comprising administering to a subject (e.g., a human subject) screened and determined to have or be at risk for a disease or condition susceptible to treatment with a compound active against KRAS a therapeutically effective amount of a tartrate salt, or composition of matter, according to any one of aspects 1.1-1.19.

[0174] Pharmaceutical preparations The tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 is typically administered to a patient in the form of a pharmaceutical composition. Accordingly, in another aspect (Aspect 4.1) of the invention, the invention provides a pharmaceutical composition comprising the tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 and a pharmaceutically acceptable excipient.

[0175] In a further aspect, there is provided:

[0176] 4.2 A pharmaceutical composition according to aspect 4.1, comprising from about 1% (w / w) to about 95% (w / w) of the tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19, and from 99% (w / w) to 5% (w / w) of a pharmaceutically acceptable excipient or combination of excipients, and optionally one or more further therapeutically active ingredients.

[0177] 4.3 A pharmaceutical composition according to aspect 4.2, comprising from about 5% (w / w) to about 90% (w / w) of the tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19, and from 95% (w / w) to 10% of a pharmaceutical excipient or combination of excipients, and optionally one or more further therapeutically active ingredients.

[0178] 4.4 A pharmaceutical composition according to embodiment 3.3, comprising from about 10% (w / w) to about 90% (w / w) of the tartrate salt or composition of matter according to any one of embodiments 1.1 to 1.19, and from 90% (w / w) to 10% of a pharmaceutical excipient or combination of excipients.

[0179] 4.5 A pharmaceutical composition according to embodiment 4.4, comprising from about 20% (w / w) to about 90% (w / w) of the tartrate salt or composition of matter according to any one of embodiments 1.1 to 1.19, and from 80% (w / w) to 10% of a pharmaceutical excipient or combination of excipients.

[0180] 4.6 A pharmaceutical composition according to aspect 3.5, comprising from about 25% (w / w) to about 80% (w / w) of the tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19, and from 75% (w / w) to 20% of a pharmaceutical excipient or combination of excipients.

[0181] The pharmaceutical compositions of the present invention may be in any form suitable for oral, parenteral, topical, intranasal, intrabronchial, ophthalmic, otic, rectal, intravaginal, or transdermal administration. If the compositions are intended for parenteral administration, they may be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration, or for direct delivery to a target organ or tissue by injection, infusion, or other delivery means.

[0182] Pharmaceutical dosage forms suitable for oral administration include tablets, capsules, caplets, pills, lozenges, syrups, solutions, sprays, powders, granules, elixirs and suspensions, sublingual tablets, sprays, wafers or patches, and buccal patches.

[0183] Thus, in a further aspect, the present invention provides:

[0184] 4.7 A pharmaceutical composition according to any one of aspects 4.1 to 4.6, which is suitable for oral administration.

[0185] 4.8 The pharmaceutical composition of embodiment 4.7, selected from tablets, capsules, caplets, pills, lozenges, syrups, solutions, sprays, powders, granules, elixirs and suspensions, sublingual tablets, sprays, wafers or patches, and buccal patches.

[0186] 4.9 The pharmaceutical composition of embodiment 4.8, wherein the composition is selected from a tablet and a capsule.

[0187] 4.10 A pharmaceutical composition according to any one of aspects 4.1 to 4.6, which is suitable for parenteral administration.

[0188] 4.11 The pharmaceutical composition of embodiment 4.10, formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration, or for direct delivery to a target organ or tissue by injection, infusion, or other delivery means.

[0189] 4.12 The pharmaceutical composition according to embodiment 4.11, which is a solution or suspension for injection or infusion.

[0190] Pharmaceutical compositions containing the tartrate salt or composition of matter according to any one of embodiments 1.1 to 1.19 (e.g., as defined in any one of embodiments 4.1 to 4.12) can be formulated according to known techniques, see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

[0191] Thus, the tablet composition (embodiment 4.9) can contain a unit dose of active compound together with an inert diluent or carrier, such as a sugar or sugar alcohol, e.g., lactose, sucrose, sorbitol, or mannitol; and / or a non-sugar-derived diluent, such as sodium carbonate, calcium phosphate, talc, calcium carbonate, or a cellulose or derivative thereof, such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and a starch, such as cornstarch. The tablet may also contain binders and granulating agents, such as polyvinylpyrrolidone, disintegrants (e.g., swellable cross-linked polymers, such as cross-linked carboxymethylcellulose), lubricants (e.g., stearates), preservatives (e.g., parabens), antioxidants (e.g., BHT), buffers (e.g., phosphate or citrate buffers), and effervescent agents, such as citrate / bicarbonate mixtures. Such excipients are well known and need not be discussed in detail here.

[0192] Capsule formulations (embodiment 4.9) may be of the hard or soft gelatin variety and may contain the active ingredient in solid, semi-solid, or liquid form. Gelatin capsules may be formed from animal gelatin or its synthetic or vegetable-derived equivalents.

[0193] Solid dosage forms (e.g., tablets, capsules, etc.) can be coated or uncoated, but typically have a coating, such as a protective film coating (e.g., wax or varnish) or a release-controlling coating. Coatings (e.g., Eudragit™-type polymers) can be designed to release the active ingredient at a desired location in the gastrointestinal tract. Thus, coatings can be selected to degrade under specific pH conditions in the gastrointestinal tract, thereby selectively releasing a composition or atropisomer of the substance in the stomach or ileum or duodenum.

[0194] Alternatively, or in addition to a coating, the drug may be presented in a solid matrix containing a release-controlling agent, e.g., a release-retarding agent that may be adapted to selectively release the composition or atropisomer of the substance under conditions of changing acidity or alkalinity in the gastrointestinal tract. Alternatively, the matrix material or release-retarding coating may take the form of an erodible polymer (e.g., a maleic anhydride polymer) that is substantially continuously eroded as the dosage form passes through the gastrointestinal tract.

[0195] Compositions for topical use include ointments, creams, sprays, patches, gels, drops, and inserts (e.g., intraocular inserts). Such compositions can be formulated according to known methods.

[0196] Compositions for parenteral administration (embodiments 4.10-4.12) are typically provided as sterile aqueous or oily solutions or fine suspensions, or may be provided in finely divided sterile powder form for extemporaneous reconstitution with sterile water for injection.

[0197] Examples of formulations for rectal or vaginal administration include pessaries and suppositories which may, for example, be formed from a shaped moldable or waxy material containing the active compound.

[0198] Compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administered in standard form using powder inhalers or aerosol dispensing devices. Such devices are well known. For administration by inhalation, powdered formulations typically contain the active compound together with an inert solid powdered diluent such as lactose.

[0199] Pharmaceutical compositions are generally presented in unit dosage form and, as such, typically contain sufficient compound to provide a desired level of biological activity. (E.g., as described in any one of Embodiments 4.1-4.9) Compositions intended for oral administration may contain from 2 milligrams to 200 milligrams, more usually from 10 milligrams to 100 milligrams, e.g., 12.5 milligrams, 25 milligrams, and 50 milligrams of active ingredient.

[0200] Pothology The active compound (a tartrate salt, or composition of matter, as described in any one of embodiments 1.1-1.19) is administered to a patient in need thereof (e.g., a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect, such as an effect as described in embodiments 3.1-3.34 above.

[0201] The tartrate salt or composition of matter according to any one of embodiments 1.1 to 1.19 is generally administered to a subject in need of such administration, such as a human or animal patient, preferably a human.

[0202] The tartrate salts or compositions of matter described in any one of embodiments 1.1-1.19 are typically administered in amounts that are therapeutically or prophylactically useful and generally non-toxic. However, in certain situations, the benefits of administering the compounds of the invention may outweigh the drawbacks of toxic or side effects, in which case it may be desirable to administer the compounds in amounts associated with some toxicity.

[0203] In one embodiment, a typical daily dose of a tartrate salt, or composition of matter, according to any one of embodiments 1.1-1.19 can range from 0.025 milligrams to 5 milligrams per kilogram of body weight, e.g., up to 3 milligrams per kilogram of body weight, more typically from 0.15 milligrams to 5 milligrams per kilogram of body weight, although higher or lower doses may be administered as needed.

[0204] In another embodiment, a typical daily dose of a tartrate salt, or composition of matter, according to any one of embodiments 1.1-1.19 can range from 0.025 milligrams to 50 milligrams per kilogram of body weight, e.g., up to 30 milligrams per kilogram of body weight, more typically from 0.15 milligrams to 50 milligrams per kilogram of body weight (e.g., 0.5 milligrams to 30 milligrams per kilogram), although higher or lower doses may be administered as needed.

[0205] By way of example, in one embodiment of the invention, an initial starting dose of 12.5 mg may be administered two to three times daily. The dose may be increased by 12.5 mg daily every three to five days until the maximum tolerated and effective dose for the individual is reached, as determined by a physician.

[0206] In another embodiment of the invention, a weekly dosing schedule can consist of an initial starting dose of 0.5 to 1.5 mg / kg (e.g., 1 mg / kg) in week 1, followed by escalation of the dose (e.g., two or three times the previous dose for three, four, or five dose escalations) in weeks 2 and beyond, depending on therapeutic effect and tolerability, up to the maximum dose. For example, a starting dose of 1 mg / kg can be administered in week 1, followed by escalation to 3 mg / kg in week 2, 9 mg / kg in week 3, and 27 mg / kg in week 4.

[0207] Ultimately, the amount of compound administered will be commensurate with the nature of the disease or physiological condition being treated, and the therapeutic benefits and presence or absence of side effects caused by a given administration regimen, and will be at the discretion of the physician.

[0208] Combination therapy The tartrate salts or compositions of matter according to any one of aspects 1.1-1.19 are believed to be useful either as the sole chemotherapeutic agent or, more generally, as combination therapy with chemotherapeutic agents or radiation therapy in the prevention or treatment of various proliferative diseases or conditions, examples of which are set forth above.

[0209] Specific examples of chemotherapeutic agents or other treatments that may be co-administered with a tartrate salt or composition of matter according to any one of embodiments 1.1-1.19 include: Topoisomerase I inhibitors (e.g., irinotecan) Antimetabolites: (e.g., cytarabine or gemcitabine) Tubulin-targeting agents (e.g., paclitaxel) DNA binders and topoisomerase II inhibitors EGFR inhibitors (e.g., gefitinib or afatinib) mTOR inhibitors (e.g., everolimus) PI3K pathway inhibitors (e.g., PI3K, PDK1) Akt inhibitors Alkylating agents (e.g., temozolomide) Monoclonal antibodies. Antihormones Signal transduction inhibitors Proteasome inhibitors DNA methyltransferase inhibitors Cytokines and retinoids Hypoxia-induced DNA damaging agents (e.g., tirapazamine) Aromatase inhibitors Anti-Her2 antibodies (see, for example, http: / / www.wipo.int / pctdb / en / wo.jsp?wo=2007056118) Anti-CD20 antibodies (e.g., rituximab) Angiogenesis inhibitors HDAC inhibitors MEK inhibitors B-Raf inhibitors ERK inhibitors HER2 small molecule inhibitors (e.g., lapatinib or afatinib) Bcr-Abl tyrosine kinase inhibitors (e.g., imatinib) CDK4 / 6 inhibitors (e.g., palbociclib) Mps1 / TTK inhibitors Aurora B inhibitor FLT3 kinase inhibitors IDH1 or IDH2 inhibitors BRD4 inhibitors Temozolomide Immune checkpoint inhibitors that block signaling components, including PD1, PDL-1, and CTLA4; and KRAS blockers, including those directed against specific mutations such as G12C (e.g., sotorasib); Bcl2 inhibitors (e.g., venetoclax, sabutoclax, or obatoclax); and Radiation therapy.

[0210] Preferred examples of chemotherapeutic agents or other treatments that may be co-administered with the tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 may be selected from: Topoisomerase I inhibitors (e.g., irinotecan) Antimetabolites: (e.g., cytarabine or gemcitabine) Tubulin-targeting agents (e.g., paclitaxel) EGFR inhibitors (e.g., gefitinib or afatinib) mTOR inhibitors (e.g., everolimus) Alkylating agents (e.g., temozolomide) Anti-CD20 antibodies (e.g., rituximab) Immune checkpoint inhibitors that block signaling components, including PD1, PDL-1, and CTLA4; and KRAS blockers, including those directed against specific mutations such as G12C (e.g., sotorasib); Bcl2 inhibitors (e.g., venetoclax, sabutoclax, or obatoclax); and Radiation therapy.

[0211] Thus, in a further aspect, the present invention provides:

[0212] 5.1 A pharmaceutical combination comprising a tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 and another therapeutically active agent.

[0213] 5.2 The pharmaceutical combination of embodiment 5.1, wherein said additional therapeutic agent is selected from the specific and preferred chemotherapeutic agents listed above.

[0214] 5.3 The pharmaceutical combination of embodiment 5.1, wherein the additional therapeutic agent is an anti-cancer agent.

[0215] 5.4 A pharmaceutical combination according to any one of aspects 5.1 to 5.3, wherein the tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19 and said further therapeutically active agent are provided in a single pharmaceutical composition or patient pack.

[0216] 5.5 A pharmaceutical composition comprising a tartrate salt or composition of matter according to any one of aspects 1.1 to 1.19, another therapeutically active agent, and at least one pharmaceutically acceptable excipient.

[0217] 5.6 A method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of a pharmaceutical combination according to any one of aspects 5.1 to 5.5. [Brief explanation of the drawings]

[0218] [Figure 1] FIG. 1 is a schematic diagram showing the R / S classification system of atropisomers. [Figure 2] 1 is a depiction of the three-dimensional structure of atropisomer A-2 of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)-ethyl]benzamide as determined by single crystal X-ray crystallographic studies. [Figure 3] Schematic stereochemistry of the two atropisomers A-1 (S) and A-2 (R) and the basis for assigning their stereochemistry using Cahn-Ingold-Prelog (CIP) sequencing rules. [Figure 4] 1 is a powder X-ray diffraction spectrum of atropisomer A-2 free base. [Figure 5] 1 is a powder X-ray diffraction spectrum of atropisomer A-2 tartaric acid Pattern A salt (bottom trace) and Pattern B salt (top and middle traces). [Figure 6] 6 shows the thermal profile of atropisomer A-2 free base, showing the differential scanning calorimetry plot (line 6A) and the thermogravimetry plot (line 6B). [Figure 7]7 shows the thermal profile of atropisomer A-2 tartaric acid Pattern A salt, showing the differential scanning calorimetry plot (line 7A) and the thermogravimetry plot (line 7B). [Figure 8] 8A shows the thermal profile of atropisomer A-2 tartrate Pattern B salt, showing the differential scanning calorimetry plot (line 8A) and the thermogravimetry plot (line 8B). [Figure 9] 1 is a plot of weight change versus relative humidity in a gravimetric vapor sorption study conducted on atropisomer A-2 tartrate Pattern B salt. [Figure 10] 1 is a bar graph showing the percentage of different mitotic phenotypes (unassembled chromosomes, multipolar spindles / abnormal cytokinesis, monopolar spindles, normal prometaphases, and subsequently generated normal metaphases) observed after treatment of U87MG cells with either atropisomer A-1 or atropisomer A-2 at a concentration of 0.03 μM. [Figure 11] 1 is a bar graph showing the number of centrioles present in HeLa cells after treatment with either atropisomer A-1 or atropisomer A-2 at a concentration of 0.02 μM. [Figure 12] 1 is a plot of plasma concentration versus time following oral and iv administration of atropisomer A-2 to mice. The lower line extending to 24 hours is for the 2 mg / kg iv dose. The other line is for the 10 mg / kg po dose. [Figure 13] 1 is a plot of plasma and brain concentrations of atropisomer A-2 versus time following oral administration (10 mg / kg) to mice. The upper line represents the brain concentration, and the lower line represents the plasma concentration. [Figure 14] 1 is a plot of tumor volume versus time in male athymic nude mice in the U87MG subcutaneous xenograft model following administration of Atropisomer A-2. [Figure 15] 1 is a graphical comparison of bioluminescent signals associated with tumor growth in male athymic nude mice in the U87-Luc orthotopic xenograft model following administration of Atropisomer A-2. [Figure 16]1 is a plot of tumor volume versus time in male athymic nude mice in the HCT116 subcutaneous xenograft model following administration of Atropisomer A-2.

[0219] example The present invention will now be illustrated, but not limited, by reference to specific embodiments described in the following examples.

[0220] Proton magnetic resonance (H NMR) spectra were recorded on a Bruker 400 instrument operating at 400 MHz in dimethyl sulfoxide-d6 (DMSO-d6) or methanol-d4 (MeOH-d4) (as indicated) at 27 °C unless otherwise noted, and are reported as follows: chemical shift δ / ppm (multiplicity s = singlet, d = doublet, dd = double doublet, dt = double triplet, t = triplet, q = quartet, m = multiplet, br = broad, number of protons). Residual protic solvent was used as an internal reference.

[0221] Liquid chromatography and mass spectrometry were performed using the systems and operating conditions shown below. Where atoms with different isotopes are present and a single mass is quoted, the mass quoted for the compound is the monoisotopic mass (i.e., 35 Cl; 79 Br, etc.).

[0222] LCMS conditions The LCMS data shown in the examples below was obtained using one of the methods described below.

[0223] LCMS method 1 LCMS was performed on a UPLC AQUITY equipped with a PDA photodiode array detector and a QDa mass detector. The column used was a C18, 2.1 x 50 mm, 1.9 µm column. The column flow rate was 1.2 mL / min, and the mobile phases used were (A) 0.1% formic acid in MilliQ water (pH = 2.70) and (B) 0.1% formic acid in water:acetonitrile (10:90). The injection volume was 4-7 µL. Samples were prepared in methanol:acetonitrile to achieve a concentration of approximately 250 ppm.

[0224] The following gradient was used for elution: [Table 1]

[0225] Mass parameters Probe: ESI capillary Source temperature: 120℃ Probe temperature: 600℃ Capillary voltage: 0.8KV (+Ve and -Ve) Cone voltage: 10&30V Ionization mode: positive and negative

[0226] HPLC analysis The HPLC data reported were obtained using the following methods.

[0227] HPLC method 1 HPLC analysis was performed on an Agilent Technologies 1100 / 1200 series HPLC system. The column used was an ACE 3 C18, 150 x 4.6 mm, 3.0 μm particle size (e.g., Hichrom, product number: ACE-111-1546). The flow rate was 1.0 mL / min. Mobile phase A was water:trifluoroacetic acid (100:0.1%), and mobile phase B was acetonitrile:trifluoroacetic acid (100:0.1%). The injection volume was 5 μL, and the following gradient was used: [Table 2]

[0228] Chiral HPLC analysis The chiral HPLC data reported was obtained using one of the methods described below.

[0229] Chiral HPLC Method 1 Chiral HPLC was analytical and performed on an Agilent Technologies 1200 Series HPLC system. The column used was a CHIRAL PAK IG, 250 x 4.6 mm, 5 μm. The column flow rate was 1.0 mL / min, and the mobile phases were: (A) 0.1% v / v DEA in n-heptane and (B) IPA:MeOH (70:30). The injection volume was 25 μL. Samples were prepared in IPA:MeOH to achieve a concentration of approximately 250 ppm, and the following isocratic method was used: [Table 3]

[0230] Chiral HPLC Method 3 Chiral HPLC was performed on an Agilent Technologies 1200 Series HPLC system. The column used was a CHIRAL PAK IG, 250 x 4.6 mm, 5 μm. The column flow rate was 1.0 mL / min, and the mobile phases were: (A) 0.1% v / v DEA in n-heptane and (B) IPA:MEOH (70:30). The injection volume was 10 μL. Samples were prepared in IPA:MeCN to achieve a concentration of approximately 250 ppm, and the following isocratic method was used: [Table 4]

[0231] Chiral HPLC Method 4 Identical conditions as in Chiral Method 3 except using the following isocratic method: [Table 5]

[0232] Chiral HPLC Method 5 Identical conditions as in Chiral Method 3 except using the following isocratic method: [Table 6]

[0233] Chiral HPLC Method 6 Chiral HPLC was analytical and performed on an Agilent Technologies 1100 / 1200 series HPLC system. The column used was a CHIRALPAK AD-H; 250 × 4.6 mm, 5.0 μm. The column flow rate was 1.0 mL / min, and the mobile phase was hexane:EtOH:TFA (90:10:0.1%). The injection volume was 5 μL. Samples were prepared in 100% EtOH to achieve a concentration of approximately 0.5 mg / mL.

[0234] Chiral HPLC Method 7 Chiral HPLC was analytical and performed on an Agilent Technologies 1100 / 1200 series HPLC system. The column used was a CHIRALPAK IA, 250 x 4.6 mm, 5.0 μm. The column flow rate was 1.0 mL / min, and the mobile phase was hexane:EtOH:ethanolamine (90:10:0.1%). The injection volume was 5 μL. Samples were prepared in 100% EtOH to achieve a concentration of approximately 0.5 mg / mL.

[0235] Preparative Chiral HPLC Method: Atropisomers were isolated using one of the following preparative chiral HPLC methods.

[0236] Preparative Chiral HPLC Method 1 Preparative chiral HPLC was performed using a CHIRALPAK IG SFC, 21 x 250 mm, 5 μm column, eluting with (A) 0.1% DEA in heptane and (B) IPA as the mobile phase, with a flow rate of 30 mL / min and the following isocratic system: [Table 7]

[0237] Preparative Chiral HPLC Method 2 Preparative chiral HPLC was performed using a CHIRALPAK IG SFC column, 21 x 250 mm, 5 μm, eluting with (A) 0.1% DEA in heptane and (B) IPA:MeOH (90:10) as the mobile phase at a flow rate of 22 mL / min using the following isocratic system: [Table 8]

[0238] Example 1 (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]benzamide [ka]

[0239] The title compound was prepared by following steps 1, 2, 3, 4b, and 5b of the synthetic route shown in Scheme 1 above.

[0240] Step 1: 4-[4-(4-chlorophenyl)-4-oxo-butanoyl]benzonitrile (6) [ka]

[0241] Zinc chloride (30.5 g, 223 mmol) was heated under vacuum until melted and then cooled to room temperature. Toluene (100 mL), tert-butanol (16.5 mL, 172 mmol), and triethylamine (24 mL, 172 mmol) were added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours, at which point the zinc chloride had completely dissolved. 4-Cyanoacetophenone (25 g, 172 mmol) and 4-chlorophenacyl bromide (40.2 g, 172 mmol) were added, and the reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was then diluted with ethyl acetate (300 mL) and washed with water (5 × 100 mL). The combined organic extracts were dried (NaSO) and evaporated under reduced pressure. The resulting residue was purified by trituration with methyl tert-butyl ether (400 mL) to give the title compound (30 g, 101 mmol, 59%).

[0242] Step 2: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzonitrile (7) [ka]

[0243] A stirred solution of 4-(4-(4-chlorophenyl)-4-oxobutanoyl)benzonitrile (30 g, 101 mmol), 2-trifluoromethylaniline (48.79 g, 303 mmol), and p-toluenesulfonic acid (1.92 g, 10.099 mmol) in dioxane (300 mL) was heated at 150° C. for 16 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by column chromatography on silica gel (60-120 mesh) using 8% ethyl acetate / hexane as eluent to give the title compound (30 g, 71 mmol, 70%).

[0244] Step 3: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (8) [ka]

[0245] To a solution of 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzonitrile (2 g, 4.739 mmol) in methanol (20 mL) was added sodium hydroxide (1.89 g, 47 mmol) in water (10 mL), and the resulting mixture was stirred at 90° C. for 24 hours. The mixture was concentrated under reduced pressure, and the resulting residue was purified by trituration with diethyl ether (10 mL) to give the title compound (1.8 g, 4.1 mmol, 86%).

[0246] Step 4b: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)-N-(2-(dimethylamino)ethyl)benzamide (9) [ka]

[0247] To a stirred solution of 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (1.8 g, 4.0 mmol) in dimethylformamide (12 mL) was added N,N-diisopropylethylamine (2.13 mL, 22 mmol), followed by (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate) (HATU) (4.65 g, 12 mmol). The reaction mixture was stirred at room temperature for 30 minutes, followed by the dropwise addition of N,N'-dimethylethylenediamine (1.08 g, 12 mmol), and stirring was continued at room temperature for 4 hours. The mixture was then poured into ice-cold water (150 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried (NaSO) and concentrated under reduced pressure. The resulting residue was purified by column chromatography on neutral alumina eluting with 6% methanol / dichloromethane to give the title compound (1.2 g, 2.3 mmol, 57%) as a mixture of atropisomers.

[0248] Step 5b: Separation of atropisomers The atropisomers of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]benzamide were resolved by chiral HPLC using preparative chiral HPLC method 1.

[0249] Two peaks were isolated: Peak 1: Example A-1, 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide - atropisomer 1 (0.3 g, 0.58 mmol, 38%, >99% ee), and: Peak 2: Example A-2, 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide - atropisomer 2 (0.31 g, 0.606 mmol, 39%, 98% ee).

[0250] The compounds may also be isolated as their hydrochloride salts.

[0251] Example 2 Further purification and characterization of atropisomers Atropisomer A-1: ​​(S)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide hydrochloride Peak 1 from Example 1, Step 5b (0.31 g, 0.606 mmol) was further purified by stirring in HPLC-grade water (30 mL), followed by sonication for 10 minutes, and extraction with ethyl acetate (3 × 30 mL). The combined organic layers were dried (NaSO), filtered, concentrated under reduced pressure, and then lyophilized to give an amorphous solid (0.290 g, 0.567 mmol, 94%), which was dissolved in dichloromethane (7.12 mL). The resulting solution was cooled to 0 °C, and 4 N HCl in dioxane (1.42 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. The mixture was concentrated and dried under high vacuum. Purification by trituration with diethyl ether (10 mL) and lyophilization afforded the title compound (0.3 g, 0.56 mmol, 98%) as an off-white solid.

[0252] 1 H NMR(DMSO-d6)δ 10.03,(brs,1H),8.62(s,1H),7.81-7.68(m,6H),7.25(d,J=8.4 Hz,2H),7.10-7.03(m,4H),6.67-6.58(m,2H),3.56-3.54(m,2H),3.20-3.18(m,2H),2.76(s,6H).LCMS(Method 1)-RT2.54,MH+512.4

[0253] Atropisomer A-2: (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide hydrochloride The hydrochloride salt of atropisomer A-2 was prepared using the same method as used for atropisomer A-1 starting from peak 2 to give the title compound (0.31 g, 0.56 mmol, 99%) as an off-white solid.

[0254] 1 H NMR(DMSO-d6)δ 9.91(brs,1H),8.69(s,1H),7.81-7.68(m,6H),7.25(d,J=8.0 Hz,2H),7.10-7.03(m,4H),6.67-6.58(m,2H),3.56-3.54(m,2H),3.20-3.18(m,2H),2.77(s,6H).LCMS(Method 1)-RT2.56,MH+512.4

[0255] Single crystal X-ray crystallography of atropisomer A-2 (see Example 3 below) showed that atropisomer A-2 is the R isomer (compound (1)), and therefore atropisomer A-1 must be the S isomer.

[0256] Chiral Analysis Analysis of the chiral properties of atropisomers A-1 and A-2 was carried out by measuring their optical rotations and their retention times obtained by chiral HPLC.

[0257] Specific Rotation Protocol: Equipment: Optically active AA-10 automatic polarimeter Wavelength: 589nm Temperature: 23℃ Cell path length: 1 dm Solvent: Chloroform (Fisher, HPLC grade) Concentration: 1.0g / 100mL

[0258] Sampling Technique: The instrument was switched on and allowed to stabilize for 30 minutes before calibration, which was then verified using an optically active quartz control plate (S / N 00049). The angular rotation at 23°C was measured at 34.16° (after first zeroing the instrument without the sample tube) using the yellow sodium D line. The quality of the sample tube was verified by zeroing the instrument, then filling the sample tube with chloroform and verifying that the instrument still read 0.00 (+ / - 0.02). The instrument was zeroed with a chloroform blank. The sample was dissolved in CHCl3 (2 mg in 2 mL), filtered, and 2 mL was pipetted into the cell to measure α.

[0259] The specific rotation was calculated from the following formula: [α]Tλ=(α×100) / (cl) [Table 9]

[0260] Classification of atropisomers Stability studies were performed on the isolated atropisomers A-1 and A-2.

[0261] To assess the interconversion of atropisomer A-1 and atropisomer A-2, chiral stability was monitored at 40° C. and 80° C. As shown by the results shown below, no interconversion was observed after 10 days of heating at either temperature. [Table 10]

[0262] protocol: 1.2×1 mg of the pure atropisomer was dissolved in 1 mL of ethanol in a sealed dram vial. 2. One set of vials was heated at 40°C and another set was heated at 80°C. 3. At the designated time points, 20 μL aliquots were taken from each stock solution (1 mL) and quenched into HPLC vials containing 80 μL of a solution of hexane:ethanol; 80:20 to obtain a final concentration of 200 ppm, and the samples were analyzed by chiral HPLC. 4. Analyses were performed at the following time points: 0 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 240 hours for samples held at 40°C, and 24 hours, 96 hours, and 240 hours for samples held at 80°C using chiral HPLC method 5.

[0263] The stability of the isolated atropisomers, A-1 and A-2, confirmed that they were class 3 atropisomers (LaPlante et al., J. Med. Chem., 54:7005-7022 (2011)).

[0264] Example 3 X-ray crystallographic analysis of atropisomer A-2 Atropisomer A-2 free base was prepared and single crystals were subjected to X-ray crystallographic studies as follows.

[0265] experiment: A single crystal of undefined morphology of atropisomer A-2 was obtained by recrystallization from methyl isobutyl ketone (MIBK). The preferred crystal was 0.19 x 0.13 x 0.04 mm 3 The crystal was selected and mounted on a Rigaku XtaLAB Syngery-S diffractometer equipped with a HyPix-6000HE detector using a MiTiGen MicroMount. The crystal was kept at a constant T = 123(2) K during data collection.

[0266] The data were generated using CuKα radiation. The maximum resolution achieved was Θ = 74.263° (0.80 Å). Data reduction, scaling, and absorption correction were performed. The final completeness was 100.00% for Θ = 74.263°. The absorption coefficient μ of the compound was 1.761 mm at wavelength (λ = 1.542 Å).-1 It was decided that:

[0267] Data were collected and processed using CrysAlisPro software, and structures were solved using the ShelXT (Sheldrick, 2015) structure solving program using the Intrinsic Phasing solution method and Olex2 (Dolomanov et al., 2009) as a graphical interface. Models were refined with ShelXL-2018 / 3 (Sheldrick, 2018), version 2018 / 3, using least-squares minimization.

[0268] The crystal structure was found to be monoclinic and was assigned to the space group P21(#4).

[0269] All non-hydrogen atoms were refined anisotropically. Hydrogen atom positions were calculated geometrically and refined using a riding model.

[0270] References: OVDolomanov and LJBourhis and RJGildea and JAKHoward and H.Puschmann, Olex2: A complete structural solution, refinement and analysis program, J.Appl.Cryst., (2009), 42, 339-341. Sheldrick,GM,Crystal structure refinement with ShelXL,Acta Cryst.,(2015),C71,3-8. Sheldrick, GM, ShelXT-Integrated space-group and crystal-structure determination, Acta Cryst., (2015), A71, 3-8.

[0271] The results of the study are shown in Tables 1-7 below. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15-1] [Table 15-2] [Table 16-1] [Table 16-2] [Table 17]

[0272] Based on the data presented below, atropisomer A-2 is believed to have the R configuration shown in Figures 2 and 3 and can therefore be designated (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)-ethyl]benzamide.

[0273] Example 4 Preparation and characterization of (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide tartrate Method 1: Small-scale preparation of tartrate salt Atropisomer A-2 free base (904.2 mg) was suspended in acetone (9.042 mL, 10 volumes) and stirred for 40 minutes at 25° C. When the solution contained no visible particulates, it was divided into 12 equal aliquots (603 μL), yielding an active content of approximately 60.3 mg per sample.

[0274] An aliquot of 247 μL of a 0.5 M tartaric acid solution in ethanol (1.05 equivalents) was added to the aliquot of the free base solution at 25° C. After stirring the mixture at 25° C. for 18 hours, a white suspension formed, and the resulting solid was then isolated by filtration (PTFE 10 micron frit cartridge) and dried in vacuo at 40° C. for approximately 72 hours. The resulting salt was labeled as tartrate salt Pattern A (solvate).

[0275] Method 2: Preparation of the tartrate salt of atropisomer A-2 using an isopropyl acetate solution Atropisomer A-2 (749.8 mg) was suspended in isopropyl acetate (15 mL, 20 volumes), and the suspension was heated to 40°C with stirring. When the solution contained no visible particulates, it was divided into 12 equal aliquots (1 mL), yielding an active content of approximately 50 mg per sample. An aliquot of 195.3 μL of a 1 M atropisomer A-2 solution in ethanol was added to an aliquot of the free base solution at 40°C. The resulting mixture was cooled to 25°C at a cooling rate of approximately 10°C / hr. A white suspension formed, and the resulting solid was then isolated by filtration and dried in vacuo at 40°C for approximately 18 hours. The resulting salt was labeled as tartrate salt pattern B.

[0276] Method 3: Preparation of the tartrate salt using a solution of atropisomer A-2 in isopropyl alcohol The tartaric acid Pattern A salt was prepared according to Method 2, except that atropisomer A-2 (750.1 mg) was first suspended in isopropyl alcohol (15 ml, 20 vol).

[0277] Method 4: Preparation of the tartrate salt using a solution of atropisomer A-2 in 2-methyl-tetrahydrofuran Method 1 was repeated, except that atropisomer A-2 (913.9 mg) was first suspended in 2-methyl-tetrahydrofuran (15 ml, 20 vol), (9.139 mL, 10 vol) and stirred at 25° C. for approximately 40 minutes, and then a 250 μl (1.05 equiv) aliquot of 1 M tartaric acid in ethanol was added to an aliquot (609 μL) of the A-2 free base solution to give the tartaric acid Pattern A salt.

[0278] Method 5: Preparation of atropisomer A-2 tartrate pattern B salt on a 500 mg scale Atropisomer A-2 free base (521.5 mg) was weighed into a glass vial and charged with isopropyl acetate (20 volumes, 10.430 ml). The mixture was heated to 40°C and stirred for 15 minutes to obtain a clear solution. Tartaric acid (1.05 equivalents, 162.5 mg) dissolved in 3 mL of tetrahydrofuran was then charged to the solution. The resulting mixture was seeded with atropisomer A-2 tartaric acid pattern B, which immediately precipitated the salt at 40°C, forming a mobile suspension. The mixture was cooled to 25°C and stirred for 20 hours. The resulting solid was isolated by filtration and dried in vacuo at 40°C to obtain atropisomer A-2 tartaric acid pattern B salt in 84% yield.

[0279] Method 6: Scale-up preparation of atropisomer A-2 tartrate pattern B salt (anhydrous form) Atropisomer A-2 free base (10.0497 g) was weighed into a Buchi flask and charged with isopropyl acetate (20 volumes, 200 ml). The mixture was heated to 40 °C to obtain a clear, particulate-free solution and stirred for 30 minutes. Tartaric acid (3.1954 g, 1.08 equiv.) dissolved in tetrahydrofuran (50 mL) was added to the solution, and the acid was added portionwise as follows: 15 mL at 40 °C; seed with atropisomer A-2 tartaric acid Pattern B salt and stir for 30 minutes; add 10 mL and stir for 1 hour; add 10 mL and stir for 30 minutes; add 15 mL and stir for 30 minutes. The white suspension was then cooled to room temperature at a cooling rate of 10 °C / hour and stirred for 18 hours. The resulting solid was isolated by filtration in vacuo, washed with isopropyl acetate (2 × 2 volumes), and dried in vacuo at 40 °C for 20 hours to give A-2 tartaric acid Pattern B salt (anhydrous) in 97% yield; HPLC purity 99.74% (HPLC Method 1), chiral purity 99.27% ​​(Chiral HPLC Method 7).

[0280] Method 7: Alternative scale-up preparation of atropisomer A-2 tartrate pattern B salt (anhydrous form) by cooling crystallization from butanol / water 96:4 Atropisomer A-2 free base (36.79 g) was weighed into a flask and charged with butanol (282.57 ml, 7.68 volumes). The mixture was heated to 80°C (a pale yellow, cloudy solution), stirred for 30 minutes, and then clarified into a Mya* vessel preheated at 80°C. L-(+)-tartaric acid (1.023 equiv., 11.0806 g) was then charged to the solution as an aqueous solution (11.77 mL, 0.32 volumes of initial API charge). The dropwise addition was carried out at 80°C while the acid solution clarified. The mixture was then cooled to 68°C over 30 minutes, seeded with 0.1% ground atropisomer A-2 tartaric acid Pattern B salt seed crystals (32.6 mg), and held for 1 hour. The mixture was then cooled to 5°C at a cooling rate of 5°C / hour and stirred at 5°C for 6 hours before isolating the solid. The solid was filtered under vacuum, washed twice with butanol, dried on the filter for 15 min, and then dried at 40 °C for 20 h to give atropisomer A-2 tartrate Pattern B salt (anhydrous) in 83% yield; HPLC purity 99.84% (HPLC Method 1), chiral purity 99.66% (Chiral HPLC Method 7).

[0281] *Note: For the aforementioned equilibration or crystallization procedures requiring temperature control and / or defined heating / cooling profiles, a Radley Mya4 Reaction Station was used. The Radley Mya4 Reaction Station is a four-zone reaction station with magnetic and overhead stirring capabilities and a temperature range of -30 to 180 °C for 2- to 400-mL-scale mixtures. The required reaction conditions were programmed via the Mya 4 Control Pad.

[0282] Characterization of the atropisomer A-2 tartrate salt The identity of the salts as 1:1 (molar ratio of free base:tartaric acid) stoichiometric salts was confirmed by their analysis, which was collected using a JEOL ECX 400 MHz spectrometer equipped with an autosampler. 1 The H NMR spectrum confirmed the identity of the compound. Samples were dissolved in suitable deuterated solvents for analysis. Data were acquired using Delta NMR Processing and Control Software version 4.3.

[0283] The tartrate salt was characterized using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), gravimetric solubility testing, and gravimetric vapor sorption testing using the techniques described below.

[0284] X-ray powder diffraction (XRPD) Powder X-ray diffraction patterns were collected on a PANalytical diffractometer using CuKα radiation (45 kV, 40 mA), a θ-θ goniometer, a focusing mirror, a divergence slit (½"), Soller slits (4 mm) in both the incident and diverging beams, and a PIXcel detector. The software used for data collection was X'Pert Data Collector, version 2.2f, and data were presented using X'Pert Data Viewer, version 1.2d. XRPD patterns were acquired under ambient conditions through a transparent foil sample stage (polyimide-Kapton, 12.7 μm thick film) using PANalytical X'Pert PRO. The data collection range was 2.994–35° 2θ, and the continuous scan rate was 0.202004° s.

[0285] Differential scanning calorimetry (DSC) DSC data were collected on a PerkinElmer Pyris 6000 DSC equipped with a 45-position sample holder. The instrument was verified for energy and temperature calibration using certified indium. A predetermined amount of sample, 0.5–3.0 mg, was placed in a pinhole-equipped aluminum pan and heated from 30–350°C at 20°C / min or as varied by experiment. 20 ml / min -1 A purge of dry nitrogen at 1000 K was maintained over the sample. Instrument control, data acquisition, and analysis were performed using Pyris Software v11.1.1 revision H.

[0286] Thermogravimetric analysis (TGA) TGA data were collected on a PerkinElmer Pyris 1 TGA equipped with a 20-position autosampler. The instrument was calibrated for temperature using certified weights and certified Alumel and Perkalloy samples. A predetermined amount of sample, 1-5 mg, was placed in a tared aluminum crucible and heated from ambient to 400 °C at 20 °C / min. -1 It was heated at 20 ml / min. -1 A nitrogen purge at 0 °C was maintained over the sample. Instrument control, data acquisition, and analysis were performed using Pyris Software v11.1.1 revision H.

[0287] weight solubility The water solubility of the salts was measured using a gravimetric solubility protocol.

[0288] 1 ml of water was charged into a crystallizing tube. The solid was weighed into a tared glass vial and added portionwise to the solution, weighing the vial after each addition until a cloudy solution was observed. The amount in mg was then calculated to give the solubility in mg / ml.

[0289] The results from the characterization studies are shown in Table 8 below. [Table 18]

[0290] Gravimetric Vapor Sorption (GVS) A GVS study was performed on atropisomer A-2 tartrate Pattern B salt using the protocol shown below.

[0291] Sorption isotherms were obtained using a Hiden Isochema moisture sorption analyzer (model IGAsorp) controlled by IGAsorp Systems Software V6.50.48. Samples were maintained at a constant temperature (25°C) by instrument control. Humidity was controlled by mixing dry and humid nitrogen streams, with a total flow rate of 250 ml / min. -1The instrument was validated for relative humidity content by measuring three calibrated Rotronic salt solutions (10-50-88%). Sample weight change was monitored as a function of humidity with a microbalance (accuracy + / - 0.005 mg). A defined amount of sample was placed in a tared mesh stainless steel basket under ambient conditions. A complete experimental cycle typically consisted of three scans (sorption, desorption, and sorption) at constant temperature (25°C) and 10% RH intervals over the range of 0-90% (60 minutes for each humidity level). This type of experiment should demonstrate the ability of the study sample to absorb (or not absorb) moisture over a well-defined set of humidity ranges.

[0292] GVS analysis (see Figure 9) showed a moisture content of approximately 0.3% before the first desorption. Between 80 and 90% RH, there was a slightly higher increase in moisture, with the solid gaining approximately 0.8% moisture. A second absorption / desorption cycle showed how moisture uptake was completely reversible, returning to 0 wt % at 0% RH. XRPD after GVS cycling at 0% and 90% RH holds for a minimum of 3 hours yielded anhydrous pattern B at both RH values.

[0293] Therefore, it can be concluded that the atropisomer A-2 tartrate pattern B salt exists as a stable solid and absorbs only surface moisture without changing its morphology.

[0294] Example 5 Further characterization of (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide tartrate solubility The atropisomer A-2 tartrate Pattern B salt was found to be highly soluble in aqueous formulations and suitable for oral delivery. After continuous stirring and gentle warming to 40 °C, concentrations of >100 mg / mL were achieved in aqueous solutions of 20% w / v (2-hydroxypropyl)-β-cyclodextrin.

[0295] powder density Bulk density was measured by placing atropisomer A-2 tartrate Pattern B salt in a 50 mL glass beaker. The compound was allowed to settle before weighing, and the volume occupied by approximately 40 mL and the bulk density were calculated.

[0296] Tap density was determined by placing 6-8 mL of atropisomer A-2 tartrate Pattern B salt into a 10 mL graduated cylinder, then repeatedly tapping and shaking the material vertically and horizontally manually using a rubber mat and a mallet for 15 minutes, or until a consistent bed with no further settling was achieved. The tap density was then calculated.

[0297] Atropisomer A-2 tartrate Pattern B salt has a powder bulk density value of 0.55 g / mL and a tapped density of about 0.659 g / mL.

[0298] The flowability of a drug powder and therefore its suitability for formulation in capsule form can be defined by its Carr index and Hausner ratio, which can be calculated from its bulk density and tapped density according to the following formulas: Carr compressibility index = 100(ρT-ρB) / ρT Hausner ratio=ρT / ρB

[0299] The Carr index and Hausner ratio can then be converted into the fluidity descriptors listed in the table below (Source: https: / / www.researchgate.net / figure / Specifications-for-Carrs-index-and-Hausner-ratio_tbl1_325365029). [Table 19] For the atropisomer A-2 tartrate Pattern B salt, the Carr index was calculated to be 16.5% and the Hausner ratio was calculated to be 1.19, indicating a "medium" flowability. Therefore, this salt form is suitable for powder administration in capsules.

[0300] stability The stability of atropisomer A-2 tartrate Pattern B salt was evaluated using two sets of storage conditions: 25°C ± 2°C / 60% RH and 40°C ± 2°C / 75% RH. The stability protocol followed ICH guidelines.

[0301] Therefore, atropisomer A-2 tartrate Pattern B salt (1 g per container) was placed in an ICH rated stability cabinet at 25° C. / 60% RH and 40° C. / 75% RH with the following filler components: -Double polythene bags (vendor -Armagrip; part number G01-PB-120), -Wide-mouth HDPE bottle with screw cap closure (vendor - Curtec; part number 4303), -Plastic tie strips (Vendor - Thomas & Betts; Part Number TY125-40-100).

[0302] After storage, the samples were removed from the stability cabinet and analyzed for appearance, water content by Karl Fischer titration (KF), and purity.

[0303] No change in water content or chemical purity by HPLC was observed over a 6-month period.No change in water content or chemical purity by HPLC was observed over a 6-month period. [Table 20]

[0304] Example 6 Alternative method for preparing (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide (atropisomer A-2) [ka]

[0305] The title compound was prepared by following steps 1, 2, 3, 4a, and 5a of the synthetic route shown above in Scheme 1. In this route, chiral resolution is performed on the carboxylic acid intermediate (8) rather than the dimethylaminoethylamide (9).

[0306] Step 1: 4-[4-(4-chlorophenyl)-4-oxo-butanoyl]benzonitrile (6) The flask was charged with tetrahydrofuran (4 mL / g) and zinc chloride (1.222 g / g, 1.3 equiv.) was added portionwise to give a mobile white suspension, which was stirred for 15 minutes. tert-Butanol (0.66 mL / g, 1 equiv.) was added portionwise, followed by triethylamine (0.96 mL / g, 1 equiv.), maintaining the temperature below 40°C. The reaction was stirred for 2 hours. 4-Cyanoacetophenone (1 g / g, 1 equiv.) and 4-chlorophenacyl bromide (1.61 g / g, 1 equiv.) were added, and the reaction mixture was stirred at 20°C (±5°C) for 48 hours or until the reaction was complete. The product was isolated by precipitation with aqueous HCl and slurry in aqueous HCl and methanol. The resulting solid was dried under vacuum (45°C) to give the title compound as a pale yellow solid.

[0307] Step 2: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzonitrile (7) 4-(4-(4-chlorophenyl)-4-oxobutanoyl)benzonitrile (1 g / g, 1 eq.) was placed in a flask and dioxane (10 mL / g) was added to give a yellow suspension. 2-Trifluoromethylaniline (1.269 mL / g, 3 eq.) was added in one portion, followed by p-toluenesulfonic acid (0.06399 g / g, 0.1 eq.), and the reaction mixture was heated at 101 °C for 40–72 h (additional portions of p-toluenesulfonic acid (0.1 eq.) were added every 8 h as needed to drive the reaction to completion). The reaction mixture was cooled to room temperature and concentrated in vacuo. The resulting oily residue was purified by slurrying in methanol (10 mL / g). The solid was isolated by filtration and dried under vacuum (45 °C) to give the title compound as a yellow solid.

[0308] Step 3: 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (8) To 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzonitrile (1 g / g, 1 eq.) in methanol (10.9 mL / g) was added aqueous sodium hydroxide (0.948 g / g, 10 eq.) (5 mL / g) dropwise over 15 minutes, and the resulting mixture was stirred at 70-76 °C for 18 hours or until complete. The reaction mixture was cooled to room temperature, acidified, and the product isolated by filtration and washed with water (5 mL / g) and acetonitrile (3 mL / g). The product was slurried in acetone / water (20 vol., 75:25) at 50-55 °C and dried under vacuum (60 °C) to give the title compound as a yellow solid.

[0309] Step 4a: Chiral resolution of (8) to (R) 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (3) 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (1 g / g, 1 equiv.) was added to a flask, followed by tetrahydrofuran (2 mL / g) and acetonitrile (0.75 mL / g). (S)-1-(4-Methoxyphenyl)-ethylamine (0.335 mL / g, 1 equiv.) was added dropwise over 5 min, and the resulting reaction mixture was stirred at 40–50 °C for 15 min and then cooled to room temperature. Acetonitrile (7.25 mL / g) was added to seed the reaction (0.0001 g / g, 99% ee, the (S)-1-(4-methoxyphenyl)-ethylamine salt of the desired atropisomer). The reaction mixture was stirred for 16 h, and the resulting solid was isolated by filtration, washing with acetonitrile. Hot (75-80 °C) slurry in acetonitrile afforded the chiral salt as a white solid (40% yield, 98.16% ee). Salt destruction in THF / water (2 / 2 vol) using 1 M HCl (2.2 equiv.) gave the acid, which was further purified by slurry in water to give the title compound (90.52 g, 97% salt destruction yield, 39% overall yield, 98.06% ee). 1H NMR (DMSO-d6) δ 12.83 (brs, 1H), 7.77-7.67 (m, 6H), 7.23-7.10 (m, 2H), 7.08-7.01 (m, 4H), 6.68 (d, J = 4.0 Hz, 1H), 6.59-6.58 (d, J = 4.0 Hz, 1H). Chiral HPLC using chiral HPLC method 6 showed a single atropisomer, RT 6.083 min, 99.02 area% (minor atropisomer, RT 7.07 min, 0.98 area%).

[0310] Chiral resolution can also be achieved using (S)-(-)-1-phenylethylamine.

[0311] Step 5a: (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)ethyl]benzamide (1) 4-(5-(4-chlorophenyl)-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (single atropisomer, (3)) (1 g / g, 1 equiv.) was dissolved in THF (5 mL / g), and N,N-dimethylethylenediamine (0.75 mL / g, 3 equiv.) was added dropwise, followed by DIPEA (1.58 mL / g, 4 equiv.). 50% T3P in THF (2.72 mL / g, 2 equiv.) was added dropwise, and the reaction mixture was stirred at 20 °C for 15 min. Additional portions of 50% T3P in THF were added until the reaction was complete. The reaction mixture was diluted with 10% brine (2 mL / g) and sodium hydroxide solution (2 mL / g) until the pH reached 8–10. The layers were separated, and the aqueous layer was extracted with ethyl acetate (2 × 5 mL / g). The combined organic layers were washed with brine, dried (MgSO), and concentrated to give the title compound (80 g, 156 mmol, 71%) as a white triboluminescent solid. Chiral HPLC using Chiral HPLC Method 7 showed a single atropisomer, RT 12.62 min, 99.32 area% (minor atropisomer, RT 10.58 min, 0.67 area%).

[0312] Example 7 biological activity A. Assay measuring the effect of atropisomers A-1 and A-2 on the viability of U87MG human glioblastoma cancer cells The following protocol was used to measure the effect of atropisomers A-1 and A-2 on the viability of U87MG cells.

[0313] U87MG cells were grown in the recommended growth medium / supplements (ATCC). Cells were seeded at 5000 cells / well in 96-well plates overnight at 37°C, 5% CO2. Cells were treated with relevant concentrations of test compounds for 72 hours. After 72 hours of incubation, viability was established using a sulforhodamine B (SRB) colorimetric assay. Viability was calculated relative to the mean of DMSO-treated control samples, and IC50 values ​​for inhibition of cell proliferation were calculated by nonlinear regression (four-parameter logistic equation) using GraphPad Prism software.

[0314] ICs obtained for atropisomers A-1 and A-2 according to the above protocol 50 The values ​​are shown in Table 9 below. [Table 21]

[0315] B. Assay measuring the effect of atropisomers A-1 and A-2 on cancer cell viability in a diverse panel of cancer cell lines A diverse cancer cell line screen was performed to identify tumor types sensitive to atropisomers A-1 and A-2. A panel of 48 cancer-derived cell lines was screened in a high-throughput proliferation assay using dilutions of atropisomers A-1 / A-2. Screened cell lines included representatives of pancreatic, colon / colorectal, lung, brain and neuronal cancers, as well as lymphoma and leukemia cell lines. Cell lines were treated with serial half-log dilutions of compound and assayed for proliferation 72 hours later using the CellTiter-Glo Assay (Promega). IC 50 Values ​​were calculated by fitting dose-response data using a nonlinear regression model. IC values ​​at micromolar concentrations for atropisomers A-1 and A-2 50 The values ​​are shown in Table 10 below. [Table 22-1] [Table 22-2] As can be seen from the data, atropisomer A-2 was a significantly more active cell growth inhibitor than atropisomer A-1 against all cell lines.

[0316] C. Assay to measure the effect of atropisomer A-2 on mitotic cells It is known that inhibiting the ability of PLK1 and PLK4 to bind to their partners via their PBDs arrests cells in mitosis. Experimentally, this can be measured by assessing the number of cells undergoing mitosis at a specific time point after treatment with a test compound by immunofluorescence detection of phosphorylated histone H3 (pH3), a mark present only in mitotic cells. PLK1 / 4-PBD inhibitors are expected to cause a dose-dependent increase in pH3-positive cells, which is reported as the mitotic index (MI)—the percentage of cells positive for this mitotic mark at a specific time point.

[0317] Distinct mitotic phenotypes are induced after inhibition of PLK1 and PLK4 in cells. Disruption of the PBD domain of PLK1 has been demonstrated to cause mitotic arrest with unassembled chromosomes, a phenotype distinct from the monopolar spindle phenotype induced by an ATP-competitive PLK1 inhibitor (Hanisch et al., 2006 Mol. Biol. Cell 17, 448-459). Centriole assembly is regulated by PLK4, and its inhibitor induces a multipolar spindle phenotype due to centrosome defects, which leads to abnormal cytokinesis (Wong et al., 2015 Science 348(6239);1155-1160).

[0318] The following protocol was used to measure the effect of atropisomer A-2 on cell arrest in mitosis and phenotypic analysis.

[0319] Mitotic index and phenotypic protocol Cells were plated at 10,000 cells / well in a 96-well plate and incubated overnight. The next day, atropisomer A-2 stock in DMSO was diluted into culture medium and then added to the cells for a maximum final DMSO concentration of 0.2%. Cells were incubated with the compounds for 24 hours and then fixed with 3.7% formaldehyde. Cells were permeabilized with 0.1% Triton X-100 and then incubated with anti-phosphohistone H3 (Ser10) antibody (Abcam ab5176). Cells were washed with PBS and then incubated with AlexaFluor 488-labeled goat anti-rabbit IgG (Invitrogen A11034) in the presence of 4 μg / ml Hoechst 33342 (Invitrogen H3570). Cells were washed in PBS and then imaged on an Arrayscan VTi HCS instrument (Thermo Fisher Scientific) using Target Activation V4 Bioapplication. A user-defined threshold was applied to identify mitotic cells based on the intensity of phospho-histone H3 staining.

[0320] GraphPad Prism was used to plot % mitotic cells against compound concentration using a least squares fit and the slope of the log(inhibitor) versus the response variable with no constraints.

[0321] From the results obtained according to the above protocol, atropisomer A-2 showed mitotic activity in HeLa and U87MG cell lines. 50 The values ​​and percentages of cells were obtained. EC 50 The values ​​are shown in Table 11 below. [Table 23] In a separate study, following the protocol described above but using a single compound concentration of 0.03 µM for each of atropisomers A-1 and A-2, the frequency of observed mitotic phenotypes in U87MG cells was manually assessed and classified into the following phenotypes for each of A-1 and A-2: unassociated chromosomes, multipolar spindles / abnormal cytokinesis, monopolar spindles, normal prometaphase, and normal metaphase. The results are shown in Figure 10.

[0322] The results, shown in Figure 10, demonstrate that atropisomer A-2 is much more effective at disrupting normal mitosis than atropisomer A-1. Thus, with A-1, 76% of cells exhibited a normal mitotic phenotype, compared to 77% in cells treated with the DMSO control, and 24% exhibited abnormal cytokinesis compared to 23% in cells treated with the DMSO control. No evidence of an unassembled chromosome phenotype was observed in either the DMSO control or atropisomer A-1-treated cells. In contrast, treatment of cells with the more active atropisomer A-2 resulted in only 17% cells with a normal mitotic phenotype, 70% cells with abnormal cytokinesis, and 13% cells with unassembled chromosomes. These phenotypes are consistent with disruption of PLK1 and PLK4 activity during mitosis.

[0323] D. Assay to measure the effect of atropisomer A-2 on centrosomes The results of Study C above indicate that atropisomer A-2 induces mitotic effects characteristic of dysregulated centrosome function. Therefore, the effects of A-2 on centrosome function were further investigated. HeLa cells stably expressing a centrin1-GFP fusion protein were seeded overnight in 96-well plates. Cells were treated with atropisomer A-2 (0.02 μM in DMSO) or DMSO control for 72 hours and then imaged using a fluorescence microscope. Multiple cell fields were captured for each treatment condition, and the images were then manually analyzed. Centrin1-GFP specifically marks centrioles as separate foci and can therefore be used to quantify the number of centrioles per cell. Therefore, for each treatment condition, 100 cells were analyzed and the number of centrioles present in each cell was recorded. The data were then separated into bins (no centrioles, one centriole, two centrioles, and more than two centrioles) and are shown in Figure 11.

[0324] From the data it can be concluded that atropisomer A-2 shows evidence of a PLK4 inhibitory phenotype in HeLa cells.

[0325] E. Assay measuring the effect of atropisomer A-2 on the viability of wild-type versus KRAS HeLa cells Assays were performed to compare the effects of atropisomer A-2 on wild-type HeLa cells and HeLa cells carrying the KRAS oncogene.

[0326] Therefore, atropisomer A-2 was tested in HeLa cells engineered to inducibly express wild-type or oncogenic KRas G12V transgenes using the FLP-in / T-Rex system (Invitrogen). Cells were cultured, transgene expression was induced with or without doxycycline, and then treated with serially diluted atropisomer A-2. After 72 hours of incubation, cell viability was assessed using Cell Titer Blue reagent (Promega) and a BMG Pherastar plate reader. The effect of PBD inhibition on cell viability of wild-type or oncogenic G12V KRAS was assessed using GraphPad Prism.

[0327] The results obtained according to the above protocol showed that atropisomer A-2 exhibited GI activity against wild-type and KRAS G12V HeLa cell lines. 50 The values ​​were determined and are shown in Table 12 below. [Table 24]

[0328] F. Kinase Selectivity Assay The evidence provided herein indicates that atropisomer A-2 binds to the PBD domains of PLK1 and PLK4, but not to the catalytic domains of PLK1 and PLK4, and should exhibit better selectivity over other kinases. Atropisomer A-2 was investigated by testing it for off-target activity against a panel of 97 kinases distributed across the kinome at a concentration of 3 μM using the DiscoverX KinomeScreen assay.

[0329] The DiscoverX KinomeScreen assay is a site-specific competitive binding assay that measures the binding affinity of compounds to kinases through the use of a solid-supported control compound that can bind or capture kinases in solution. In the absence of a kinase inhibitor test compound, all of the kinases bind to the solid support. When a kinase inhibitor test compound is added to the assay mix, the amount of kinase that binds to the solid support decreases, and the degree of decrease depends on the potency of the test compound as a kinase inhibitor. The potency of a test compound against a kinase can be expressed as the percentage of kinase that binds to the solid support (control percentage) at a given concentration of the test compound; the lower the percentage, the stronger the kinase-binding ability of the test compound. Thus, a control percentage value of 100% would indicate that the test compound does not bind to any kinases, since all of the kinases are bound to the solid support. Conversely, a control percentage value of 0% would indicate that the test compound binds to all kinases, since none are bound to the solid support.

[0330] protocol: For most assays, kinase-tagged T7 phage strains were grown in parallel in 24-well blocks in E. coli hosts derived from the BL21 strain.

[0331] Escherichia coli (E. coli) were grown to logarithmic phase, infected with T7 phage from a frozen stock (multiplicity of infection = 0.4), and incubated with shaking at 32°C until lysis (90–150 min). The lysate was centrifuged (6,000 × g) and filtered (0.2 μm) to remove cellular debris. The remaining kinase was produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 min at room temperature to generate affinity resins for kinase assays. The ligand-bearing beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and reduce nonspecific phage binding. Binding reactions were assembled by combining the kinase, ligand-bearing affinity beads, and test compound in 1x binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6mM DTT). Test compounds were prepared as 40x stocks in 100% DMSO and diluted directly into the assay. All reactions were performed in a polypropylene 384-well plate with a final volume of 0.02 ml. The assay plate was incubated at room temperature with shaking for 1 hour, and the affinity beads were washed with wash buffer (1x PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1x PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluate was measured by qPCR.

[0332] Compounds that bind to the kinase active site and directly (sterically) or indirectly (allosterically) prevent the kinase from binding to the immobilized ligand will reduce the amount of kinase captured on the solid support. Conversely, test molecules that do not bind to the kinase will have no effect on the amount of kinase captured on the solid support.

[0333] The strength of binding of the test molecule to the kinase can be expressed as percent of control (%Ctrl).

[0334] Percent Control (%Ctrl) Compound(s) were screened at a concentration of 3000 nM and the results of the primary screening binding interactions are reported as "%Ctrl" on the following pages, where a lower number indicates a stronger hit in the matrix. %Ctrl calculation

number

[0335] The %Ctrl values ​​for atropisomer A-2 against the panel kinases are shown in Table 13 below. [Table 25-1] [Table 25-2]

[0336] The results against 97 kinases demonstrate that atropisomer A-2 has little to no binding activity against a wide range of kinases and is therefore less susceptible to problems associated with off-target kinase inhibition.

[0337] In the case of PLK1 and PLK4, atropisomer A-2 showed little to no binding affinity to the catalytic domains of these kinases (%Control values ​​of 97% and 100%, respectively). Therefore, we conclude that the activity profiles demonstrating PLK1 / PLK4 inhibitory activity demonstrated in the above examples result from binding to the non-catalytic Polo-box domains of PLK1 and PLK4.

[0338] G. Determination of Oral Bioavailability and Brain Exposure in Mouse PK Atropisomer A-2 was evaluated in an in vivo mouse model to determine brain and plasma concentrations after po and iv administration.

[0339] The following protocol was followed: Male CD-1 mice were administered atropisomer A-2 either iv (2 mg / kg) or po (10 mg / kg).

[0340] Eight samples from the iv leg at 2, 10, 30 min, 1, 2, 4, 8, and 24 (if iv) and nine samples from the po leg at 15, 30 min, 1, 2, 4, 8, 24, 48, and 72 h were taken for analysis.

[0341] Atropisomer A-2 was formulated in 10% DMSO / 95% hydroxypropyl-β-cyclodextrin (20% w / v in water) for iv and po administration to N=3 mice per time point.

[0342] After dosing, a final blood sample was collected from each animal and delivered to a labeled polypropylene tube containing anticoagulant (EDTA). Samples were kept on wet ice for a maximum of 30 minutes until sampling of all animals in the cohort was completed. Blood samples were centrifuged for plasma (4°C, 21,100 g for 5 minutes), and the resulting plasma was transferred to the corresponding labeled tube. The terminal brain of each PO-treated animal was excised, rinsed with saline, and placed in a pre-weighed, labeled polypropylene tube. Samples were reweighed before storage.

[0343] Quantitative bioanalysis was performed using liquid chromatography and mass spectrometry was carried out, and the results are shown in Tables 14 and 15 below and in Figures 12 and 13.

[0344] Oral bioavailability [Table 26] [Table 27] The results demonstrate that atropisomer A-2 is highly absorbed after oral administration to mice.

[0345] Brain exposure [Table 28] The results of the brain exposure study, shown in Table 16, demonstrate that atropisomer A-2 has high brain exposure after oral administration to mice with an AUC B:P ratio of 3.3.

[0346] H. In Vivo Efficacy Atropisomer A-2 demonstrates efficacy in glioblastoma mouse models when tumors are implanted subcutaneously and orthotopically, as demonstrated by the studies described below.

[0347] (i) In vivo anticancer activity in the U87MG subcutaneous xenograft model Male athymic nude mice bearing U87MG tumors were given oral doses of 100 mg / kg atropisomer A-2 on days 1, 4, and 7, and tumor volumes were measured over a 20-day period. Tumor volumes were also measured in a control group of tumor-bearing mice that received vehicle only at the same time points. As shown in Figure 14, the treated group exhibited significantly reduced tumor volumes compared to controls (3.85% T / C on day 13).

[0348] (ii) In vivo anticancer activity in the U87-Luc orthotopic xenograft model U87-Luc cells were intracerebrally implanted into the brains of male athymic nude mice, and tumor growth was monitored by bioluminescence signals. In the treatment group, animals received an oral dose of 100 mg / kg of atropisomer A-2 on days 1, 4, 7, 10, and 13. Animals in the control group received vehicle only. The results, shown in Figure 15, demonstrate a decrease in tumor signal in the treatment group versus the control group on day 15.

[0349] (iii) In vivo anticancer activity in HCT116 tumor-bearing mice Atropisomer A-2 demonstrated efficacy in a KRAS mutant colorectal cancer model as follows:

[0350] Male athymic nude mice bearing HCT116 xenograft tumors were given oral doses of 100 mg / kg atropisomer A-2 on days 1, 8, and 15, and tumor volumes were measured over a 3-week period. Tumor volumes were also measured in a control group of tumor-bearing mice that received vehicle only at the same time points.

[0351] The results, shown in Figure 16, demonstrate a significant effect on tumor growth at 20 days (TGI 60%).

[0352] Pharmaceutical preparations (i) Tablet formulation A tablet composition containing the tartrate salt or composition of matter according to any one of embodiments 1.1-1.19, or the examples above, is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as a diluent and 3 mg of magnesium stearate as a lubricant and compressing to form a tablet by known methods.

[0353] (ii) Capsule formulation A capsule formulation is prepared by mixing 100 mg of any one of embodiments 1.1-1.19, or the tartrate salt or composition of matter described in the examples above, with 100 mg of lactose and filling the resulting mixture into a standard opaque hard gelatin capsule.

[0354] (iii) Injection Preparation I A parenteral composition for administration by injection can be prepared by dissolving the tartrate salt or composition of matter according to any one of embodiments 1.1 to 1.19, or as described in the examples above, in water containing 10% propylene glycol to obtain a concentration of 1.5% by weight of active compound. The solution is then filter-sterilized, filled into an ampoule and sealed.

[0355] (iv) Injection Preparation II A parenteral composition for injection is prepared by dissolving the tartrate salt or composition of matter according to any one of embodiments 1.1 to 1.19 or the examples above (2 mg / ml) and mannitol (50 mg / ml) in water, sterile filtering the solution and filling into sealable 1 ml vials or ampoules.

[0356] (v) Injection preparation III A formulation for iv delivery by injection or infusion can be prepared by dissolving the composition of matter described in any one of embodiments 1.1-1.19, or in the examples above, in 20 mg / ml water. The vial is then sealed and sterilized by autoclaving.

[0357] (vi) Injectable preparation IV Formulations for iv delivery by injection or infusion can be prepared by dissolving the composition of matter described in any one of embodiments 1.1-1.19, or in the examples above, in water containing 20 mg / ml of buffer (e.g., 0.2 M acetate pH 4.6). The vial is then sealed and sterilized by autoclaving.

[0358] (vii) Subcutaneous injection formulation A composition for subcutaneous administration is prepared by mixing the composition of matter described in any one of embodiments 1.1-1.19, or in the examples above, with pharmaceutical grade corn oil to a concentration of 5 mg / ml. The composition is sterilized and filled into a suitable container.

[0359] (viii) freeze-dried preparation Aliquots of the composition of matter described in any one of aspects 1.1-1.19, or in the examples above, are placed in 50 ml vials and lyophilized. During lyophilization, the composition is frozen at (-45°C) using a one-step freezing protocol. The temperature is raised to -10°C for annealing, then lowered to -45°C for freezing, followed by primary drying at +25°C for approximately 3400 minutes, followed by secondary drying in incremental steps at 50°C. The pressure during primary and secondary drying is set at 80 mTorr.

[0360] equivalent The foregoing examples are presented for the purpose of illustrating the present invention and should not be construed as imposing any limitations on the scope of the present invention. It will be readily apparent that numerous modifications and variations can be made to the specific embodiments of the invention described and illustrated above without departing from the principles underlying the invention. All such modifications and variations are intended to be encompassed by this application.

Claims

1. (R)-4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2-(dimethylamino)-ethyl]benzamide (+)-L-tartrate, with a molar ratio between acid and base of 1:

1.

2. (+)-L-tartrate salt of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide having the formula (2): 【Chemistry 1】

3. (+)-L-tartrate salt of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide, in which there is a 1:1 molar ratio between acid and base and 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is in the form of a single atropisomer.

4. The (+)-L-tartrate salt of claim 3, wherein the single atropisomer is the atropisomer of formula (1). 【Chemistry 2】

5. The (+)-L-tartrate salt of claim 3, wherein the single atropisomer is the R atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide.

6. A (+)-L-tartrate salt according to any one of claims 1 to 5, which is crystalline.

7. The (+)-L-tartrate salt according to claim 6, which is anhydrous.

8. 8. The composition of matter comprising the (+)-L-tartrate salt of claim 1, wherein either (a) a single atropisomer is the only atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide present in the composition, or (b) less than 10% by molar amount of any other atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is present relative to the single atropisomer.

9. 9. The composition of matter of claim 8, wherein either (a) a single atropisomer is the only atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide present in the composition, or (b) less than 0.1% by molar amount of any other atropisomer of 4-[5-(4-chlorophenyl)-1-[2-(trifluoromethyl)-phenyl]pyrrol-2-yl]-N-[2(dimethylamino)ethyl]benzamide is present relative to the single atropisomer.

10. A pharmaceutical composition comprising the (+)-L-tartrate salt of any one of claims 1 to 7, or a composition of matter of any one of claims 8 and 9, and a pharmaceutically acceptable excipient.

11. The pharmaceutical composition of claim 10 for the treatment of cancer.

12. A pharmaceutical composition comprising the (+)-L-tartrate salt according to any one of claims 1 to 7 or a composition of matter according to claim 8 or 9, and another therapeutically active agent.

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