Crystalline forms of (s)-1-(1-(3-chlorphenyl)-2-(dimethylamino) ethyl)-4-(5-morpholino-1h-pyrrolo [2, 3-b] pyridin-3-yl) pyridin-2 (1h)-one, their preparation and their use as ERK kinase inhibitors

Abstract

The present invention relates to crystalline forms of (s)-1-(1-(3-chlorphenyl)-2-(dimethylamino) ethyl)-4-(5-morpholino-1h-pyrrolo [2, 3-b] pyridin-3-yl) pyridin-2 (1h)-one of the following formula (I): (I)

Description

Technical Field

[0001] This invention relates to a novel crystalline form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one as an inhibitor of ERK kinases (ERK1 and ERK2), its preparation, and its therapeutic use. Background Technology

[0002] ERK proteins belong to the RAS / RAF / MEK / ERK pathway, which plays a major role in cell cycle, proliferation, growth, and survival. The RAS / RAF / MEK / ERK pathway is activated by growth factors through their receptor tyrosine kinases, which in turn activate the GTPase RAS. RAS then activates RAF proteins. RAF then activates MEK, and MEK activates ERK.

[0003] Finally, this enables the phosphorylation of many substrates that play a key role in metabolism, protein synthesis, cell proliferation, and survival.

[0004] RAF mutations specifically lead to overactivation of this RAS / RAF / MEK / ERK pathway and are responsible for 7% of all human cancers (Davies et al., Nature. 2002; Garnett et al., Cancer Cell. 2004).

[0005] In fact, RAF mutations are commonly found in melanoma (27-70%), thyroid cancer (36-53%), colorectal cancer (5-22%), and ovarian cancer (30%). Similarly, RAS mutations occur in almost 30% of cancers and are present in pancreatic cancer (90%), lung cancer (35%), colorectal cancer (45%), and liver cancer (30%) (Downward, Nat. Rev. Cancer. 2003).

[0006] Therefore, proteins along the RAS / RAF / MEK / ERK pathway represent potential targets for cancer therapy. In fact, pharmaceutical companies are focusing on upstream kinases (RAF, MEK).

[0007] However, resistance eventually develops after current treatment with RAF and MEK inhibitors (Lito et al., Nat. Med. 2013; Caunt et al., Nat. Rev. Cancer, 2015).

[0008] Furthermore, most resistance to MEK or RAF inhibitors induces ERK reactivation through different mechanisms (e.g., MEK mutation, B-RAF amplification, C-RAF mutation, etc.) (Little et al., Oncogene. 2013).

[0009] Furthermore, RAF or MEK inhibition suppresses the negative feedback of ERK, which restores upward signaling and ultimately restores ERK activity (Lito et al., Nat. Med., 2013).

[0010] Given the drug resistance that has emerged after treatment with RAF and MEK inhibitors, it is necessary to develop new treatment options.

[0011] ERK signaling, in addition to its key role in proliferative diseases, has been described as being associated with neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease, and Huntington's disease (Cheung et al., Sci. STKE. 2004; Bodai et al., Bioessays., 2012) and inflammation, such as its role in the pathogenesis of rheumatoid arthritis (Thalhamer et al., Rheumatology. 2008).

[0012] Therefore, this invention relates to the development of ERK inhibitors for the treatment of a variety of diseases.

[0013] Several ERK inhibitors have been described in the prior art. Therefore, US 8,697,697 B2 describes substituted pyrazole derivatives as inhibitors of ERK2 kinase activity.

[0014] Pyrrolo[2,3-b]pyrazine derivatives have been reported as ERK inhibitors in international patent application WO 2014 / 060395 A1, and azaindole derivatives have been reported as ERK inhibitors in international patent application WO 2017 / 085230 A1.

[0015] The (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one of formula (I) described below is an inhibitor of ERK kinases (ERK1 and ERK2). It is particularly useful as an anticancer agent.

[0016] (I)

[0017] This invention relates to a novel crystal form of this compound.

[0018] In fact, the identification of novel crystal forms of known active ingredients can be particularly significant for drug development.

[0019] Polymorphism occurs when the same chemical entity crystallizes in different lattice arrangements, resulting in distinct thermodynamic properties and stability specific to each polymorph. When the chemical entity is a pharmaceutical product, its ability to exist in more than one crystal form can profoundly impact its shelf life (stability), solubility, formulation properties, and / or processing properties. Therefore, from a quality perspective, it is crucial to ensure that the manufacturing process achieves the specific polymorph permitted for market release by regulatory agencies and to control the formation of other polymorphs with different thermodynamic properties and stability.

[0020] Therefore, there is a need to provide novel crystalline or polymorphic forms of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one, which particularly possesses good stability properties. Summary of the Invention

[0021] The present invention specifically relates to a novel crystalline form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one, its preparation and its use as an inhibitor of ERK kinase activity.

[0022] The compounds of the present invention are novel crystal forms with at least enhanced antiproliferative activity and good stability.

[0023] The crystal form according to the present invention is also characterized by its low toxicity, high permeability, and selective kinase inhibition.

[0024] In summary, the crystal form according to the present invention has significant advantages due to its drug-like properties.

[0025] The first subject of this invention relates to the crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one of formula (I):

[0026] (I)

[0027] Unexpectedly, the inventors discovered that the free base of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one can exist in a specific crystal form.

[0028] After extensive searching and screening tests, the inventors have determined that the crystal form of the compound of formula (I) selectively targets the active site of ERK kinase and can be used as an effective inhibitor of ERK kinase activity.

[0029] Advantageously, this novel crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one exhibits very good stability, as shown in the examples.

[0030] Therefore, this new crystal form can be stored for a long time without requiring specific conditions.

[0031] This excellent stability also facilitates processing.

[0032] Furthermore, the reproducibility of the compound is advantageously ensured by defining a specific crystal form and thus controlling its synthesis through the acquisition of the crystal form according to the invention.

[0033] As described below, this compound can be used to treat conditions or diseases in which alterations in ERK activity have a positive therapeutic effect, particularly cancer.

[0034] Another topic relates to methods for preparing the crystal form of compound (I).

[0035] Another topic relates to the crystal form of the compound of formula (I), and in particular its use in pharmaceuticals or pharmaceutical compositions.

[0036] Another subject relates to the use of the crystal form according to the invention as an inhibitor of ERK kinase activity, particularly as an inhibitor of ERK1 and / or ERK2 kinase activity.

[0037] Abbreviations and Definitions

[0038] In the context of this invention, the following abbreviations and empirical formulas are used:

[0039] ACN Acetonitrile

[0040] ATP adenosine 5'-triphosphate

[0041] Brij-35 Polyoxyethylene glycol dodecyl ether

[0042] C18 column reverse C18 column

[0043] CaCl2 (calcium chloride)

[0044] CMC (carboxymethyl cellulose)

[0045] DABCO 1,4-diazabicyclo[2.2.2]octane

[0046] DCM dichloromethane

[0047] DMF (dimethylformamide)

[0048] DMEM DuPont Modified Eagle Medium

[0049] DMSO (dimethyl sulfoxide)

[0050] DSC Differential Scan Calorimetry

[0051] ℃ Celsius

[0052] ee enantiomer excess

[0053] EGTA (Ethanoic acid)

[0054] Eq equivalent

[0055] Et2O diethyl ether

[0056] EtOAc (ethyl acetate)

[0057] EtOH (ethanol)

[0058] FBS Fetal Bovine Serum

[0059] FT-IR Fourier Transform Infrared Spectroscopy

[0060] g gram

[0061] h hours

[0062] HBSS Hanks balanced salt solution

[0063] HCl hydrochloric acid

[0064] HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)

[0065] hERG, the human Ether-à-go-go related gene

[0066] HPLC (High Performance Liquid Chromatography)

[0067] IR infrared spectrum

[0068] K2CO3 (potassium carbonate)

[0069] KCl (potassium chloride)

[0070] KF potassium fluoride

[0071] KOH (potassium hydroxide)

[0072] LC liquid chromatography

[0073] LC-MS (Liquid Chromatography / Mass Spectrometry)

[0074] LiAlH4 lithium aluminum hydride

[0075] LiHMDS Bis(trimethylsilyl)aminolithium

[0076] M moles / liter

[0077] MeCN Acetonitrile

[0078] MeOH (methanol)

[0079] mg

[0080] MH+ quasi-molecular ion (positive ion mode in mass spectrometry)

[0081] MHz

[0082] MS mass spectrometry

[0083] µl

[0084] MgCl2 (magnesium chloride)

[0085] ml

[0086] mmol millimole

[0087] mol

[0088] MPA Mobile Phase A

[0089] MPB Mobile Phase B

[0090] NaCl (Sodium Chloride)

[0091] Na2CO3 (Sodium carbonate)

[0092] NaHCO3 (Sodium bicarbonate)

[0093] NaOH (sodium hydroxide)

[0094] Na2SO4 Sodium sulfate

[0095] NH4Cl ammonium chloride

[0096] NMR (Nuclear Magnetic Resonance)

[0097] RH (Relative Humidity)

[0098] RuPhos 2-Dicyclohexylphosphino-2',6'-diisopropoxybiphenyl

[0099] RuPhos Pd G2 Chlorine(2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II)

[0100] SDS Sodium lauryl sulfate

[0101] TBAF Tetrabutylammonium Fluoride

[0102] TEA Triethylamine

[0103] TG thermogravimetric analysis

[0104] THF Tetrahydrofuran

[0105] U(H)PLC Ultra-High Performance Liquid Chromatography

[0106] UV ultraviolet rays

[0107] XRPD X-ray powder diffraction

[0108] In the context of this invention, "kinase inhibitor" is intended to refer to a compound that reduces or inhibits the activity of a targeted kinase compared to the activity measured without the inhibitor.

[0109] In the context of this invention, the term "prevent" or "prevention" in relation to an event is intended to mean reducing the risk of the event occurring.

[0110] As used in this article, the term “ambient temperature” or “room temperature” refers to a temperature between 15°C and 30°C, and more specifically between 18°C ​​and 25°C.

[0111] Other features, properties, and advantages of the present invention will become more apparent from the following description and examples. Attached Figure Description

[0112] Figure 1 X-ray powder diagram of the crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one (see Example 11).

[0113] Figure 2 It is the crystalline form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one. 1 H NMR spectrum (see Example 11). Detailed Implementation

[0114] As described above, the crystal form according to this disclosure exhibits very good stability.

[0115] The crystal form of the present invention

[0116] As described above, one object of the present invention is the crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one of formula (I):

[0117] (I)

[0118] The crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one can be characterized, for example, by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC).

[0119] According to a preferred embodiment, the crystalline form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one has a powder X-ray diffraction pattern showing peaks at the following angles (in degrees 2-θ): 14.72; 15.28; 17.18; 22.88; 23.08 and 24.00. (±0.2 each time), and optionally also showing the following additional peaks in degrees 2-θ angle: 12.03; 17.42; 22.10; 22.47; 25.07 and 25.90 (±0.2 each time); and even optionally showing the following additional peaks in degrees 2-θ angle: 3.36; 12.49; 12.91; 13.42; 13.93 and 19.64 (±0.2 each time), as Figure 1 As shown in the powder X-ray diffraction pattern and / or with a single endothermic peak at an onset temperature of 281 °C (±2 °C).

[0120] The characteristic X-ray powder diffraction pattern of the crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one can be found in Figure 1 The characteristic signals are summarized in the table below:

[0121]

[0122] According to a preferred embodiment, the crystalline form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one exhibits a powder X-ray diffraction pattern showing at least one peak, particularly at least two peaks, preferably at least five peaks, more preferably at least ten peaks, expressed in degrees 2-θ angles, selected from 3.36; 3.36; 12.03; 12.49; 12.91; 13.42; 13.93; 14.72; 15.28; 17.18; 17.42; 19.64; 22.10; 22.47; 22.88; 23.08; 24.00; 25.07 and 25.90 (±0.2 each time).

[0123] According to a preferred embodiment, the crystal form of the (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one of the present invention has an XRPD substantially similar to that of... Figure 1 The XRPD shown in the image.

[0124] Preparation of crystal form

[0125] Compound (I) can be prepared according to the method described in Example 1.

[0126] This article also provides a method for preparing the crystalline form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)one according to the present invention, the method comprising the following steps:

[0127] a) Suspending (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one in a solvent or solvent mixture, preferably in MeCN / H2O (70 / 30);

[0128] b) Optionally, evaporate the solvent at a temperature between 0°C and the boiling point of the solvent or mixture of solvents selected in step a);

[0129] c) Optionally, a solvent or solvent mixture may be added.

[0130] d) Apply a temperature program;

[0131] e) Optional filtering; and

[0132] f) The obtained crystals may be washed with a solvent or a mixture of solvents.

[0133] g) Then optionally dry to obtain the desired crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one.

[0134] According to a preferred embodiment, the solvents used in steps a), c), and f) are any solvent commonly used in the crystallization step, particularly organic solvents, and more particularly selected from water, alcohol solvents (e.g., 1-propanol, 2-propanol, ethanol, methanol, 1-butanol, and 2-butanol), glycol solvents (e.g., propylene glycol), ketone solvents (e.g., acetone, methyl ethyl ketone, and methyl isobutyl ketone), ether solvents (e.g., 1,4-dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentylmethyl ether, and methyl tert-butyl ether), acetate solvents (e.g., ethyl acetate and isopropyl acetate), aromatic solvents (e.g., toluene), hydrocarbon solvents (e.g., n-heptane), chlorinated solvents (e.g., dichloromethane and chloroform), organic sulfur solvents (e.g., dimethyl sulfoxide (DMSO)), amine and amide solvents (e.g., N-methyl-2-pyrrolidone (NMP), dimethylacetamide, and acetonitrile), and mixtures thereof.

[0135] According to the preferred embodiment, the solvent used in step a) is MeCN / H2O (70 / 30).

[0136] According to the preferred embodiment, the solvent used in step c) is MeCN / H2O (70 / 30).

[0137] According to the preferred embodiment, the solvent used in step f) is Et2O.

[0138] According to one implementation, the solvent used in steps a) and c) is the same.

[0139] According to another embodiment, the solvents used in steps a) and c) are different.

[0140] According to one implementation, the solvent used in steps a) and f) is the same.

[0141] According to another embodiment, the solvents used in steps a) and f) are different.

[0142] application

[0143] As previously described and clearly illustrated by the following examples, the crystal form according to the invention can be used as an inhibitor of ERK kinase activity.

[0144] According to a first aspect, the crystal form of the present invention is used as an inhibitor of ERK2 kinase activity, preferably as a selective inhibitor of ERK2 kinase activity.

[0145] More specifically, the crystal forms of the present invention are used for the prevention and / or inhibition and / or treatment of diseases or conditions mediated by ERK kinase activity, particularly ERK2 kinase activity.

[0146] Therefore, the present invention provides a method for preventing and / or treating diseases or conditions mediated by ERK kinase activity, comprising at least the step of administering at least an effective amount of at least one crystal form according to the invention to an individual in need of such treatment.

[0147] The present invention also provides a crystal form of the invention for use in preventing and / or inhibiting and / or treating, preferably preventing and / or treating, more preferably treating diseases or conditions mediated by ERK kinase activity, preferably ERK2 kinase activity.

[0148] The present invention also provides the use of the crystal form of the present invention for the prevention and / or inhibition and / or treatment, preferably prevention and / or treatment, more preferably treatment of diseases or conditions mediated by ERK kinase activity, preferably ERK2 kinase activity.

[0149] According to one embodiment, the disease or condition may be selected from cancer, metastases, and human immunodeficiency virus (HIV), and is preferably selected from cancer and metastases.

[0150] More specifically, the disease or condition may be selected from glioblastoma, multiple myeloma, cancer, leukemia (especially myeloid leukemia (AML), lymphocytic leukemia, myelogenous leukemia (CML), or lymphoblastic leukemia), myelodysplastic syndrome, Kaposi's sarcoma, cutaneous angiosarcoma, solid tumors, lymphoma (especially non-Hodgkin's lymphoma), melanoma (especially malignant melanoma), bladder cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, endometrial cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, head and neck cancer, liver cancer, ovarian cancer (especially serous ovarian cancer), seminoma, cancers of the respiratory tract and chest, thyroid cancer (especially papillary or follicular thyroid cancer), or other tumors expressing ERK.

[0151] According to another implementation, the disease or condition may be selected from neoplastic conditions, allergic conditions, inflammatory conditions, autoimmune conditions, malaria-related diseases, mast cell-related diseases, graft-versus-host disease, metabolic syndrome, CNS-related diseases, neurodegenerative diseases, pain conditions, substance abuse conditions, prion diseases, heart disease, fibrotic diseases, idiopathic arterial hypertension (IPAH), or essential pulmonary hypertension (PPH).

[0152] According to another embodiment, the crystal form of the present invention can be used to prevent and / or inhibit and / or treat human immunodeficiency virus (HIV).

[0153] The crystal form of the present invention can be used alone or in combination with chemotherapeutic agents or radiotherapy regimens.

[0154] Therefore, according to one embodiment, the method of the present invention may include the steps of applying the crystal form according to the invention and the chemotherapeutic agent separately, sequentially, or simultaneously.

[0155] Examples of chemotherapeutic agents suitable for use in this invention may be mentioned from the following: alkylating agents, intercalating agents, antimicrotubule agents, antimitotic agents, antimetabolites, antiproliferative agents, antibiotics, immunomodulators, anti-inflammatory drugs, kinase inhibitors, anti-angiogenic agents, anti-angiogenic agents, estrogens, and androgens.

[0156] Radiation therapy can be administered by exposing individuals in need to a source of ionizing radiation, such as X-rays, gamma rays, or beta rays.

[0157] According to another aspect of the invention, it relates to a pharmaceutical composition comprising at least one crystal form according to the invention and at least one pharmaceutically acceptable excipient.

[0158] The crystal form according to the invention can be used to prepare drugs, particularly drugs for inhibiting ERK kinase activity, preferably ERK2 kinase activity.

[0159] Therefore, according to another aspect of the invention, it relates to a drug comprising at least one crystal form according to the invention.

[0160] The term "pharmaceutically acceptable" refers to a substance that can be used to prepare a pharmaceutical composition, which is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes substances acceptable for veterinary and human pharmaceutical use.

[0161] The pharmaceutical composition may more specifically contain an effective dose of at least one crystal form according to the invention.

[0162] "Effective dose" refers to an amount sufficient to produce positive changes in a condition to be regulated or treated, but low enough to avoid serious side effects. Effective doses can vary depending on factors such as the drug effect achieved or the specific condition being treated, the age and physical condition of the end user, the severity of the condition being treated / prevented, the duration of treatment, the nature of any other treatments, the specific compound or composition used, and the route of administration.

[0163] The crystal form according to the invention can be administered at an effective dose by any method of administration acceptable in the art.

[0164] In one embodiment, the crystal form of the present invention can be used in compositions intended for administration via oral, nasal, sublingual, otopathic, ophthalmic, topical, rectal, vaginal, urethral, ​​or parenteral injection routes.

[0165] The route of administration and the galen formulation will be adjusted by those skilled in the art according to the desired drug effect.

[0166] In a preferred embodiment, the crystal form of the present invention can be used in compositions intended for oral administration.

[0167] Those skilled in the art of therapeutic formulations can determine the effective therapeutic dose of the crystal form of the present invention for a given indication without extensive experimentation and relying on personal knowledge.

[0168] Depending on the dosage, galen dosage form, route of administration, etc., the pharmaceutical compositions of the present invention can be formulated with any known suitable pharmaceutically acceptable excipient.

[0169] As used herein, "pharmaceuticalally acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. Unless any conventional excipient is incompatible with the active compound, the present invention envisions its use in pharmaceuticals or pharmaceutical compositions.

[0170] The drugs or drug compositions of the present invention may be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols, sprays, ointments, gels, creams, sticks, lotions, pastes, soft gelatin capsules and hard gelatin capsules, suppositories, sterile injection solutions, sterile packaged powders, etc.

[0171] According to one embodiment, the pharmaceutical composition of the present invention may be intended to be administered separately, sequentially, or simultaneously with an agent for the prevention and / or treatment of disease conditions (particularly cancer conditions), said agent being different from the compound of formula (I) of the present invention.

[0172] This application also includes novel kit-of-parts for cancer treatment.

[0173] The multi-component kit according to the present invention may comprise (i) a crystal form according to the present invention, and (ii) at least one agent for the prevention and / or treatment of cancer conditions, said agent being different from said crystal form. The agent for the prevention and / or treatment of cancer conditions may be a chemotherapeutic agent or a radiotherapy agent.

[0174] The invention will be better understood by referring to the following embodiments, which are for illustrative purposes only and should not be construed as limiting the invention in any way.

[0175] Example

[0176] apparatus and analytical method for synthesis used in the embodiments

[0177] Unless otherwise stated, the following devices and analysis methods are used in the embodiments.

[0178] 1. X-ray powder diffraction (XRPD)

[0179] XRPD analysis was performed on a PANalytical X'pert pro equipped with a PIXcel detector (128 channels), scanning the sample from 3° to 35°2θ. The material was gently ground to release any aggregates and loaded into a porous plate with a Kapton or Mylar polymer film for sample support. The porous plate was then placed in a diffractometer for analysis using Cu K radiation (α1λ = 1.54060 Å; α2 = 1.54443 Å; β = 1.39225 Å; α1:α2 ratio = 0.5), run in transmission mode (step size 0.0130°2θ, step time 18.87 s), using a 40 kV / 40 mA generator setting. The data were visualized and images generated using the HighScore Plus 4.7 desktop application (PANalytical, 2017).

[0180] 2. Differential Scanning Calorimetry (DSC)

[0181] Approximately 1–5 mg of material was weighed into an aluminum DSC pan and loosely sealed with an aluminum cap. The sample pan was then loaded into a TA Instruments Discovery DSC 2500 differential scanning calorimeter equipped with an RC90 cooler. The sample and reference were heated to a maximum of 300 °C at a scan rate of 10 °C / min, and the resulting heat flux response was monitored. The sample was then cooled back to 20 °C and then reheated to a maximum of 300 °C at a rate of 10 °C / min. Nitrogen was used as the purge gas at a flow rate of 50 cm⁻¹. 3 / min.

[0182] 3. Nuclear Magnetic Resonance (NMR)

[0183] Equipment: Bruker 400 MHz.

[0184] Method: Use DMSO-d5 as an internal reference in DMSO-d6. 1 H NMR spectra, chemical shifts are expressed in parts per million (ppm), and signals are represented as follows: s = singlet, d = doublet, t = triplet, q = quartet, sept = septet, dd = double doublet, dt = double triplet, m = multiplet or broad singlet, br = broad peak, H = proton.

[0185] 4. Mass spectrometry

[0186] Equipment: Waters Micromass ZQ (simple quadrupole).

[0187] Quality testing method: Electrospray positive ion mode (ESI+), quality range: 50-800 uma.

[0188] 5. Rapid chromatography

[0189] Equipment: Biotage SP, equipped with an automatic collector and UV detection (2 wavelengths).

[0190] Normal phase column: 120 g or 300 g Biotage external dry loading cartridge kit, filled with Sigma-Aldrich 40-63 µm silica gel.

[0191] 6. Liquid Chromatography

[0192] Equipment: Waters Alliance 2695 HPLC system, equipped with an autosampler and a Waters 2996 diode array detector.

[0193] Column: Macherey-Nagel Nucleoshell RP18 plus (5 μm, 4 mm x 100 mm).

[0194] Column temperature: 40℃.

[0195] Solvents: A (H2O 99.9%, H2CO2 0.1%); B (CH3CN 99.9%, H2CO2 0.1%).

[0196] Flow rate: 1 mL / min.

[0197] Gradient (A / B v / v): 90 / 10 (t=0min), 90 / 10 (t=1min), 0 / 100 (t=7min), 0 / 100 (t=10min).

[0198] Detection range: 210-400 nm.

[0199] 7. Chiral chromatography

[0200] Chiral column: Daicel ChiralPak IG (Amylose-based) 20 μm, 4.6 mm x 100 mm.

[0201] Chiral column: Daicel ChiralPak IG (Amylose-based) 5 μm, 4.6 mm x 250 mm.

[0202] Column temperature: 25℃.

[0203] Analysis of the final compound (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one (isocratic conditions): solvent: 50% heptane / 40% EtOH (containing 0.1% Et3N) and 10% DCM, flow rate: 1 mL / min.

[0204] 8. Three-month stability study

[0205] The compound was placed for stability studies to investigate its chemical and physical stability over a total of three months. The evaluation conditions were:

[0206] - Ambient light and temperature (sealed vial) at 2 weeks, 1 month and 3 months.

[0207] - At 2 weeks, 1 month and 3 months, 80°C (sealed vial)

[0208] - At 2 weeks, 1 month and 3 months, 25°C / 60%RH (open vial)

[0209] - At 2 weeks, 1 month and 3 months, 40°C / 75%RH (open vial)

[0210] The samples were analyzed by XRPD and HPLC at each time point.

[0211] Example 1: Synthesis of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one (compound N°1)

[0212]

[0213] Step 1: 1-Chloro-3-vinylbenzene

[0214]

[0215] 10 g (71.1 mmol) of 3-chlorobenzaldehyde was dissolved in 50 mL of anhydrous THF, and the solution was cooled to -10 °C using an ice / acetone bath. 17.3 g (48.4 mmol, 1.2 equivalents) of methyltriphenylphosphonium bromide was added, followed by 2.1 g (52.4 mmol, 1.3 equivalents) of sodium hydride (60% paraffin oil solution). The suspension was then stirred overnight at room temperature under argon. The mixture was diluted with 100 mL of Et₂O, and the precipitate was filtered through diatomaceous earth. The filtrate was evaporated under reduced pressure to give an orange residue. Finally, the crude mixture was purified by rapid chromatography using a silica gel column and an Et₂O / pentane mixture (3 / 97) as eluent. 4.05 g of the title compound was obtained.

[0216] Yield: 41%.

[0217] MH+: Non-ionizable.

[0218] Step 2: 2-(3-chlorophenyl)ethylene oxide

[0219]

[0220] 4.05 g (29.2 mmol) of 1-chloro-3-vinylbenzene (as described in the previous steps) was dissolved in 6 mL of 1,4-dioxane and 18 mL of water. The solution was cooled to 0 °C, and 584 µl (10.2 mmol, 1 equivalent) of acetic acid was added, followed by 1.99 g (11.2 mmol, 1.1 equivalent) of N-bromosuccinimide. The reaction mixture was stirred at 0 °C for 5 min, and then at room temperature for 2 h. The mixture was then cooled to 0 °C again, and an aqueous solution of NaOH 2N (35.7 mmol, 3.5 equivalent) was slowly added. The solution was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure, and the resulting aqueous phase was extracted three times with DCM. The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a silica gel column and a DCM / hexane mixture as eluent (2 / 98). 3.85 g of the title compound was obtained.

[0221] Yield: 85%.

[0222] MH+: Non-ionizable.

[0223] Step 3: 1-(3-chlorophenyl)-2-(dimethylamino)ethyl-1-ol

[0224]

[0225] To a solution of 4.15 g (26.9 mmol) 2-(3-chlorophenyl)ethylene oxide (as described in the previous steps) in 14 mL of EtOH (96%), 7.38 mL (14.76 mmol, 2 equivalents) of dimethylamine (2 M, in THF) was added. The resulting clear solution was heated under microwave irradiation at 80 °C for 30 min. The reaction mixture was then concentrated under vacuum and diluted with water. The solution was extracted three times with DCM. The combined organic layers were dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 4.06 g of the title compound was obtained.

[0226] Yield: 76%.

[0227] MH+: 200.2; 202.3 (M; M+2).

[0228] Step 4: 2-Chloro-2-(3-Chlorophenyl)-N,N-Dimethylethyl-1-amine

[0229]

[0230] 4.06 g (20.3 mmol) of 1-(3-chlorophenyl)-2-(dimethylamino)ethyl-1-ol (as described in the previous steps) was dissolved in 15 mL of DCM and placed at 0 °C. 2.1 mL (15.1 mmol, 3 equivalents) of triethylamine was added, followed by 0.781 mL (10.1 mmol, 2 equivalents) of methanesulfonyl chloride. The reaction was stirred at 0 °C for 2 h under argon. Water was then added, and the mixture was decanted. The aqueous layer was extracted twice with DCM. The combined organic layers were dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude compound was used directly in the next step without further purification. 4.41 g of the title compound was obtained.

[0231] Yield: 99%.

[0232] MH+: 218.4; 220.4 (M; M+2).

[0233] Step 5: 4-Bromo-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)pyridine-2(1H)-one

[0234]

[0235] At 0 °C, a solution of 4.41 g (20.3 mmol) of 2-chloro-2-(1H)-one and 1.39 g (4.28 mmol, 1 equivalent) of cesium carbonate in 10 mL of anhydrous DMF was added. The solution was then stirred at room temperature for 2 h. EtOAc was added, and the mixture was washed four times with water and once with brine. The organic layer was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a deactivated silica gel column and a hexane / EtOAc mixture as eluent. 5.02 g of the title compound was obtained.

[0236] Yield: 70%.

[0237] MH+: 355.2; 357.2 (M; M+2).

[0238] Step 6: 1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one

[0239]

[0240] Under argon atmosphere, 2 g (5.6 mmol) of 4-bromo-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)pyridin-2(1H)-one (as described in the previous step) and 2.45 g (5.01 mmol, 1.3 equivalents) of 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborphane-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-5-yl)morpholine (as described in step 3) were dissolved in 13 ml of MeCN. Then, 13 ml of 2M Na2CO3 solution was added to obtain a two-phase mixture, which was purged with argon for 15 min. 135 mg (0.19 mmol, 0.05 equivalents) of bis(triphenylphosphine)palladium dichloride was added, and the solution was purged with argon for an additional 15 min. The reaction was stirred at 70 °C for 2 h under argon atmosphere. The reaction mixture was then diluted with water and EtOAc, followed by decantation. The aqueous layer was extracted twice with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 2.68 g of the title compound was obtained.

[0241] Yield: 75%.

[0242] MH+: 632.8; 634.8 (M; M+2).

[0243] Step 7: 1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one

[0244]

[0245] Under argon atmosphere, 2.68 g (4.2 mmol) of 1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one (as described in the previous step) was dissolved in 15 mL of anhydrous THF. Then, 10 mL (10 mmol, 3 equivalents) of TBAF solution (1 M, in THF) was added, and the reaction was stirred at 66 °C for 1 h under argon atmosphere. The solvent was removed under reduced pressure, and 100 mL of saturated NaHCO3 solution was added. The mixture was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 617 mg of the racemic mixture was obtained.

[0246] Yield: 30%.

[0247] MH+: 478.5; 480.6 (M; M+2).

[0248] 1H NMR (DMSO-d6, 400 MHz): δ 12.06 (br s, 1H); 8.17 (d, J=2.4Hz, 1H); 8.10 (d, J=2.3Hz, 1H); 7.76 (d, J=8.0Hz, 1H); 7.70 (d, J=2.4Hz, 1H); 7.47 (s,1H); 7.44-7.32 (m, 3H); 6.72-6.65 (m, 2H); 6.23-6.13 (m, 1H); 3.84-3.73 (m,4H); 3.34-3.23 (m, 1H); 3.20-3.08 (m, 4H); 2.78-2.67 (m, 1H); 2.21 (s, 6H).

[0249] Step 8: (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one

[0250]

[0251] The enantiomers obtained in the preceding steps were separated by rapid chromatography using a Chiralflash IG column and a hexane / EtOH / DCM / 0.1% TEA mixture as the mobile phase. The first eluted fraction was the (-)(R)-enantiomer, followed by the (+)(S)-enantiomer, with ee > 98%. Starting with 617 mg of the racemic mixture, 227 mg of the title compound was obtained.

[0252] MH+: 478.5; 480.6 (M; M+2).

[0253] 1H NMR (DMSO-d6, 400 MHz): δ 12.06 (br s, 1H); 8.17 (d, J=2.4Hz, 1H); 8.10 (d, J=2.3Hz, 1H); 7.76 (d, J=8.0Hz, 1H); 7.70 (d, J=2.4Hz, 1H); 7.47 (s,1H); 7.44-7.32 (m, 3H); 6.72-6.65 (m, 2H); 6.23-6.13 (m, 1H); 3.84-3.73 (m,4H); 3.34-3.23 (m, 1H); 3.20-3.08 (m, 4H); 2.78-2.67 (m, 1H); 2.21 (s, 6H).

[0254] Example 2: Synthesis of the comparative compound (S)-1-(2-amino-1-(3-chlorophenyl)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one (compound A)

[0255]

[0256] Step 1: 2-(3-chlorophenyl)-2-((trimethoxy)oxy)acetonitrile

[0257]

[0258] Under argon atmosphere, 399 mg (36 mmol, 1 equivalent) of DABCO was added to 5 g (36 mmol, 1 equivalent) of 3-chlorobenzaldehyde in 50 mL of anhydrous DCM, followed by 4.45 mL (36 mmol, 1 equivalent) of trimethylsilyl cyanide. The resulting mixture was stirred at 40 °C for 2 h. The reaction mixture was then diluted with DCM, washed twice with water, and once with brine. The organic layer was dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude compound was used directly in the next step without further purification. 7.80 g of the title compound was obtained.

[0259] Yield: 91%.

[0260] MH+: Non-ionizable.

[0261] Step 2: 2-Amino-1-(3-chlorophenyl)ethyl-1-ol

[0262]

[0263] 1.85 g (49 mmol, 1.5 equivalent) of LiAlH4 was added fractionally to a solution of 7.80 g (33 mmol, 1 equivalent) of 2-(3-chlorophenyl)-2-((trimethoxy)oxy)acetonitrile (as described in the previous step) in 80 mL of anhydrous Et2O, which was placed in an ice / water bath at 0 °C. The resulting mixture was stirred at 0 °C for 1 h. Then, ice was slowly added to the reaction at 0 °C until no more gas was formed, and finally 100 mL of water was added. The mixture was stirred at room temperature for 30 min, and the precipitate was filtered through diatomaceous earth and washed twice with Et2O. The filtrate was decanted, and the aqueous layer was extracted twice with Et2O. The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude compound was used directly in the next step without further purification. 5.95 g of the title compound was obtained.

[0264] Yield: Quantitative.

[0265] MH+: 172.3; 174.3 (M; M+2).

[0266] Step 3: (2-(3-chlorophenyl)-2-hydroxyethyl) tert-butyl carbamate

[0267]

[0268] Add 8.32 g (38 mmol, 1.1 equivalent) of di-tert-butyl dicarbonate to a solution of 5.95 g (35 mmol, 1 equivalent) of 2-amino-1-(3-chlorophenyl)ethyl-1-ol (as described in the previous steps) in 60 mL of THF, and stir the resulting mixture at room temperature for 1 h. Then remove the solvent under reduced pressure, and dilute the mixture with 100 mL of EtOAc. Wash the organic layer twice with water and once with brine, dry with Na₂SO₄, filter, and evaporate under reduced pressure. Purify the crude mixture by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 6.78 g of the title compound was obtained.

[0269] Yield: 72%.

[0270] MH+: 272.6; 274.7 (M; M+2).

[0271] Step 4: 2-((tert-butoxycarbonyl)amino)-1-(3-chlorophenyl)ethyl methanesulfonate

[0272]

[0273] 6.78 g (25 mmol) of (2-(3-chlorophenyl)-2-hydroxyethyl)carbamate tert-butyl ester (as described in the previous step) was dissolved in 15 ml of DCM and placed at 0 °C. 2.1 ml (15.1 mmol, 3 equivalents) of triethylamine was added, followed by 0.781 ml (10.1 mmol, 2 equivalents) of methanesulfonyl chloride. The reaction was stirred at 0 °C for 2 h under argon. Water was then added, and the mixture was decanted. The aqueous layer was extracted twice with DCM. The combined organic layers were dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude compound was used directly in the next step without further purification. 9.28 g of the title compound was obtained.

[0274] Yield: Quantitative.

[0275] MH+ (dimer): 700.2; 702.2 (M; M+2).

[0276] Step 5: (2-(4-bromo-2-oxopyridin-1(2H)-yl)-2-(3-chlorophenyl)ethyl)tert-butyl carbamate

[0277]

[0278] At 0 °C, a solution of 8.73 g (25 mmol) of 2-((tert-butoxycarbonyl)amino)-1-(3-chlorophenyl)ethyl methanesulfonate (as described in the previous step) in 5 mL of anhydrous DMF was added to a mixture of 0.744 g (4.28 mmol, 1 equivalent) of 4-bromopyridin-2-(1H)-one and 1.39 g (4.28 mmol, 1 equivalent) of cesium carbonate in 10 mL of anhydrous DMF. The solution was then stirred at room temperature for 2 h. EtOAc was added, and the mixture was washed four times with water and once with brine. The organic layer was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a deactivated silica gel column and a hexane / EtOAc mixture as eluent. 5.56 g of the title compound was obtained.

[0279] Yield: 52%.

[0280] MH+: 427.7; 429.7; 431.7(M; M+2; M+4).

[0281] Step 6: 4-(1H-pyrrolo[2,3-b]pyridin-5-yl)morpholine

[0282]

[0283] 947 mg (2.03 mmol, 0.01 equivalent) of RuPhos and 1.58 g (2.03 mmol, 0.01 equivalent) of RuPhosPd G2 were dissolved in 487 mL of LiHMDS (1 M, THF, 487 mmol, 2.4 equivalent). Then, 40 g (203 mmol, 1 equivalent) of 5-bromo-1H-pyrrolo[2,3-b]pyridine and 21.1 mL (244 mmol, 1.2 equivalent) of morpholine were added under argon atmosphere, and the solution was heated at 66 °C for 1 h30. The reaction mixture was then cooled to room temperature and added dropwise to 1.2 L of saturated NH4Cl solution, maintaining the temperature below 10 °C in an ice-water bath. The mixture was stirred at this temperature for 10 min and then decanted. The aqueous layer was extracted three times with DCM. The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure to give 44.4 g of a brown solid. The crude product was ground in a mixture of 200 ml EtOAc and hexane (3 / 7) for 1 h. The solid was filtered, washed with a mixture of 200 ml EtOAc and hexane (1 / 9), and dried under vacuum to give 38.98 g of light brown powder.

[0284] Yield: 94%.

[0285] MH+: 204.3 (M+1).

[0286] Step 7: 4-(1-Toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-5-yl)morpholine

[0287]

[0288] Under argon atmosphere, 38.98 g (192 mmol, 1 equivalent) of 4-(1H-pyrrolo[2,3-b]pyridin-5-yl)morpholine (as described in the previous step) was dissolved in 390 mL of anhydrous DMF. The solution was cooled to 0 °C, and 11.5 g (288 mmol, 1.5 equivalent) of sodium hydride (60% paraffin oil solution) was slowly added. The mixture was stirred at this temperature for 10 min, and then stirred at room temperature for 40 min. The mixture was cooled to 0 °C again, and 47.5 g (249 mmol, 1.3 equivalent) of toluenesulfonyl chloride was slowly added under argon atmosphere, and the reaction mixture was stirred at 0 °C for 1 h, and then stirred at room temperature for 1 h. The mixture was added dropwise to 800 g of ice / water and stirred for 1 h. A precipitate was obtained, which was filtered and washed several times with cold water. The precipitate was then dissolved in 1.2 L of DCM, and the solution was washed twice with saturated NaHCO3 solution, twice with water, and once with brine. The organic layer was dried with Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was ground in 500 ml of a mixture of EtOAc and hexane (5 / 95) for 3 h. The solid was filtered, washed with hexane, and dried under vacuum to give 62.56 g of a grayish-white solid.

[0289] Yield: 91%.

[0290] MH+: 358.6 (M+1).

[0291] 1H NMR (DMSO-d6, 400 MHz): δ 8.18 (d, J=2.7Hz, 1H); 7.92 (d, J=8.3Hz, 2H); 7.78 (d, J=4.0Hz, 1H); 7.52 (d, J=2.7Hz, 1H); 7.39 (d, J=8.3Hz, 2H); 6.68 (d, J=4.0Hz, 1H); 3.76-3.71 (m, 4H); 3.12-3.07 (m, 4H); 2.33 (s, 3H).

[0292] Step 8: 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-5-yl)morpholine

[0293]

[0294] Under argon atmosphere, 62.56 g (175 mmol, 1 equivalent) of 4-(1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-5-yl)morpholine (as described in the previous step) was suspended in 512 mL of Me-THF. Then, 48.9 g of bis(pinacolyl)diboron (193 mmol, 1.1 equivalent), 1.88 g of 4,4'-di-tert-butylbiphenyl (7 mmol, 0.036 equivalent), and 2.32 g of (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (3.5 mmol, 0.018 equivalent) were added. The reaction mixture was heated under argon atmosphere and refluxed for 45 min. The reaction mixture was then cooled to -10 °C in an ice / acetone bath and carefully quenched with MeOH (350 mL). The solution was stirred at room temperature for 15 min and evaporated under vacuum to give a brown oil. The dark oily substance was then dissolved in 1 L of DCM, washed three times with water and once with brine. The organic layer was evaporated under reduced pressure to obtain a black paste. 2 L of Et2O was added, and the mixture was stirred at room temperature for 15 min. The mixture was filtered through diatomaceous earth and evaporated under reduced pressure to obtain 95 g of brown solid foam. The crude mixture was finally purified by rapid chromatography using a silica gel column and an EtOAc / hexane mixture as eluent, yielding 75.5 g of the title compound.

[0295] Yield: 89%.

[0296] MH+: 484.6 (M+1).

[0297] 1H NMR (DMSO-d6, 400 MHz): δ 8.21 (d, J=2.5Hz, 1H); 8.01 (d, J=8.3Hz, 2H); 7.94 (s, 1H); 7.50 (d, J=2.4Hz, 1H); 7.41 (d, J=8.0Hz, 2H); 3.78-3.72(m, 4H); 3.12-3.07 (m, 4H); 2.33 (s, 3H); 1.30 (s, 12H).

[0298] Step 9: (2-(3-chlorophenyl)-2-(4-(5-morpholino-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxopyridin-1(2H)-yl)ethyl)tert-butyl carbamate

[0299]

[0300] Under argon atmosphere, 2.8 g (6.50 mmol, 1 equivalent) of tert-butyl 2-(4-bromo-2-oxopyridin-1(2H)-yl)-2-(3-chlorophenyl)ethyl)carbamate (as described in step 5), 3.16 g (6.50 mmol, 1 equivalent) of 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-5-yl)morpholine (as described in the previous step), and 905 mg (6.50 mmol, 1 equivalent) of K2CO3 were placed in 140 ml MeCN. The mixture was purged with argon for 15 min, and 459 mg (0.65 mmol, 0.1 equivalent) of bis(triphenylphosphine)palladium dichloride was added. The mixture was purged for an additional 15 min, and then the reaction was stirred at 80 °C for 1 h under argon. The reaction mixture was diluted with 140 mL of EtOAc and washed three times with water. The organic layer was dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 4.79 g of the title compound was obtained.

[0301] Yield: Quantitative.

[0302] Step 10: (2-(3-chlorophenyl)-2-(4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxopyridin-1(2H)-yl)ethyl)tert-butyl carbamate

[0303]

[0304] 1.0 g (1.4 mmol) of tert-butyl 2-(2-(3-chlorophenyl)-2-(4-(5-morpholino-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxopyridin-1(2H)-yl)ethyl)carbamate (as described in the previous steps) was suspended in 5 ml of MeCN and 2.5 ml of 2M Na₂CO₃ solution. The mixture was stirred at 120 °C for 1 h in a sealed tube under microwave irradiation (150 W). The mixture was cooled to room temperature, diluted with EtOAc, and washed three times with water. The organic layer was dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was finally purified by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 644 mg of the racemic mixture was obtained.

[0305] Yield: 82%.

[0306] MH+: 550.6; 552.6 (M; M+2).

[0307] Step 11: 1-(2-amino-1-(3-chlorophenyl)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one

[0308]

[0309] To a solution of 644 mg (1.17 mmol, 1 equivalent) of tert-butyl 2-(2-(3-chlorophenyl)-2-(4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxopyridin-1(2H)-yl)ethyl)carbamate (as described in the previous step) in 6 mL of DCM, placed in an ice / water bath at 0 °C, 3 mL of trifluoroacetic acid was added. The solution was stirred at 0 °C for 1 h 30, then the solvent was removed under reduced pressure, and the mixture was diluted with 100 mL of saturated NaHCO3 solution. The solution was extracted three times with DCM. The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a C18 column and a water / MeOH mixture as eluent. 340 mg of the title compound was obtained.

[0310] Yield: 64%.

[0311] MH+: 450.7; 452.7 (M; M+2).

[0312] 1H NMR (DMSO-d6, 400 MHz): δ 12.06 (br s, 1H); 8.17 (d, J=2.5Hz, 1H); 8.09 (s, 1H); 7.75 (d, J=8.1Hz, 1H); 7.70 (d, J=2.6Hz, 1H); 7.44-7.29 (m,4H); 6.74-6.67 (m, 2H); 5.90 (t, J=7.5Hz, 1H); 3.84-3.72 (m, 4H); 3.34-3.25(m, 2H); 3.18-3.09 (m, 4H); 1.60 (br s, 2H).

[0313] Step 12: (S)-1-(2-amino-1-(3-chlorophenyl)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one

[0314]

[0315] The enantiomers obtained in the preceding steps were separated by rapid chromatography using a Chiralflash IG column and a hexane / EtOH / DCM / 0.1% TEA mixture as the mobile phase. The first eluted fraction was the (-)(R)-enantiomer, followed by the (+)(S)-enantiomer, with ee > 98%. Starting with 120 mg of the racemic mixture, 33 mg of the title compound was obtained.

[0316] MH+: 450.7; 452.7 (M; M+2).

[0317] 1H NMR (DMSO-d6, 400 MHz): δ 12.06 (br s, 1H); 8.17 (d, J=2.5Hz, 1H); 8.09 (s, 1H); 7.75 (d, J=8.1Hz, 1H); 7.70 (d, J=2.6Hz, 1H); 7.44-7.29 (m,4H); 6.74-6.67 (m, 2H); 5.90 (t, J=7.5Hz, 1H); 3.84-3.72 (m, 4H); 3.34-3.25(m, 2H); 3.18-3.09 (m, 4H); 1.60 (br s, 2H).

[0318] Example 3: Synthesis of the comparative compound (S)-1-(1-(3,4-dichlorophenyl)-2-(methylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one (compound B).

[0319]

[0320] Step 1: 2-(3,4-Dichlorophenyl)-2-((trimethoxy)oxy)acetonitrile

[0321]

[0322] Under argon atmosphere, 399 mg (36 mmol, 1 equivalent) of DABCO was added to 2.5 g (14.3 mmol) of 3,4-dichlorobenzaldehyde in 50 mL of anhydrous DCM, followed by 4.45 mL (36 mmol, 1 equivalent) of trimethylsilyl cyanide. The resulting mixture was stirred at 40 °C for 2 h. The reaction mixture was then diluted with DCM, washed twice with water and once with brine. The organic layer was dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude compound was used directly in the next step without further purification. 3.70 g of the title compound was obtained.

[0323] Yield: 94%.

[0324] MH+: Non-ionizable.

[0325] Step 2: 2-Amino-1-(3,4-dichlorophenyl)ethyl-1-ol

[0326]

[0327] 1.85 g (49 mmol, 1.5 equivalent) of LiAlH4 was added fractionally to a solution of 3.70 g (13.5 mmol) of 2-(3,4-dichlorophenyl)-2-((trimethylsilyl)oxy)acetonitrile (as described in the previous step) in 80 mL of anhydrous Et2O, which was placed in an ice / water bath at 0 °C. The resulting mixture was stirred at 0 °C for 1 h. Then, ice was slowly added to the reaction at 0 °C until no more gas was formed, and finally 100 mL of water was added. The mixture was stirred at room temperature for 30 min, and the precipitate was filtered through diatomaceous earth and washed twice with Et2O. The filtrate was decanted, and the aqueous layer was extracted twice with Et2O. The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude compound was used directly in the next step without further purification. 1.66 g of the title compound was obtained.

[0328] Yield: 60%.

[0329] MH+: 206.1; 208.2; 210.1 (M; M+2; M+4).

[0330] Step 3: (2-(3,4-dichlorophenyl)-2-hydroxyethyl) tert-butyl carbamate

[0331]

[0332] Add 8.32 g (38 mmol, 1.1 equivalent) of di-tert-butyl dicarbonate to a solution of 1.66 g (8.03 mmol) of 2-amino-1-(3,4-dichlorophenyl)ethyl-1-ol (as described in the previous steps) in 60 mL of THF, and stir the resulting mixture at room temperature for 1 h. Then remove the solvent under reduced pressure, and dilute the mixture with 100 mL of EtOAc. Wash the organic layer twice with water and once with brine, dry with Na₂SO₄, filter, and evaporate under reduced pressure. Purify the crude mixture by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 2.55 g of the title compound was obtained.

[0333] Yield: Quantitative.

[0334] MH+: 306.3; 308.3 (M; M+2).

[0335] Step 4: 2-((tert-butoxycarbonyl)amino)-1-(3,4-dichlorophenyl)ethyl methanesulfonate

[0336]

[0337] 2.46 g (8.03 mmol) of (2-(3,4-dichlorophenyl)-2-hydroxyethyl) tert-butyl carbamate (as described in the previous step) was dissolved in 15 ml of DCM and placed at 0 °C. 2.1 ml (15.1 mmol, 3 equivalents) of triethylamine was added, followed by 0.781 ml (10.1 mmol, 2 equivalents) of methanesulfonyl chloride. The reaction was stirred at 0 °C for 2 h under argon. Water was then added, and the mixture was decanted. The aqueous layer was extracted twice with DCM. The combined organic layers were dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude compound was used directly in the next step without further purification. 3.23 g of the title compound was obtained.

[0338] Yield: Quantitative.

[0339] MH+ (dimer): 767.5; 769.4; 771.6 (M; M+2; M+4).

[0340] Step 5: (2-(4-bromo-2-oxopyridin-1(2H)-yl)-2-(3,4-dichlorophenyl)ethyl)tert-butyl carbamate

[0341]

[0342] At 0 °C, a solution of 3.09 g (8.03 mmol) of 2-((tert-butoxycarbonyl)amino)-1-(3,4-dichlorophenyl)ethyl methanesulfonate (as described in the previous step) in 5 mL of anhydrous DMF was added to a mixture of 0.744 g (4.28 mmol, 1 equivalent) of 4-bromopyridin-2-(1H)-one and 1.39 g (4.28 mmol, 1 equivalent) of cesium carbonate in 10 mL of anhydrous DMF. The solution was then stirred at room temperature for 2 h. EtOAc was added, and the mixture was washed four times with water and once with brine. The organic layer was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a deactivated silica gel column and a hexane / EtOAc mixture as eluent. 1.74 g of the title compound was obtained.

[0343] Yield: 47%.

[0344] MH+: 461.3; 463.3; 465.2 (M; M+2; M+4).

[0345] Step 6: (2-(4-bromo-2-oxopyridin-1(2H)-yl)-2-(3,4-dichlorophenyl)ethyl)(methyl)carbamate tert-butyl ester

[0346]

[0347] Under argon atmosphere, 87 mg (2.18 mmol, 1 equivalent) of sodium hydride (60% paraffin oil solution) was added to a solution of 840 mg (1.82 mmol, 1 equivalent) of 2-(4-bromo-2-oxopyridin-1(2H)-yl)-2-(3,4-dichlorophenyl)ethyl)carbamate (as described in the previous step) in 9 mL of anhydrous DMF. Then, 170 µl (2.73 mmol, 1.5 equivalent) of iodomethane was added. The solution was stirred at 0 °C for 1 h30, and then diluted with 100 mL of EtOAc. The solution was washed four times with water and once with brine. The organic layer was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a silica gel column and an EtOAc / hexane mixture as eluent. 750 mg of the title compound was obtained.

[0348] Yield: 86%.

[0349] MH+: 475.4; 477.3; 479.3 (M; M+2; M+4).

[0350] Step 7: (2-(3,4-dichlorophenyl)-2-(4-(5-morpholino-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxopyridin-1(2H)-yl)ethyl)(methyl)carbamate tert-butyl

[0351]

[0352] Under argon atmosphere, 750 mg (1.57 mmol) of (2-(4-bromo-2-oxopyridin-1(2H)-yl)-2-(3,4-dichlorophenyl)ethyl)(methyl)carbamate tert-butyl ester (as described in the previous step) and 2.45 g (5.01 mmol, 1.3 equivalents) of 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborhecyclopentan-2-yl)-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-5-yl)morpholine (as described in step 3) were dissolved in 13 ml of MeCN. Then, 13 ml of 2M Na2CO3 solution was added to obtain a two-phase mixture, which was purged with argon for 15 min. 135 mg (0.19 mmol, 0.05 equivalents) of bis(triphenylphosphine)palladium dichloride was added, and the solution was purged with argon for an additional 15 min. The reaction mixture was stirred at 70 °C for 2 h under argon atmosphere. The reaction mixture was then diluted with water and EtOAc, followed by decantation. The aqueous layer was extracted twice with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude mixture was purified by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 1.27 g of the title compound was obtained.

[0353] Yield: Quantitative.

[0354] MH+: 753.4; 755.2 (M; M+2).

[0355] Step 8: (2-(3,4-dichlorophenyl)-2-(4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxopyridin-1(2H)-yl)ethyl)(methyl)carbamate tert-butyl

[0356]

[0357] 1.27 g (1.69 mmol) of tert-butyl (2-(3,4-dichlorophenyl)-2-(4-(5-morpholino-1-toluenesulfonyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxopyridin-1(2H)-yl)ethyl)(methyl)carbamate (as described in the previous steps) was suspended in 5 mL of MeCN and 2.5 mL of 2M Na₂CO₃ solution. The mixture was stirred at 120 °C for 1 h in a sealed tube under microwave irradiation (150 W). The mixture was cooled to room temperature, diluted with EtOAc, and washed three times with water. The organic layer was dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The crude product was finally purified by rapid chromatography using a silica gel column and a DCM / MeOH mixture as eluent. 730 mg of the racemic mixture was obtained.

[0358] Yield: 72%.

[0359] MH+: 598.7; 600.5 (M; M+2).

[0360] Step 9: 1-(1-(3,4-dichlorophenyl)-2-(methylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one

[0361]

[0362] Add 3 ml of trifluoroacetic acid to a solution of 730 mg (1.20 mmol) of (2-(3,4-dichlorophenyl)-2-(4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)-2-oxopyridin-1(2H)-yl)ethyl)(methyl)carbamate (as described in the previous step) in 6 ml of DCM. Stir the solution at 0 °C for 1 h 30, then remove the solvent under reduced pressure. Dilute the mixture with 100 ml of saturated NaHCO3 solution. Extract the solution three times with DCM. Dry the combined organic layers with Na2SO4, filter, and evaporate under reduced pressure. Purify the crude mixture by rapid chromatography using a C18 column and a water / MeOH mixture as eluent. 543 mg of the racemic mixture is obtained.

[0363] Yield: 89%.

[0364] MH+: 498.6; 500.5 (M; M+2).

[0365] 1H NMR (DMSO-d6, 400 MHz): δ 12.08 (br s, 1H); 8.17 (d, J=2.5Hz, 1H); 8.10 (d, J=2.6Hz, 1H); 7.76 (d, J=7.3Hz, 1H); 7.69 (d, J=2.5Hz, 1H); 7.66-7.61 (m, 2H); 7.37-7.30 (m, 1H); 6.74-6.66 (m, 2H); 6.08-5.98 (m, 1H); 3.84-3.73 (m, 4H); 3.34-3.24 (m, 1H); 3.22-3.14 (m, 1H); 3.14-3.09 (m, 4H); 2.30(s, 3H); 1.94 (br s, 1H).

[0366] Step 10: (S)-1-(1-(3,4-dichlorophenyl)-2-(methylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one

[0367]

[0368] The enantiomers obtained in the preceding steps were separated by rapid chromatography using a Chiralflash IG column and a hexane / EtOH / DCM / 0.1% TEA mixture as the mobile phase. The first eluted fraction was the (-)(R)-enantiomer, followed by the (+)(S)-enantiomer, with ee > 98%. Starting with 150 mg of the racemic mixture, 54 mg of the title compound was obtained.

[0369] MH+: 498.6; 500.5 (M; M+2).

[0370] 1H NMR (DMSO-d6, 400 MHz): δ 12.08 (br s, 1H); 8.17 (d, J=2.5Hz, 1H); 8.10 (d, J=2.6Hz, 1H); 7.76 (d, J=7.3Hz, 1H); 7.69 (d, J=2.5Hz, 1H); 7.66-7.61 (m, 2H); 7.37-7.30 (m, 1H); 6.74-6.66 (m, 2H); 6.08-5.98 (m, 1H); 3.84-3.73 (m, 4H); 3.34-3.24 (m, 1H); 3.22-3.14 (m, 1H); 3.14-3.09 (m, 4H); 2.30(s, 3H); 1.94 (br s, 1H).

[0371] Example 4: ERK2 (MAPK1) enzyme assay

[0372] To evaluate the ability of compound N°1 according to Example 1 to inhibit ERK2 enzyme activity, the Z'-Lyte biochemical assay of Life Technologies was used according to the manufacturer's instructions. Briefly, a black 384-well plate containing 100 nl of 100X compound solution in 100% DMSO, 2.4 µl of kinase buffer, 5 µl of 2X MAPK1 (ERK2) / Ser / Thr O3 mixture, and 2.5 µl of 4X ATP solution was used. The plate was shaken for 30 seconds and incubated at room temperature for 60 minutes. Then, 5 µl of 1:1024 diluted developing reagent A was added. The plate was shaken for 30 seconds and incubated at room temperature for 60 minutes. Fluorescence was read using a plate reader. In this assay, ERK2 enzyme at a concentration of 0.4 µg / ml (5.74 nM) was used at ATP Km (100 µM). The kinase buffer consisted of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA. The IC50 of the compound was determined using a 3-fold serial dilution. 50 (Repeat the 10-point titration twice).

[0373] ERK2 inhibitory activity (IC50) of compound N°1 in Example 1 50 The concentration is 2.1 nM. Therefore, it exhibits the ability to inhibit ERK2 enzyme activity.

[0374] Example 5: Cell line proliferation assay

[0375] The ability of compound N°1 to inhibit cell proliferation according to Example 1 was determined using cell line assays. A375 cells (malignant melanoma) were grown to approximately 80% confluence and seeded at 3000 cells / 100 µl / well in 96-well flat-bottomed plates of DMEM containing 10% FBS. Cells were incubated at 37°C for 24 h in 5% CO2. 100 µl of the compound solution was added to the cells, and the cells were incubated at 37°C for 72 h. The total volume of the medium was 200 µl / well. The compound was screened in 0.15% DMSO (final), with two replicates using 10 titration points. The negative control wells consisted only of solvent (0.15% DMSO in 10% FBS DMEM). After 72 hours of compound treatment, 1% SDS (final) was added to the positive control wells at 37°C for 15 min. Then, the culture medium was discarded and replaced with 100 µl / well of MTT solution (3-[4,5-dimethylthiazolyl-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma, Cat#M5655) at a concentration of 0.5 mg / ml in 10% FBS DMEM. Cells were incubated at 37°C for 4 hours. The MTT reaction was stopped and homogenized by adding 100 µl / well of SDS in 10% 0.01M HCl. After 16 hours at 37°C, absorbance was measured at 570 nm using a Bio-Tek microplate reader (PowerWave HT). The percentage of proliferation inhibition was calculated using a negative control (0.15% DMSO) as 0% growth inhibition and a positive control (1% SDS) as 100% growth inhibition. IC50 50 The concentration (inducing half-maximal growth inhibition) was determined by nonlinear regression analysis of the inhibition curves generated from the average replicates (using a sigmoid dose-response curve with a variable Hill slope, with the top capped at a constant value of 100 and the bottom capped at values ​​from 0 to 50). Analysis was performed using GraphPad Prism software.

[0376] Based on the cell proliferation inhibitory activity of compound N°1 in Example 1 (“MTT A375 IC…”), 50 The concentration was 35 nM. Therefore, it exhibited the ability to inhibit the proliferation of A375 cells.

[0377] Example 6: hERG channel inhibition assay

[0378] The inhibition of hERG channels by compound N°1 according to Example 1 was evaluated. The compound was used as a 10 mM stock solution in DMSO, then diluted to 30 µM with HEPES-buffered saline. A 6-point concentration-response curve was generated using a 3.16-fold serial dilution of the highest tested concentration. Electrophysiological recordings were performed in a Chinese hamster ovary cell line stably expressing the full-length ion channel. Single-cell ion currents were measured in a whole-cell configuration using a Patchliner (Nanion Technologies) at room temperature (21–23 °C). The internal solution for hERG contained (mM): 120 KF, 20 KCl, 10 EGTA, 10 HEPES, buffered to pH 7.3. The external solution (HEPES-buffered saline) contained (mM): 138 NaCl, 4.5 KCl, 1.8 CaCl2, 1.0 MgCl2, 10 HEPES, 10 glucose, buffered to pH 7.4. The voltage protocol is shown below. The current was measured starting from a step change and held at that current. The compound was then incubated for 2 minutes to reach a stable state before adding the next concentration.

[0379]

[0380] Based on the hERG channel inhibition performance (“hERG IC”) of compound N°1 in Example 1 50 The value is 10.9 µ. Therefore, it exhibits safe hERG properties through patch-clamp testing.

[0381] Example 7: CYP 3A4 Inhibition Test

[0382] To evaluate the ability of compound N°1 according to Example 1 to inhibit CYP 3A4 enzyme activity, the test compound (0.1 μM-25 μM) was incubated with cryopreserved human hepatocytes for 10 min in the presence of the specific CYP3A4 probe substrate midazolam.

[0383] 1-Hydroxymidazolam was monitored by LC-MS / MS, and the IC50 was calculated using the reduction in metabolite formation compared to the solvent control. 50 value.

[0384] CYP 3A4 inhibitory activity of the compound (“CYP 3A4 IC50”) 50 The following table reports:

[0385]

[0386] Compound N°1 according to Example 1 exhibits low inhibition of CYP 3A4. In fact, its IC50 value is [missing information]. 50The value is greater than 5 µM. In comparison, the IC50 value of compound A is greater than 5 µM. 50 The value is 0.74 µM. Therefore, the control compound A is a highly efficient inhibitor of CYP 3A4.

[0387] Example 8: Kinase Profile

[0388] To evaluate the kinase selectivity of compound n°1(S) according to Example 1, Life Technologies’ Z'-Lyte biochemical assay and Adapta / Lanthascreen binding assay were used according to the manufacturer’s instructions. The inhibition rates of 500 nM test compounds against the following 58 kinases were determined: ABL1, ACVR1B (ALK4), AKT2 (PKB β), AMPK (A1 / B2 / G3), AURKA (Aurora Kinase A), AXL, BRAF, BTK, CAMK2B (CaMKII β), CDK2 / cyclin A, CHEK1 (CHK1), CLK1, CSNK1A1 (CK1 α 1), CSNK2A1 (CK2 α 1), DAPK3 (ZIPK), DYRK1A, EGFR (ErbB1), EPHB3, ERBB2 (HER2), FGFR2, FLT3, FRAP1 (mTOR), GSK3B (GSK3 β), IGF1R, and IKBKB (IKK). β), INSR, IRAK4, JAK2, KDR (VEGFR2), KIT, LCK, MAP2K1 (MEK1), MAPK10 (JNK3), MAPK11 (p38 β), MAPKAPK2, MARK2, MET (cMet), NEK2, NTRK1 (TRKA), PAK2 (PAK65), PDGFRB (PDGFR β), PDK1Direct, PHKG2, PIK3CA / PIK3R1 (p110 α / p85 α), PIM1, PLK1, PRKACA (PKA), PRKCA (PKC α), PTK2 (FAK), RET, ROCK1, RPS6KA1 (RSK1), RPS6KB1 (p70S6K), SRC, STK3 (MST2), SYK, TEK (Tie2), TYRO3 (RSE). Except for 100 mM MAPK10 (JNK3) and BRAF and MAP2K1 (MEK1) as binding assays, ATP concentrations were expressed as apparent Km for all kinases.

[0389] The results are reported in the table below:

[0390]

[0391] Compound n°1 according to Example 1 exhibited a favorable kinase selectivity score in the representative kinase profile. In fact, only one of the 58 kinases was inhibited with an inhibition percentage greater than 80%, and a maximum of 10 kinases were inhibited with an inhibition percentage greater than 50%.

[0392] In contrast, compounds A and B showed poorer selectivity scores. In fact, compounds A and B inhibited 15 / 58 and 10 / 58 kinases with inhibition percentages of over 80%, respectively, and 26 / 58 and 20 / 58 kinases with inhibition percentages of over 50%.

[0393] Example 9: Permeability Test

[0394] The Caco 2 cell line, obtained from ATCC, was used for in vitro transport studies. Cells were isolated every other day at a separation ratio of 1:3 to 1:5 and grown in DuPont modified Eagle medium (GlutaMAX I, 4,500 mg / L D-glucose, sodium pyruvate) supplemented with 10% FBS in the presence of antibiotics. For transport studies, cells were seeded at a density of 60,000 cells / well onto polycarbonate Transwell membranes (Millipore). The medium was changed 24 h after seeding, and the cells were cultured for another 21 days before transport assays were performed. Donor solutions were prepared by diluting a stock solution of the test compound in transport medium (HBSS buffer containing 10 mM HEPES, pH 7.4). The receiving solution was the same HBSS buffer containing 10 mM HEPES, pH 7.4. Transport of the test compound (5 µM) was measured repeatedly in two directions [apex to lateral base (A→B) and lateral base to apex (B→A)].

[0395] The permeability coefficient for membrane transport of the test compound was determined using the following equation: Papp (cm / sec) = (Vr / C0)(1 / S)(dC / dt); Papp = apparent permeability, Vr = volume of culture medium in the receiving chamber, C0 = PAR of the test drug in the receiving chamber, S = surface area of ​​the monolayer, dC / dt = change in PAR of the drug in the receiving chamber over time. The effluent ratio was defined as: effluent ratio = Papp BA / Papp AB. Bioanalysis was performed by LC-MS / MS.

[0396] The results are reported in the table below:

[0397]

[0398] Compound n°1 from Example 1 exhibits excellent Caco-2 permeability parameters. In fact, it has a value higher than 10.10.-6 The Papp AB value is cm / s. Furthermore, its outflow ratio is less than 2.

[0399] In contrast, compounds A and B exhibited poor Caco-2 permeability parameters, with low Papp AB values ​​(below 10.10). -6 (cm / s) and high outflow ratio (above 3).

[0400] Example 10: PK Experiment

[0401] To determine the absolute oral bioavailability of compound n°1 according to Example 1, the compound was suspended in 0.5% CMC carrier and administered to a group of 5-6 week old male BalbC mice. For intravenous injection, a single dose of 1 mg / kg was administered via the tail vein, and blood samples were collected at 0.12, 0.25, 0.5, 1, 2, 4, 8, and 24 h post-injection. For oral administration, a single dose of 20 mg / kg was administered, and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 10, and 24 h post-gavage. Plasma separated from the blood samples was stored at -20°C until analysis. The vials containing the study samples were removed and thawed to room temperature. 50 µl of the sample was added to 200 µl of acetonitrile containing the internal standard (tolbutamide; 25 ng / mL) and vortexed for 5 min, then centrifuged at 14,000 rpm for 5 min at 4°C. 200 µl of supernatant was separated and transferred to an HPLC vial for analysis. For prodrug pharmacokinetics, only the active drug was quantitatively analyzed by HPLC.

[0402] The results are reported in the table below:

[0403]

[0404] Compound n°1 according to Example 1 exhibits favorable PK parameters. In fact, it has an oral bioavailability of over 8.9, which would allow the target molecular concentration to be achieved in plasma.

[0405] Example 11: Preparation of the crystal form of compound N°1 according to Example 1

[0406] 7 g of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one according to Example 1 was suspended in 1.2 L of solvent MeCN / H2O (70 / 30).

[0407] After cooling to room temperature and incubating overnight at 4°C, the sample was filtered to obtain crystals.

[0408] 4.1 g of pure (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one was obtained.

[0409] Repeat the same procedure 6 times with a scale of 7 g to 9 g (total volume of 1.3 L to 1.8 L of solvent).

[0410] After all recrystallizations, 33.4 g of product was obtained and homogenized in Et2O (400 ml) to give 33.4 g of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one with LC purity of 100% and LC chirality of 99.5%.

[0411] Collect all recrystallization filtrate to obtain 26 g of product, which is then recrystallized in three batches to obtain 16.8 g of product.

[0412] The product was ground again in 300 ml Et2O to give 16.5 g (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one with LC purity of 100% and LC chirality of 99.3%.

[0413] Characterization of compounds

[0414] The following results were obtained by characterizing the compound:

[0415] XRPD analysis revealed crystalline material, termed diffraction pattern 1 (see...). Figure 1 ).

[0416] - 1 H NMR analysis also showed that the material was consistent with the structure (see...). Figure 2 ).

[0417] - Thin-layer chromatography: Rf (retention factor) = 0.25 (solvent, 10% MeOH in DCM solution).

[0418] - LC purity: 100%.

[0419] - LC chirality: 99.5%.

[0420] - MH+: 478.7; 480.7 (M; M+2).

[0421] Three-month stability study

[0422] The table below presents the results of the three-month stability study.

[0423] After two weeks of storage under each condition, the purity of the material remained unchanged compared to the input purity (compound N°1: 98.56). Chemical stability also remained unchanged.

[0424] The purity was slightly lower, but within 0.5% of the area, which may be related to variations in the analytical method and sample preparation, as the stability was good after three months compared to T=0. The diffraction pattern showed no change, indicating that the physical and chemical purity was stable after one month.

[0425] At the three-month time point, the purity of the sample remained constant relative to the input material. The diffraction pattern showed no change, indicating that the physical and chemical purity was stable after three months under the studied conditions.

[0426]

[0427] Stability studies over three months showed that the crystal form was highly stable under environmental and accelerated aging conditions for three months. Purity did not decrease while the crystal form was retained, indicating that the (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one crystal form possesses excellent stability.

[0428] Therefore, the three-month stability study of the crystal form showed that the compound has high purity and maintains its crystal form.

Claims

1. A crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one of formula (I): (I)， The crystalline form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one exhibits a powder X-ray diffraction pattern showing at least one peak, particularly at least two peaks, preferably at least five peaks, more preferably at least ten peaks, expressed in degrees 2-θ angles, selected from 3.36; 12.03; 12.49; 12.91; 13.42; 13.93; 14.72; 15.28; 17.18; 17.42; 19.64; 22.10; 22.47; 22.88; 23.08; 24.00; 25.07 and 25.90 (±0.2 each time).

2. The crystalline form according to any one of the preceding claims, wherein the crystalline form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one has a powder X-ray diffraction pattern showing peaks at the following angles in degrees 2-θ: 14.72; 15.28; 17.18; 22.88; 23.08 and 24.00 (±0.2 each).

3. The crystal form according to the preceding claim, wherein the crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one has a powder X-ray diffraction pattern that also shows the following additional peaks in degrees 2-θ: 12.03; 17.42; 22.10; 22.47; 25.07 and 25.90 (±0.2 each).

4. The crystal form according to the preceding claim, wherein the crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one has a powder X-ray diffraction pattern that also shows the following additional peaks in degrees 2-θ: 3.36; 12.49; 12.91; 13.42; 13.93 and 19.64 (±0.2 each).

5. The crystal form according to any one of the preceding claims, having a single endothermic peak with an onset temperature of 281°C (±2°C).

6. A method for preparing a crystal form according to any one of the preceding claims, comprising the following steps: a) Suspending (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one in a solvent or solvent mixture, preferably in MeCN / H2O (70 / 30); b) Optionally, evaporate the solvent at a temperature between 0°C and the boiling point of the solvent or mixture of solvents selected in step a); c) Optionally, a solvent or solvent mixture may be added. d) Apply temperature program; e) Optional filtering; and f) Optionally, the obtained crystals are washed with a solvent or a mixture of solvents. g) Then optionally dry to obtain the desired crystal form of (S)-1-(1-(3-chlorophenyl)-2-(dimethylamino)ethyl)-4-(5-morpholino-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2(1H)-one.

7. The crystal form according to any one of claims 1 to 5, for the prevention and / or inhibition and / or treatment of diseases or conditions mediated by ERK kinase activity, particularly ERK2 kinase activity.

8. The crystal form according to claim 7, wherein the disease or condition is selected from cancer and metastatic tumors.

9. The crystal form according to claim 7 or 8, wherein the disease or condition is selected from glioblastoma, multiple myeloma, cancer, leukemia, especially myeloid leukemia (AML), lymphocytic leukemia, myeloid cell leukemia, myeloid leukemia (CML) or lymphoblastic leukemia, myelodysplastic syndrome, Kaposi's sarcoma, cutaneous angiosarcoma, solid tumor, lymphoma, especially non-Hodgkin's lymphoma, melanoma, especially malignant melanoma, bladder cancer, breast cancer, gastric cancer, colon cancer, colorectal cancer, endometrial cancer, lung cancer, including non-small cell lung cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, head and neck cancer, liver cancer, ovarian cancer, especially serous ovarian cancer, seminoma, cancers of the respiratory tract and chest, thyroid cancer, especially papillary or follicular thyroid cancer, and other tumors expressing ERK.

10. The crystal form according to claim 7, wherein the disease or condition is selected from neoplastic diseases, allergic diseases, inflammatory diseases, autoimmune diseases, malaria-related diseases, mast cell-related diseases, graft-versus-host disease, metabolic syndrome, CNS-related diseases, neurodegenerative diseases, pain conditions, substance abuse diseases, prions, heart disease, fibrotic diseases, idiopathic arterial hypertension (IPAH), and essential pulmonary hypertension (PPH).

11. The crystal form according to claim 7, used for prevention and / or inhibition and / or treatment of human immunodeficiency virus (HIV).

12. Use of the crystal form according to any one of claims 1 or 5 for the prevention and / or inhibition and / or treatment of diseases or conditions mediated by ERK kinase activity, preferably ERK2 kinase activity.

13. A drug comprising at least one crystal form according to any one of claims 1 or 5.

14. A pharmaceutical composition comprising at least one crystal form according to any one of claims 1 to 5, and at least one pharmaceutically acceptable excipient.

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