Salt form of heterocyclic antitumor compound, pharmaceutical composition comprising same, and use thereof

Abstract

The present disclosure provides a salt of a heterocyclic antitumor compound, and a crystal form thereof. In addition, the present disclosure also relates to a use of the salt of the compound and the crystal form thereof in the treatment of tumors or cancers and related diseases and conditions. The salt and the crystal form thereof provided by the present disclosure have considerable clinical application value, and can serve as excellent candidate forms for subsequent drug development.

Description

A salt form of a heterocyclic antitumor compound, a pharmaceutical composition comprising the compound, and its uses.

[0001] Citation of relevant applications

[0002] This disclosure claims priority to Chinese patent application No. 202411833718.9, filed on December 13, 2024, entitled "A Salt Form of a Heterocyclic Antitumor Compound and Its Use Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of medicinal chemistry, and more specifically, to a salt form of compound (R)-1-(4-(4-(1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)amino)-2-methyl-8,9-dihydroimidazo[1',2':1,6]pyrido[2,3-d]pyrimidin-6-yl)-3,6-dihydropyridin-1(2H)-yl)acetone as an SOS1 inhibitor, and pharmaceutical compositions comprising the same. Furthermore, this disclosure also relates to the use of a salt form of compound I and pharmaceutical compositions comprising the same in the treatment of SOS1-related diseases and conditions. Background Technology

[0004] Currently, three genes in the RAS family are known: KRAS (Kirsten rat sarcoma virus oncogene homolog), NRAS (neuroblastoma RAS virus oncogene homolog), and HRAS (Harvey rat sarcoma virus oncogene). RAS family proteins are a class of small GTPases and were the first oncogenes identified in human tumors. RAS family proteins possess weak intrinsic GTPase activity and a slow nucleotide exchange rate. For example, binding to the GTPase activating protein (GAP) of NF1 increases the GTPase activity of RAS family proteins.

[0005] Mutations in RAS enzymes are closely associated with tumorigenesis, and the types of RAS mutations differ across different types of tumors. In human tumors, KRAS mutations (e.g., amino acids G12, G13, Q61, A146) are the most common, accounting for approximately 85%, while NRAS (e.g., amino acids G12, G13, Q61, A146) and HRAS (e.g., amino acids G12, G13, Q61) account for 12% and 3%, respectively. Alterations in RAS family proteins (e.g., mutations, overexpression, gene amplification) have also been described as mechanisms of resistance to cancer drugs such as the EGFR antibodies cetuximab and panitumumab, and the EGFR tyrosine kinase inhibitor osimertinib. In oncogenic RAS mutants, GAP activity is weakened or significantly reduced, leading to permanent activation, which is the basis of oncogenic RAS signaling. Direct inhibition of RAS has proven extremely challenging and difficult to drug because GTP has a picomolar affinity for its binding site, lacks other well-defined pockets, and RAS interacts with GEF, GAP, and effectors through extended and flat protein-protein interactions. Therefore, inhibiting RAS activation by targeting the upstream guanine nucleotide exchange factor protein SOS may offer new hope.

[0006] There are two human isotypes of SOS: SOS1 and SOS2, but most research has focused on SOS1. Human SOS1 consists of 1333 amino acids (15 kDa) and is composed of an N-terminal histone-like domain, a Dbl homology (DH) domain, a pleckstrin homology (PH) domain, a helical linker (HL), a Ras exchanger motif (Rem) domain, and a Cdc25 domain, as well as a C-terminal region. PH, Rem, and Cdc25 are SOS... cat Components of the core catalytic domain.

[0007] Over the past few decades, the interaction between RAS family proteins and SOS1 has gained increasing recognition. Furthermore, recent studies have focused on the rational design and combination of screening platforms to screen and identify small molecule inhibitors of SOS1—compounds that bind to SOS1 and inhibit protein-protein interactions with RAS family proteins. For example, WO2021105960A1 describes several cyclic SOS1 inhibitors.

[0008] Although some small molecules of SOS1 inhibitors have been disclosed, no SOS1 inhibitors have been developed and marketed yet. Therefore, it is still urgent to develop new compounds with market potential, better efficacy, and better pharmacokinetic results. Summary of the Invention

[0009] Technical problems faced

[0010] Chinese patent application 202211629162.2 (filed on December 16, 2022) and international patent application PCT / CN2022 / 139447 (filed on December 16, 2022) disclose compounds as SOS1 inhibitors and their preparation methods, including (R)-1-(4-(4-(1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)amino)-2-methyl-8,9-dihydroimidazo[1',2':1,6]pyrido[2,3-d]pyrimidin-6-yl)-3,6-dihydropyridin-1(2H)-yl) ethyl ketone (hereinafter referred to as compound I or compound of formula I, the structure of which is shown below) and its preparation method.

[0011] Compound I is an effective SOS1 inhibitor. In vitro enzymatic inhibition assays have shown that compound I has a strong inhibitory effect on SOS1. Moreover, compound I also shows significant inhibitory activity on the proliferation of NCI-H358 cells. Therefore, it may be a promising compound for the prevention and / or treatment of SOS1-mediated diseases.

[0012] Currently, there are no reports on the crystal form and salt form of compound I. Therefore, it is necessary to further screen the crystal form and salt form of compound I to develop a pharmaceutical form suitable for large-scale production, providing more and better options for subsequent drug development.

[0013] Technical solutions to the problem

[0014] On the one hand, this disclosure provides a salt of compound I.

[0015] In some embodiments of this disclosure, the salt of compound I is an acid addition salt formed by compound I and an inorganic acid (including but not limited to hydrochloric acid, sulfuric acid, phosphoric acid, etc.).

[0016] In some embodiments of this disclosure, the salt of compound I is an acid addition salt formed by compound I and an organic acid (including but not limited to maleic acid, fumaric acid, citric acid, etc.).

[0017] In some embodiments of this disclosure, the salt of compound I is compound I fumarate.

[0018] In some specific embodiments of this disclosure, the salt formation ratio of compound I in the fumarate of compound I to fumaric acid is 1:1.5; preferably, the salt formation ratio is a molar ratio.

[0019] On the other hand, this disclosure also provides a compound I fumarate (hereinafter referred to as compound I fumarate crystal) existing in crystalline form.

[0020] In some embodiments of this disclosure, the compound I fumarate crystal has crystal form I.

[0021] In some specific embodiments of this disclosure, the crystal form I uses Cu-Kα radiation, and the X-ray powder diffraction (XRPD) spectrum, expressed in 2θ angles, has characteristic peaks at 18.2±0.2°, 21.7±0.2°, 24.1±0.2°, and 25.6±0.2°.

[0022] Preferably, the X-ray powder diffraction pattern of crystal form I also has characteristic peaks at one or more of the following locations: 10.9±0.2°, 12.4±0.2°, 21.1±0.2°, and 31.0±0.2°.

[0023] More preferably, the X-ray powder diffraction pattern of crystal form I also has characteristic peaks at one or more of the following locations: 14.6±0.2°, 16.9±0.2°, and 22.5±0.2°.

[0024] More preferably, the crystal form I has an X-ray powder diffraction pattern substantially as shown in FIG4.

[0025] In some specific embodiments of this disclosure, the differential scanning calorimetry (DSC) spectrum of crystal form I has an endothermic peak at 217.3℃±10℃.

[0026] Preferably, the differential scanning calorimetry spectrum of crystal form I has an endothermic peak at 217.3℃±5℃.

[0027] More preferably, the crystal form I has a differential scanning calorimeter essentially as shown in FIG5.

[0028] In some specific embodiments of this disclosure, the thermogravimetric analysis (TGA) spectrum of crystal form I shows a weight loss of approximately 0.9052%.

[0029] Preferably, the crystal form I has a thermogravimetric analysis spectrum substantially as shown in FIG6.

[0030] In some specific embodiments of this disclosure, the salt formation ratio of compound I to fumaric acid in the compound I fumarate crystal is 1:1.5; preferably, the salt formation ratio is a molar ratio.

[0031] In another aspect, this disclosure provides a pharmaceutical composition comprising the above-mentioned compound I fumarate.

[0032] In some embodiments of this disclosure, the pharmaceutical composition comprises the above-described fumarate crystals of compound I having crystal form I.

[0033] In some specific embodiments of this disclosure, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0034] In another aspect, this disclosure provides the use of the salt of the above-mentioned compound I, the fumarate of the above-mentioned compound I, the crystals of the above-mentioned compound I fumarate having crystal form I, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating diseases mediated by SOS1.

[0035] In some embodiments of this disclosure, the SOS1-mediated disease is cancer or a tumor-related disease.

[0036] In some specific embodiments of this disclosure, the SOS1-mediated disease is lung cancer (such as non-small cell lung cancer), pancreatic cancer, colorectal cancer, or bladder cancer (such as transitional cell carcinoma of the bladder).

[0037] This disclosure also provides the use of the salt of the above-mentioned compound I, the above-mentioned compound I fumarate, the above-mentioned compound I fumarate crystal having crystal form I, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating cancer or tumor-related diseases.

[0038] In some embodiments of this disclosure, the cancer or tumor-related disease is a solid tumor.

[0039] In some specific embodiments of this disclosure, the cancer or tumor-related disease is lung cancer (such as non-small cell lung cancer), pancreatic cancer, colorectal cancer, or bladder cancer (such as transitional cell carcinoma of the bladder).

[0040] This disclosure also provides a salt of the above-mentioned compound I, the above-mentioned compound I fumarate, the above-mentioned compound I fumarate crystal having crystal form I, or the above-mentioned pharmaceutical composition for treating diseases mediated by SOS1.

[0041] In some embodiments of this disclosure, the SOS1-mediated disease is cancer or a tumor-related disease.

[0042] In some specific embodiments of this disclosure, the SOS1-mediated disease is lung cancer (such as non-small cell lung cancer), pancreatic cancer, colorectal cancer, or bladder cancer (such as transitional cell carcinoma of the bladder).

[0043] This disclosure also provides a salt of the above-mentioned compound I, the above-mentioned compound I fumarate, the above-mentioned compound I fumarate crystal having crystal form I, or the above-mentioned pharmaceutical composition for the treatment of cancer or tumor-related diseases.

[0044] In some embodiments of this disclosure, the cancer or tumor-related disease is a solid tumor.

[0045] In some specific embodiments of this disclosure, the cancer or tumor-related disease is lung cancer (such as non-small cell lung cancer), pancreatic cancer, colorectal cancer, or bladder cancer (such as transitional cell carcinoma of the bladder).

[0046] This disclosure also provides a method for treating SOS1-mediated diseases, comprising administering to an individual in need of such treatment (including, but not limited to, a patient) a therapeutically effective dose of a salt of the aforementioned compound I, the aforementioned compound I fumarate, the aforementioned compound I fumarate crystals having crystal form I, or the aforementioned pharmaceutical composition.

[0047] In some embodiments of this disclosure, the SOS1-mediated disease is cancer or a tumor-related disease.

[0048] In some specific embodiments of this disclosure, the SOS1-mediated disease is lung cancer (such as non-small cell lung cancer), pancreatic cancer, colorectal cancer, or bladder cancer (such as transitional cell carcinoma of the bladder).

[0049] This disclosure also provides a method for treating cancer or tumor-related diseases, comprising administering to an individual in need of such treatment (including, but not limited to, a patient) a therapeutically effective dose of a salt of the aforementioned compound I, the aforementioned compound I fumarate, the aforementioned compound I fumarate crystal having crystal form I, or the aforementioned pharmaceutical composition.

[0050] In some embodiments of this disclosure, the cancer or tumor-related disease is a solid tumor.

[0051] In some specific embodiments of this disclosure, the cancer or tumor-related disease is lung cancer (such as non-small cell lung cancer), pancreatic cancer, colorectal cancer, or bladder cancer (such as transitional cell carcinoma of the bladder).

[0052] For the treatment of cancer or tumor-related diseases, the salt of compound I, the fumarate of compound I, the crystal of compound I fumarate having crystal form I, or pharmaceutical compositions thereof disclosed herein may be co-administered with other therapeutic agents (e.g., chemotherapy drugs, biological therapeutic agents, etc.) or in combination with other therapeutic means, including but not limited to radiotherapy. Beneficial effects

[0053] This disclosure provides for the first time the crystal form (or free base crystal form I) and fumarate form (or fumarate crystal form I) of compound I. The fumarate crystal form I provided in this disclosure has superior performance in terms of solubility, stability, and hygroscopicity, and has good clinical application value, making it an excellent alternative for subsequent drug development. Attached Figure Description

[0054] Figure 1 shows the XRPD spectrum of compound I in its free alkali crystal form I.

[0055] Figure 2 shows the DSC spectrum of compound I in its free alkali crystal form I.

[0056] Figure 3 shows the TGA spectrum of compound I in its free alkali crystal form I.

[0057] Figure 4 shows the XRPD spectrum of compound I fumarate crystal form I.

[0058] Figure 5 shows the DSC spectrum of compound I fumarate crystal form I.

[0059] Figure 6 shows the TGA spectrum of compound I fumarate crystal form I.

[0060] Figure 7 shows the crystal form I of compound I fumarate. 1 H-NMR spectrum. Detailed Implementation

[0061] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. The following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0062] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0063] Example 1 of this disclosure was determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS) and / or high-performance liquid chromatography (HPLC). The instrument used for NMR was a Bruker AVANCE III 600MHz, the instrument used for LC-MS was a WATERS ACQUITY UPLC H-Class PLUS and / or SQD2, and the instrument used for HPLC was a WATERS e2695_2998 and / or Agilent 1100.

[0064] The instruments and detection parameters used in the salt / salt type screening disclosed herein are as follows:

[0065] Example 1: Synthesis of Compound I

[0066] (1) Synthesis of intermediate 5-1

[0067] 4,6-Dichloro-2-methylpyrimidin-5-carboxaldehyde (2.00 g, 10.47 mmol) was dissolved in tetrahydrofuran (20 mL), followed by the addition of ethoxyformylmethylenetriphenylphosphine (5.47 g, 15.71 mmol) and triethylamine (2.12 g, 20.94 mmol). The reaction mixture was incubated at 80 °C for 6 h, and LC-MS monitoring showed no residue of the starting material. The reaction mixture was then purified by column chromatography (n-hexane:ethyl acetate = 20:1–10:1, v / v) to give intermediate 5-1 (1.71 g, 6.54 mmol, yield 63%). ESI-MS: m / z 260.85 [M+H] + .

[0068] (2) Synthesis of intermediate 5-2

[0069] Intermediate 5-1 (1.71 g, 6.54 mmol) was dissolved in N,N-dimethylformamide (20 mL), followed by the addition of (2-aminoethyl)carbamate tert-butyl ester (1.26 g, 7.85 mmol) and triethylamine (1.32 g, 13.08 mmol). The reaction mixture was allowed to react at room temperature for 12 h. LC-MS monitoring showed no residue of starting material. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated sodium chloride (30 mL × 2), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by thin-layer chromatography (n-hexane:ethyl acetate = 10:1-3:1, v / v) to give intermediate 5-2 (2.30 g, 5.99 mmol, yield 92%). ESI-MS: m / z 385.15 [M+H] + .

[0070] (3) Synthesis of intermediate 5-3

[0071] Intermediate 5-2 (2.30 g, 5.99 mmol) was dissolved in methanol (30 mL), followed by the addition of sodium methoxide (3.6 mL, 17.97 mmol, 30% w / w in methanol). The reaction mixture was allowed to react at room temperature for 12 h. A solid precipitated from the reaction solution. LC-MS monitoring showed no residual starting material. Water (10 mL) was added to the reaction solution, and the mixture was filtered to obtain a white solid intermediate 5-3 (1.60 g, 4.79 mmol, yield 80%). ESI-MS: m / z 335.07 [M+H] + .

[0072] (4) Synthesis of intermediate 5-4

[0073] Intermediate 5-3 (1.60 g, 4.79 mmol) was dissolved in dichloromethane (30 mL), followed by the addition of bromine (1.15 g, 7.19 mmol). The reaction mixture was allowed to react at room temperature for 12 h, and LC-MS monitoring showed no residue of the starting material. The reaction mixture was then purified directly by column chromatography (n-hexane:ethyl acetate = 5:1-3:1, v / v) to give intermediate 5-4 (1.80 g, 4.36 mmol, yield 91%). ESI-MS: m / z 412.98 / 415.00 [M+H] + .

[0074] (5) Synthesis of intermediate 5-5

[0075] Intermediate 5-4 (1.80 g, 4.36 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of trifluoroacetic acid (5 mL). The reaction was carried out at room temperature for 4 h, and LC-MS monitoring showed no residue of the starting material. The solvent was removed under reduced pressure, and the residue was added to water (15 mL), the pH was adjusted to 8-9 with saturated sodium carbonate aqueous solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated sodium chloride (20 mL × 2), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give intermediate 5-5 (0.90 g, 2.88 mmol, yield 66%). ESI-MS: m / z 312.92 / 314.91 [M+H] + .

[0076] (6) Synthesis of intermediates 5-6

[0077] Intermediate 5-5 (0.90 g, 2.88 mmol) was dissolved in toluene (20 mL), followed by the slow addition of trimethylaluminum (1.9 mL, 3.75 mmol, 2.0 M in toluene). The reaction mixture was reacted at 120 °C for 5 h, and LC-MS monitoring showed no residue. The reaction solution was quenched with saturated ammonium chloride aqueous solution, and toluene was removed by vacuum distillation. The residue was added to water (15 mL), extracted with ethyl acetate (20 mL × 3), and the organic phases were combined, washed with saturated sodium chloride (20 mL × 2), and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give intermediate 5-6 (0.59 g, 2.01 mmol, 70% yield). ESI-MS: m / z 294.87 / 296.83 [M+H] + .

[0078] (7) Synthesis of intermediate 5-7

[0079] Intermediate 5-6 (0.59 g, 2.01 mmol) was dissolved in dichloromethane (10 mL). Boron tribromide (2.52 g, 10.05 mmol) was slowly added under ice bath conditions. After addition, the mixture was removed from the ice bath, and the reaction was carried out at room temperature for 72 h. LC-MS monitoring showed no residue of the starting material. The reaction was quenched by slow addition of sodium carbonate solution under ice bath conditions, and a solid precipitated. Filtration yielded intermediate 5-7 (0.46 g, 1.64 mmol, yield 82%). ESI-MS: m / z 280.82 / 282.84 [M+H] + .

[0080] (8) Synthesis of intermediate 7-1

[0081] Intermediate 5-7 (100 mg, 0.36 mmol) was dissolved in N,N-dimethylformamide (15 mL), followed by the addition of (R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethylamine hydrochloride (121 mg, 0.54 mmol), benzotriazol-1-oxytris(dimethylamino)phosphine hexafluorophosphate (205 mg, 0.47 mmol), and 1,8-diazabicycloundec-7-ene (163 mg, 1.08 mmol). The reaction mixture was allowed to react at room temperature for 8 h. LC-MS monitoring showed no residue of the starting material. Water (30 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated sodium chloride (30 mL × 2), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by thin-layer chromatography (dichloromethane:methanol = 20:1, v / v) to give intermediate 7-1 (110 mg, yield 68%). ESI-MS: m / z 451.93 / 453.93 [M+H] + .

[0082] (9) Synthesis of Compound I

[0083] Intermediate 7-1 (99 mg, 0.22 mmol) was dissolved in dioxane (30 mL), followed by the addition of 1-acetyl-5,6-dihydro-2H-pyridine-4-boronic acid pinacol ester (68 mg, 0.27 mmol), tetrakis(triphenylphosphine)palladium (23 mg, 0.02 mmol), and cesium carbonate (143 mg, 0.44 mmol). The entire system was stirred at 100 °C for 3 h. TLC monitoring showed no residue of the starting material. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was purified by column chromatography (dichloromethane:methanol = 60:1-15:1, v / v) to give compound I (69 mg, 0.14 mmol, yield 64%). ESI-MS: m / z 497.24 [M+H] + . 1 HNMR (600MHz, DMSO-d6): δ8.23(s,1H),8.00(s,1H),7.64(dd,J=7.2Hz,J=7.2Hz,1H ),7.50(dd,J=7.2Hz,J=7.2Hz,1H),7.32-7.29(m,1H),7.23(t,J=54.0Hz,1H),6.82 (d,J=87.6Hz,1H),5.75-5.70(m,1H),4.22-4.02(m,4H),3.95-3.92(m,2H),3.67-3 .62(m,2H),2.50(s,3H),2.26(s,3H),2.06(d,J=24.6Hz,2H),1.56(d,J=6.6Hz,3H).

[0084] Example 2: Preparation of free basal form I of compound I

[0085] Compound I (50 mg) was added to ethyl acetate (10 mL), stirred at room temperature, and allowed to stand overnight. After filtration, the obtained solid was dried to obtain crystals of compound I. The crystal form corresponding to this crystal is the free base crystal form I of compound I. The XRPD detection results of this crystal form I are shown in Figure 1, and the diffraction peak data are shown in the table below. The DSC and TGA detection results are shown in Figures 2 and 3, respectively.

[0086] Example 3: Preparation of Compound I Fumarate Crystal Form I

[0087] Compound I (approximately 300 mg) was dissolved in tetrahydrofuran (3.0 mL) at 50 °C to obtain solution 1. Fumaric acid (approximately 77.4 mg) was dissolved in ethanol (1.2 mL) at 50 °C to obtain solution 2. Solution 2 was added dropwise to solution 1 at room temperature to obtain a suspension. The suspension was stirred overnight at room temperature. The suspension was centrifuged, and the resulting solid was vacuum dried overnight at 40 °C to obtain crystals of compound I fumarate. The crystal form corresponding to this crystal is crystal form I of compound I fumarate. The XRPD detection results of this crystal form I are shown in Figure 4, and the diffraction peak data are shown in the table below. The DSC and TGA detection results are shown in Figures 5 and 6. 1 The H-NMR detection results are shown in Figure 7. According to the integration results, the salt formation ratio of compound I to fumaric acid is 1:1.5.

[0088] In addition to fumaric acid, the preliminary research work disclosed herein also investigated the salt formation of organic acids such as acetic acid, propionic acid, benzenesulfonic acid, L-malic acid, succinic acid, L-tartaric acid, and pamoic acid, as well as inorganic acids such as phosphoric acid and sulfuric acid.

[0089] First, based on comparisons of properties such as crystallinity, solvation, and melting point, fumarate, benzenesulfonate, phosphate, and sulfate were preliminarily determined to be suitable for subsequent crystal form preparation studies. Second, based on comparisons of sample purity and polymorphism number, fumarate and sulfate were selected for subsequent salt form evaluation studies. Finally, based on comparisons of properties such as solubility, hygroscopicity, and stability, the dominant salt form of compound I was ultimately determined to be compound I fumarate crystal form I.

[0090] Bioactivity assay experiment 1:

[0091] 1.3D Cell Proliferation Inhibition Assay

[0092] The cell proliferation inhibition assay was used to detect the inhibitory effect of the compound on the proliferation and growth of SOS1-mediated tumor cell lines at the in vitro 3D cell level. 3D inspection method.

[0093] 1.1 Reagents and Materials

[0094] NCI-H358: Non-small cell lung cancer (NSCLC) with KRAS G12C mutation;

[0095] 3D Cell Viability Assay, Promega, G9683;

[0096] RPMI 1640medium,Gibco,A10491-01;

[0097] FBS, Gibco, 10099141C;

[0098] 1.2 Test Procedure

[0099] 1.2.1 Cell Culture

[0100] Day 1, NCI-H358 cells were passaged into T75 cell culture flasks;

[0101] Day 3, remove the culture medium, wash once with DPBS, and use 2 mL of TrypLE at room temperature or 37°C. TM The cells were digested with Express Enzyme until they detached; 5 mL of fresh culture medium was added, and the cells were centrifuged at 1000 rpm for 5 min; the supernatant was discarded, and the cells were resuspended in 5 mL of fresh culture medium. After cell counting, 40 μL / well was seeded into 3D cell plates (Echo Qualified 384-Well Polypropylene Microplate 2.0, Clear, Flat Bottom).

[0102] 1.2.2 Inhibition of 3D cell proliferation

[0103] Day 1: Dissolve the test compound in DMSO to prepare a 10 mM stock solution; dilute 1000 times with DMSO solution, then perform 3-fold serial dilutions, 10 concentration gradients, with an initial concentration of 10 μM; add 200 nL of the compound to the culture plate.

[0104] Day 8, add 40 μL / well of 3D CTG reagent and use Envision to detect the signal value.

[0105] 1.3 Data Analysis

[0106] Fitting compound IC using a nonlinear regression equation 50 value;

[0107] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope));

[0108] X: Log of cpd concentration;

[0109] Y: Percent inhibition (%inh)

[0110] 1.4 Results of NCI-H358 cell proliferation inhibition activity

[0111] The compound of Example 1 was tested according to the above methods, and its inhibitory activity against NCI-H358 cell proliferation was measured using IC50 data. 50 Less than 50 nM.

[0112] Bioactivity assay experiment 2:

[0113] 1.3D NCI-H358, NCI-H520, NCI-H1373, UM-UC-3, GP2d, A549 cell proliferation inhibition assay

[0114] The in vitro proliferation inhibitory effects of the disclosed compound on human non-small cell lung cancer cells (NCI-H358, NCI-H1373, A549 and NCI-H520), human colorectal cancer cells (GP2d) and human bladder transitional cell carcinoma cells (UM-UC-3) were investigated, and its inhibitory effects on the above cell proliferation were compared with those of MRTX0902 and BI3406.

[0115] 1.1 Experimental Materials and Equipment

[0116] Cell lines:

[0117] NCI-H358: Non-small cell lung cancer (NSCLC) with KRAS G12C mutation;

[0118] NCI-H520: KRAS wild-type non-small cell lung cancer (NSCLC);

[0119] NCI-H1373: Non-small cell lung cancer (NSCLC) with KRAS G12C mutation;

[0120] UM-UC-3: KRAS G12C-mutated bladder transitional cell carcinoma (BTCC);

[0121] GP2d: Colorectal cancer (CRC) with KRAS G12D mutation;

[0122] A549: KRAS G12S-mutant non-small cell lung cancer (NSCLC).

[0123] Reagents and consumables:

[0124] F12K Medium, Hyclone, SH30526.01;

[0125] DMEM Medium, Gibco, 11965-092;

[0126] 1640Medium,Gibco,A10491-01;

[0127] EMEM Medium, ATCC, 30-2003;

[0128] Penicillin-Streptomycin, Gibco, 15140-122;

[0129] Fetal Bovine Serum, Gibco, 10099-141C;

[0130] Phosphate Buffered Saline (PBS), Gibco, 10010-031;

[0131] TrypLE, Gibco, 12604-021;

[0132] DMSO, Sigma, D8418-1L;

[0133] BI-3406, MCE, HY-125817;

[0134] MRTX0902, Zepi Biotechnology (Shijiazhuang) Co., Ltd., Zepi20221025

[0135] 3D Cell Viability assay kit, Promega, G9683;

[0136] 384 spheroid microplates, Corning, 3830;

[0137] Ultra-Low attachment 96well plate, Corning, 7007;

[0138] White Opaque 96-well Microplate, PerkinElmer, 6005680.

[0139] Instruments and equipment:

[0140] Plate shaker, QILINBEIER, QB-9002;

[0141] Centrifuge, Eppendorf, 5810R;

[0142] CO2 incubator, Thermo Scientific, 371;

[0143] Microscope, OLYMPUS, CKX41;

[0144] Echo Qualified 384-Well Polypropylene, LABCYTE, PP-0200;

[0145] Echo, LABCYTE, 655;

[0146] CountessII,Gibco,AMQAX1000;

[0147] Envision,PerkinElmer,2105.

[0148] 1.2 Test Procedure

[0149] 1.2.1 Cell Culture

[0150] a) NCI-H358, NCI-H520, and NCI-H1373 cells were cultured in 1640 medium with 1% antibiotics and 10% FBS, and cultured at 37°C and 5% CO2.

[0151] b) A549 cells were cultured in F12K medium with 1% antibiotics and 10% FBS, and cultured at 37°C and 5% CO2.

[0152] c) GP2d cells were cultured in DMEM medium with 1% antibiotics and 10% FBS, and cultured at 37°C and 5% CO2.

[0153] d) UM-UC-3 cells were cultured in EMEM medium with 1% antibiotics and 10% FBS, and cultured at 37°C and 5% CO2.

[0154] 1.2.2 Cell Plating

[0155] Cells are cultured routinely until they reach 80%-90% saturation. Once the required number of cells is reached, the cells are harvested.

[0156] Resuspend the cells in the appropriate culture medium, count them, and prepare cell suspensions of suitable density. Specifically, NCI-H358 cells were seeded at 2000 per well on a 384 Well Plate; NCI-H520 cells at 2000 per well on a 384 Well Plate; A549 cells at 1000 per well on a 384 Well Plate; NCI-H1373 cells at 1000 per well on a 96 Well Plate; GP2d cells at 1250 per well on a 96 Well Plate; and UM-UC-3 cells at 1000 per well on a 384 Well Plate.

[0157] 1.2.3 Preparation of Compounds

[0158] a) Gradient dilution of positive compounds (MRTX0902 and BI-3406):

[0159] 384-well plate dilution system: Compounds MRTX0902 and BI-3406 were dissolved in DMSO to prepare a 30 mM stock solution. MRTX0902 and BI-3406 were then diluted to 10 mM with DMSO and subsequently serially diluted 3-fold to obtain 10 concentration gradients (concentration gradients of 10000, 3333, 1111, 370, 123, 41.2, 13.7, 4.57, 1.52, and 0.51 μM). Using a nanoliter pipetting system (Echo 650 Liquid Handler), 40 nL of the above-mentioned serially diluted compounds were added to each well of a 384-well cell culture plate and centrifuged at 1000 rpm for 1 min. The concentration gradients of the compounds in the final reaction system (40 μL) were 10000, 3333, 1111, 370, 123, 41.2, 13.7, 4.57, 1.52, and 0.51 nM.

[0160] 96-well plate dilution system: MRTX0902 and BI-3406 were diluted to 2000 μM with DMSO solvent and then serially diluted 3-fold to obtain 10 concentration gradients (2000, 667, 222, 74.1, 24.7, 8.23, 2.74, 0.91, 0.30, 0 μM). Using an Echo 650 Liquid Handler, 500 nL of the above-mentioned serially diluted compounds were added to each well of a 96-well cell culture plate and centrifuged at 1000 rpm for 1 min. The concentration gradients of the compounds in the final reaction system (100 μL) were 10000, 3333, 1111, 370, 123, 41.2, 13.7, 4.57, 1.52, 0 nM.

[0161] b) Gradient dilution of the compounds disclosed herein:

[0162] 384-well plate dilution system: The compound from Example 1 was dissolved in DMSO to prepare a 27.4 mM stock solution, diluted to 9.13 mM with DMSO, and then serially diluted 3-fold to obtain 10 concentration gradients (9135, 3044, 1014, 338, 112, 37.6, 12.5, 4.18, 1.39, and 0.46 μM, respectively). Using an Echo 650 Liquid Handler, 40 nL of the serially diluted compound was added to each well of a 384-well cell culture plate. After centrifugation at 1000 rpm for 1 min, the concentration gradient of the compound in the final reaction system (40 μL) was 9135, 3045, 1015, 338, 113, 37.6, 12.5, 4.18, 1.39, and 0.46 nM, respectively.

[0163] 96-well plate dilution system: The compound of Example 1 was diluted to 1827 μM with DMSO solvent and then serially diluted 3-fold, resulting in 10 concentration gradients (concentration gradients of 1827, 609, 203, 67.7, 22.6, 7.52, 2.51, 0.84, 0.28, and 0 μM). Using a nanoliter pipetting system (Echo 650 Liquid Handler), 500 nL of the above-diluted compound was added to each well of a 96-well cell culture plate. After centrifugation at 1000 rpm for 1 min, the concentration gradients of the compound in the final reaction system (100 μL) were 9135, 3045, 1015, 338, 113, 37.6, 12.5, 4.18, 1.39, and 0 nM.

[0164] 1.2.4 CTG Method Detection

[0165] Cells were cultured at 37°C in a 5% CO2 incubator for 7 days:

[0166] a) Add 20 μL of CTG reagent (CelltiterGlo 3D kit) to each well of a 384-well plate, centrifuge at 1000 rpm for 1 min, place on a shaker and shake for 20 min, then incubate at room temperature in the dark for 120 min.

[0167] b) Add 50 μL of CTG reagent (CelltiterGlo 3D kit) to each well of a 96-well plate, centrifuge at 1000 rpm for 1 min, place on a rapid shaker for 20 min, incubate at room temperature in the dark for 120 min, and then transfer 100 μL to a White Opaque 96-well Microplate.

[0168] c) Use an Envision instrument to read the chemiluminescence signal value.

[0169] 1.2.5 Data Analysis

[0170] The IC of the compound is obtained using the following nonlinear fitting formula. 50 (Half-maximal inhibitory concentration):

[0171] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope));

[0172] Y: Inhibition rate; X: Log value of compound concentration

[0173] Inhibition rate (%) = 100 - (Compound well reading - Low-read control well reading) / (High-read control well reading - Low-read control well reading) * 100%

[0174] High-reading control wells: Cells were treated with 40 nL DMSO (NCI-H358, NCI-H520, A549, UM-UC-3); Cells were treated with 500 nL DMSO (NCI-H1373, GP2d).

[0175] Low read control wells: Cell-free culture medium

[0176] 1.3 Test Results

[0177] The cell proliferation inhibition activity data of the compounds in Example 1 were tested according to the above methods and are shown in the table below:

[0178] 2.3D MIA PaCa-2, NCI-H441 and DLD-1 cell proliferation inhibition assay

[0179] The in vitro proliferation inhibitory effects of the disclosed compound on human pancreatic cancer cells (MIA PaCa-2), human non-small cell lung cancer cells (NCI-H441), and human colorectal cancer cells (DLD-1) were investigated, and the inhibitory effects of the compound on the proliferation of the above three cell lines were compared with those of MRTX0902.

[0180] 2.1 Experimental Materials and Equipment

[0181] Cell lines:

[0182] MIA PaCa-2: Human pancreatic cancer cells (PAC) with KRAS G12C mutation;

[0183] H441: KRAS G12V-mutated non-small cell lung cancer (NSCLC);

[0184] DLD-1: Human colorectal cancer (CRC) with KRAS G13D mutation.

[0185] Reagents and consumables:

[0186] DMEM basic (1×): 500mL, Gibco, lot number: 8122483;

[0187] Horse serum: 500mL, Inner Mongolia Jinyuankang Company, batch number: 20200701;

[0188] Penicillin-Streptomycin, Gibco, 15140-122;

[0189] RPMI Medium 1640 basic (1×): 500mL, Gibco, lot number: 8122656;

[0190] FETAL BOVINE SERUM (FBS): 500mL, Gibco, Lot No.: 2528128CP;

[0191] 0.25% Trypsin-EDTA (1×): 500mL, Gibco, batch number: 2360145;

[0192] PBS pH 7.4 (1×): 500 mL, Gibco, lot number: 8121763;

[0193] DMSO (cell culture grade): 500mL, AMRESCO, batch number: 21A2756279;

[0194] 3D Cell Viability Assay: 100mL, Promega, Batch No.: 0000537948;

[0195] MRTX0902, Zepi Biotechnology (Shijiazhuang) Co., Ltd., Zepi20221025;

[0196] Low adsorption plate: 96 wells, Corning, batch number: 02722003.

[0197] Instruments and equipment:

[0198] Model 371 CO2 incubator: Thermo, equipment number: ZS01-0746;

[0199] IX70-142 Inverted Microscope: Olympus, Equipment No.: 0243;

[0200] HFsafe-1500LC Biosafety Cabinet: Shanghai Lishen Scientific Instruments Co., Ltd., Equipment No.: ZS01-0747;

[0201] TD6 centrifuge: Changsha Xiangrui Centrifuge Co., Ltd., Equipment No.: ZS01-0750;

[0202] MS105 electronic balance: METTLER TOLEDO, equipment number: ZY01-0136;

[0203] Varioskan Flash fluorescence microplate reader: Thermo, device number: 0845.

[0204] 2.2 Test Procedure:

[0205] 2.2.1 Preparation of Compounds

[0206] In the MIA PaCa-2 cell proliferation inhibition assay, the final concentration of the compound disclosed herein was set at 9134.57 nM as the initial concentration, and a total of 9 concentrations were set by 5× descending dilutions, namely 9134.57, 1826.91, 365.38, 73.08, 14.62, 2.92, 0.58, 0.12, and 0.02 nM. The final concentration of MRTX0902 was set at 10 μM, and nine concentrations were obtained through 3× descending dilutions: 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, and 1.52 nM. In the DLD-1 cell proliferation inhibition assay, the final concentration of the compound was set at 9134.57 nM, and nine concentrations were obtained through 3× descending dilutions: 9134.57, 3044.86, 1014.95, 338.32, 112.77, 37.59, 12.53, 4.18, and 1.39 nM. The final concentration of MRTX0902 was set at 10 μM, with a total of 9 concentrations obtained through 3× descending dilutions: 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, and 1.52 nM. In the NCI-H441 proliferation inhibition assay, the final concentration of the compound disclosed in this study was set at 9134.57 nM, with a total of 9 concentrations obtained through 5× descending dilutions: 9134.57, 1826.91, 365.38, 73.08, 14.62, 2.92, 0.58, 0.12, and 0.02 nM. The final concentration of MRTX0902 was set at 10 μM as the starting concentration, and a total of 9 concentrations were set with 5× descending dilutions, namely 10000, 2000, 400, 80, 16, 3.2, 0.64, 0.13 and 0.03 nM.

[0207] 2.2.2 Cell Culture and Detection

[0208] MIA PaCa-2 cells were cultured in high-glucose DMEM containing 10% FBS and 2.5% horse serum; NCI-H441 and DLD-1 cells were cultured in RPMI Medium 1640 basic containing 10% FBS. All cells were cultured at 37°C and 5% CO2.

[0209] Cell lines in the logarithmic growth phase were inoculated into low-adsorption 96-well plates at a certain number (100 μL / well). 100 μL of culture medium containing different concentration gradients of the disclosed compound or MRTX0902 was added to each well. Three replicates were set for each drug concentration, along with corresponding blank wells (culture medium only) and normal wells (drug concentration of 0). After 7 days of drug treatment, the plates were equilibrated at room temperature for 30 min. 120 μL of supernatant was aspirated from each well, and 50 μL of CellTiter-Glo assay reagent was added. The plates were mixed by shaking at room temperature in the dark for 20 min, and the luminescence value was detected using a chemiluminescence immunoassay.

[0210] The cell growth inhibition rate was calculated using the following formula:

[0211] Inhibition rate (%) = (luminescence value) 正常孔 -Luminescence value 给药孔 ) / (luminous value) 正常孔 -Luminescence value 空白孔 )×100%;

[0212] Based on the inhibition rate at each concentration, the half-maximal inhibitory concentration (IC50) of the drug was fitted using a nonlinear regression equation. 50 .

[0213] 2.3 Test Results

[0214] The cell proliferation inhibition activity data of the compounds in Example 1 were tested according to the above methods and are shown in the table below:

[0215] Test Example 1: Solubility Test

[0216] At room temperature, weigh the sample and add solvent (water) to it in portions, stirring or sonicating to aid dissolution until the sample is visually dissolved. Record the amount of solvent consumed. If the sample is not completely dissolved at a specific concentration, its solubility is expressed as "<" the specific concentration.

[0217] Table 1. Solubility Test

[0218] As can be seen from the above tests, compound I, fumarate crystal form I, has better solubility than free alkali crystal form I.

[0219] Test Example 2: Hygroscopicity Test

[0220] Table 2. DVS Detection

[0221] As can be seen from the above tests, compound I, fumarate crystal form I, in this disclosure has only slight hygroscopicity, and has better hygroscopic properties than free alkali crystal form I.

[0222] Test Example 3: Solid State Stability Test

[0223] The solid-state stability of compound I fumarate crystal form I in Example 3 was tested according to the test items and test conditions in the table below:

[0224] Table 3. Solid-state stability test of compound I fumarate crystal form I

[0225] As can be seen from the above tests, compound I fumarate crystal form I in this disclosure has excellent solid-state stability under various conditions (high temperature, long-term, accelerated, light irradiation).

[0226] The embodiments of this disclosure have been described above. However, this disclosure is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A salt of compound I as shown in the following formula, characterized in that, The salt is an acid addition salt of compound I and an organic acid, preferably compound I fumarate.

2. A fumarate of compound I as shown in the following formula, characterized in that, The salting ratio of compound I in the fumarate to fumaric acid is 1:1.5; preferably, the salting ratio is a molar ratio.

3. A fumarate crystal of compound I as shown in the following formula, characterized in that, The crystal has crystal form I, and the X-ray powder diffraction pattern of crystal form I, expressed in 2θ angles, has characteristic peaks at 18.2±0.2°, 21.7±0.2°, 24.1±0.2° and 25.6±0.2° when irradiated with Cu-Kα. Preferably, the X-ray powder diffraction pattern of crystal form I also has characteristic peaks at one or more of the following locations: 10.9±0.2°, 12.4±0.2°, 21.1±0.2°, and 31.0±0.2°. More preferably, the X-ray powder diffraction pattern of crystal form I also has characteristic peaks at one or more of the following locations: 14.6±0.2°, 16.9±0.2°, and 22.5±0.2°. More preferably, the crystal form I has an X-ray powder diffraction pattern substantially as shown in FIG4.

4. The fumarate crystal of compound I as described in claim 3, characterized in that, The differential scanning calorimetry spectrum of crystal form I shows an endothermic peak at 217.3℃±10℃; Preferably, the differential scanning calorimetry spectrum of crystal form I has an endothermic peak at 217.3℃±5℃; More preferably, the crystal form I has a differential scanning calorimeter essentially as shown in FIG5.

5. The fumarate crystal of compound I as described in claim 3 or 4, characterized in that, Thermogravimetric analysis of crystal form I showed a weight loss of approximately 0.9052%; Preferably, the crystal form I has a thermogravimetric analysis spectrum substantially as shown in FIG6.

6. The fumarate crystal of compound I according to any one of claims 3-5, characterized in that, The salting ratio of compound I in the fumarate crystal to fumaric acid is 1:1.5; preferably, the salting ratio is a molar ratio.

7. A pharmaceutical composition comprising a salt of compound I as claimed in claim 1, a fumarate of compound I as claimed in claim 2, or fumarate crystals of compound I as claimed in any one of claims 3-6; Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

8. Use of the salt of compound I as claimed in claim 1, the fumarate of compound I as claimed in claim 2, the fumarate crystals of compound I as claimed in any one of claims 3-6, or the pharmaceutical composition as claimed in claim 7 in the preparation of a medicament for treating SOS1-mediated diseases; Preferably, the SOS1-mediated disease is cancer or a tumor-related disease; More preferably, the SOS1-mediated disease is lung cancer (such as non-small cell lung cancer), pancreatic cancer, colorectal cancer, or bladder cancer (such as transitional cell carcinoma of the bladder).

9. Use of the salt of compound I as claimed in claim 1, the fumarate of compound I as claimed in claim 2, the fumarate crystals of compound I as claimed in any one of claims 3-6, or the pharmaceutical composition as claimed in claim 7 in the preparation of a medicament for treating cancer or tumor-related diseases; Preferably, the cancer or tumor-related disease is a solid tumor; More preferably, the cancer or tumor-related disease is lung cancer (such as non-small cell lung cancer), pancreatic cancer, colorectal cancer, or bladder cancer (such as transitional cell carcinoma of the bladder).

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