Futibatinib degradation products and uses thereof

Abstract

The application discloses Futibatinib degradation products and application thereof, and belongs to the technical field of medicines. The application obtains two degradation products and determines the chemical structures of the degradation products by systematically investigating the degradation behavior of Futibatinib under different stress conditions, and by separating, purifying and identifying the degradation products. The application is helpful to perfect the impurity spectrum and degradation pathway of Futibatinib, and provides important basis for quality control, stability evaluation, production process optimization and impurity reference preparation of the crude drug and preparation of Futibatinib. Meanwhile, the obtained degradation products can be used as important research objects for structure-activity relationship research of Futibatinib, and provide reference for discovery and optimization of relevant active compounds, and therefore have good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the degradation products of Futibatinib and their applications. Background Technology

[0002] Futibatinib (TAS-120) is an oral, irreversible FGFR1–4 inhibitor. Unlike traditional reversible ATP-competitive FGFR inhibitors, Futibatinib forms a covalent bond with the ATP-binding site of the FGFR kinase domain, thereby achieving sustained and stable FGFR signaling inhibition. Based on the positive results of the Phase II clinical trial FOENIX-CCA2, the U.S. Food and Drug Administration (FDA) granted accelerated approval to Futibatinib in 2022 for the treatment of previously treated, unresectable, locally advanced, or metastatic intrahepatic cholangiocarcinoma carrying FGFR2 fusions or rearrangements. Related clinical studies have shown that Futibatinib not only possesses durable antitumor activity but also exhibits good safety and tolerability. Furthermore, due to its irreversible covalent binding mechanism, Futibatinib maintains good inhibitory activity against some FGFR mutations that lead to resistance to reversible FGFR inhibitors. These characteristics make it an important targeted drug in the treatment of FGFR2-abnormal cholangiocarcinoma.

[0003] Process impurities generated during drug manufacturing and degradation impurities formed during storage are significant factors affecting drug quality, safety, and stability. According to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q3A guidance, impurities originating from manufacturing processes or degradation transformations should be rigorously monitored and their structures identified, as these impurities may affect drug efficacy, safety, and stability. Forced degradation studies are an important tool for evaluating the intrinsic stability of drugs, and can be used to establish stability indicator analytical methods and reveal potential degradation pathways.

[0004] This invention systematically investigates the degradation behavior of Futibatinib under different stress conditions, and separates, purifies, and identifies the degradation products. Two different degradation products were obtained. The obtained degradation products can serve as important research objects for the study of the structure-activity relationship of Futibatinib, and provide a reference for the discovery and optimization of related active compounds. Summary of the Invention

[0005] One objective of this invention is to provide a Futibatinib degradation product selected from any of the following structures: .

[0006] A second objective of this invention is to provide a method for preparing the aforementioned Futibatinib degradation product, comprising the following steps: Step 1: Dissolve Futibatinib raw material in methanol, add sodium hydroxide solution to react, after the reaction is completed, add hydrochloric acid solution to terminate the reaction, adjust the pH of the reaction solution to 7.0, remove residual methanol by rotary evaporation, and obtain crude product containing salt. Step 2: Dissolve the crude product in methanol-water solution to prepare a sample solution, filter it through an organic filter membrane, and use it for preparative liquid chromatography separation. Collect the elution fractions with retention times of 2.2–2.6 min and 7.7–9.0 min to obtain the degradation product.

[0007] Furthermore, in step 1, the concentration of Futibatinib active pharmaceutical ingredient is 1 mg / mL, the concentration of sodium hydroxide solution is 10 mol / L, and the concentration of hydrochloric acid solution is 10 mol / L.

[0008] Furthermore, in step 1, the volume ratio of methanol, sodium hydroxide solution, and hydrochloric acid solution is 100 mL: 50 mL: 50 mL.

[0009] Furthermore, the reaction conditions in step 1 are 45–55 °C for 6–8 h.

[0010] Furthermore, in step 2, the concentration of the methanol aqueous solution is 60%, and the concentration of the sample solution is 25 mg / mL.

[0011] Furthermore, the chromatographic conditions for preparing the liquid chromatography separation in step 2 are as follows: Column: Agilent Eclipse XDB-C 18 ; Column temperature: room temperature; Flow rate: 6 mL / min; Detection wavelength: 300 nm; Injection volume: 200 μL; Running time: 25 min; Mobile phase A is ultrapure water, and mobile phase B is methanol. The gradient elution program is as follows: 0–1 min: 48% B → 48% B; 1–12 min: 48% B → 85% B; 12–16 min: 85% B → 85% B; 16–17 min: 85% B → 48% B; 17–25 min: 48% B → 48% B.

[0012] A third objective of this invention is to provide the application of the above-mentioned Futibatinib degradation products in the preparation of tumor drugs.

[0013] Furthermore, the tumor is selected from breast cancer, pancreatic cancer, lung cancer, and stomach cancer.

[0014] This invention systematically investigated the degradation behavior of Futibatinib under different stress conditions. Forced degradation results showed that Futibatinib exhibited good stability under oxidative, thermal, and photothermal conditions, while undergoing significant degradation under alkaline conditions. Two previously unreported degradation impurities, named Fut-1 and Fut-2, were successfully separated and purified from the alkaline degradation system using preparative liquid chromatography. Combined with HRMS, 1 H NMR, 13 The structures of the two degradation products were identified using analytical methods such as C1NMR and HMBC. The results showed that the structure of Fut-1 was: ( S )-3-[(3,5-dimethoxyphenyl)ethynyl]-1-(pyrrolidine-3-yl)-1 H -pyrazolo[3,4- d The structure of pyrimidine-4-amine, Fut-2 is: ( S )-1-[3-(4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1 H -pyrazolo[3,4- d [1-pyrimidin-1-yl]pyrrolidone-1-yl]-3-methoxyprop-1-one. Further cytotoxicity evaluation results showed that Fut-1 still possessed strong antitumor activity and exhibited proliferation inhibition comparable to or even superior to the parent drug in some cell lines; while the activity of Fut-2 was significantly reduced.

[0015] This invention systematically investigated the degradation behavior of Futibatinib under different stress conditions, separated, purified, and structurally identified its degradation products, obtaining two degradation products and clarifying their chemical structures. These findings contribute to improving the study of Futibatinib's impurity profile and degradation pathways, providing important evidence for the quality control, stability evaluation, production process optimization, and preparation of impurity reference standards for its active pharmaceutical ingredients and formulations. Furthermore, the obtained degradation products can serve as important research objects for structure-activity relationship studies of Futibatinib, providing a reference for the discovery and optimization of related active compounds, thus demonstrating promising industrial application prospects. Attached Figure Description

[0016] Figure 1 The HPLC chromatograms of Futibatinib products under the following conditions are shown: no degradation (A), acid degradation (B), alkaline degradation (C), oxidative degradation (D), high-temperature degradation (E), and photodegradation (F).

[0017] Figure 2In the diagram, A is the HPLC chromatogram of crude Futibatinib after alkali degradation, B is the preparative HPLC chromatogram of crude Futibatinib after alkali degradation, C is the preparative HPLC chromatogram of Fut-1a after secondary purification, D is the UV spectrum of Futibatinib, Fut-1 and Fut-2, E is the HPLC chromatogram of Fut-1, and F is the HPLC chromatogram of Fut-2.

[0018] Figure 3 In the diagram, A is the HRMS plot of Fut-1, and C... 19 H 20 N6O2 + [M+H] + The calculated HRMS (ESI) m / z value is 365.1681, and the detected value is 365.1768; B is the HRMS plot of Fut-2, C 23 H 26 N6O4 + [M+H] + The calculated HRMS (ESI) m / z value is 451.2049, and the detected value is 451.2167.

[0019] Figure 4 In the diagram, A represents Futibatinib. 1 H NMR spectrum, 1 H NMR (400 MHz, DMSO-d6) δ 8.28(s, 1H), 6.95 – 6.89 (m, 2H), 6.71 – 6.54 (m, 2H), 6.18 (ddd, J = 16.7, 6.8,2.4 Hz, 1H), 5.69 (ddd, J = 17.2, 10.3, 2.4 Hz, 1H), 5.58 – 5.43 (m, 1H), 4.04(ddd, J = 11.0, 8.8, 4.8 Hz, 1H), 3.94 – 3.75 (m, 8H), 3.74 – 3.59 (m, 1H), 2.49 – 2.27 (m, 2H); B is Fut-1 1 H NMR spectrum, 1 H NMR (400 MHz, DMSO-d6) δ 10.01(s, 1H), 9.69 (s, 1H), 8.31 (d, J = 5.6 Hz, 1H), 8.13 (s, 1H), 6.92 (d, J= 2.3Hz, 2H), 6.63 (t, J = 2.2 Hz, 1H), 5.67 – 5.54 (m, 1H), 3.87 – 3.70 (m, 7H), 3.68 – 3.48 (m, 3H), 2.47 (dt, J = 15.3, 7.7 Hz, 1H), 2.41 – 2.32 (m, 1H); C is Fut-2 1 H NMR spectrum, 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.6 Hz, 1H), 6.92(s, 2H), 6.62 (t, J = 2.2 Hz, 1H), 5.63 – 5.34 (m, 1H), 4.02 (dt, J = 16.9, 8.5Hz, 1H), 3.92 – 3.64 (m, 9H), 3.64 – 3.48 (m, 3H), 3.23 (d, J = 15.2 Hz, 3H), 2.56 (t, J = 6.5 Hz, 1H), 2.45 (dd, J = 13.5, 6.4 Hz, 1H), 2.37 (td, J = 9.5, 4.7Hz, 1H).

[0020] Figure 5 In the above, A is Fut-1. 13 C NMR spectrum, 13 C NMR (101 MHz, DMSO-d6) δ 160.84, 158.13, 156.97, 153.63, 126.56, 122.93, 110.12, 102.97, 101.21, 94.06, 80.79, 55.98, 55.32, 48.60, 44.66, 30.89; B is Fut-2 13 C NMR spectrum, 13C NMR (101 MHz, DMSO-d6) δ 169.25, 169.13, 160.82, 158.22, 156.94, 153.57, 126.11, 126.03,123.06, 110.09, 102.95, 101.21, 93.79, 93.73, 81.02, 68.37, 58.42, 58.38,56.29, 55.94, 55.02, 50.94, 50.34, 45.44, 44.62, 34.67, 34.42, 31.53, 29.89.

[0021] Figure 6 In the image, A is the HMBC spectrum of Fut-1, and B is the HMBC spectrum of Fut-2.

[0022] Figure 7 The inhibitory effects of different concentrations of Futibatinib, Fut-1, and Fut-2 on HepG2 (A), MCF-7 (B), PANC-1 (C), A549 (D), and MKN-1 (E) cell lines were investigated. Detailed Implementation

[0023] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0026] 1. Forced Degradation Research Futibatinib active pharmaceutical ingredient (provided by Shanghai Houbo Biotechnology Co., Ltd.) was subjected to acid degradation, alkali degradation, oxidative degradation, high-temperature degradation, and photodegradation treatments, respectively, under the following specific conditions: (1) Undegraded: Accurately weigh about 25 mg of Futibatinib, place it in a 50 mL volumetric flask, dissolve it in 50% methanol aqueous solution and make up to volume.

[0027] (2) Acid degradation test: Accurately weigh approximately 25 mg of Futibatinib and place it in a 50 mL stoppered conical flask. Add 25 mL of methanol and sonicate until completely dissolved. Then add 12.5 mL of 1 mol / L hydrochloric acid solution, stopper tightly, and react in a 50 ℃ water bath for 24 h. After the reaction is complete, remove the flask and cool to room temperature. Adjust the pH to neutral with 2 mol / L sodium hydroxide solution. Quantitatively transfer the reaction solution to a 50 mL volumetric flask, dilute to the mark with 50% methanol aqueous solution, and shake well to obtain the test solution. Prepare a blank solution without Futibatinib using the same method.

[0028] (3) Alkaline degradation: Accurately weigh approximately 25 mg of Futibatinib and place it in a 50 mL stoppered conical flask. Add 25 mL of methanol and sonicate until completely dissolved. Add 12.5 mL of 0.5 mol / L sodium hydroxide solution and react at room temperature for 1 h. After the reaction is complete, adjust the pH to neutral with 1 mol / L hydrochloric acid solution. Quantitatively transfer the reaction solution to a 50 mL volumetric flask, dilute to the mark with 50% methanol aqueous solution, and shake well to obtain the test solution. Prepare a blank solution without Futibatinib using the same method.

[0029] (4) Oxidative degradation: Accurately weigh approximately 25 mg of Futibatinib and place it in a 50 mL stoppered conical flask. Add 25 mL of methanol to dissolve it, then add 25 mL of 30% hydrogen peroxide solution. Seal the flask tightly and react in a 40 °C water bath for 7 days. Remove the flask and allow it to cool to room temperature. Transfer the entire reaction solution to a 50 mL volumetric flask and dilute to the mark with 50% methanol aqueous solution. Shake well. Prepare a blank solution simultaneously.

[0030] (5) High-temperature degradation: Place an appropriate amount of sample in a weighing bottle and place it in a 100 ℃ forced-air drying oven for 7 days. After taking it out, accurately weigh about 25 mg of the degradation product, place it in a 50 mL volumetric flask, dissolve it in 50% methanol aqueous solution and make up to volume.

[0031] (6) Photodegradation: Prepare the sample solution according to the method under “Undegraded” and place it under a fluorescent lamp with a light intensity of 4500 Lx for 30 days.

[0032] All samples were analyzed by HPLC, with an injection volume of 10 μL each time. The HPLC analysis method is as follows: Accurately weigh an appropriate amount of Futibatinib and its degradation products, dissolve them in methanol and sonicate to prepare a sample solution with a concentration of 0.5 mg / mL. Chromatographic analysis was performed using a Shimadzu HPLC system equipped with a diode array detector (DAD). The chromatographic conditions were as follows: Agilent C 18The chromatographic column was 250 mm × 4.6 mm, 5 μm; the column temperature was 35 ℃; the flow rate was 1.0 mL / min; the detection wavelength was 300 nm; the injection volume was 10 μL; and the run time was 50 min. Mobile phase A was a mixture of 2 mmol / L potassium dihydrogen phosphate solution containing 0.4% triethylamine and acetonitrile (90:10, v / v); mobile phase B was acetonitrile. A gradient elution program was used: 0–2 min, 10% B → 10% B; 2–35 min, 10% B → 45% B; 35–40 min, 45% B → 60% B; 40–41 min, 60% B → 10% B; 41–50 min, 10% B → 10% B.

[0033] The purity of the purchased Futibatinib active pharmaceutical ingredient was determined by HPLC. The results showed that only one main peak was observed in the sample, corresponding to the main component of Futibatinib, with a chromatographic purity of 99.72%. The HPLC chromatogram of the undegraded sample is shown below. Figure 1 In the middle A, the peak with a retention time of 28.457 min is the main peak of Futibatinib. Chromatograms of samples obtained under different stress conditions are shown below. Figure 1 In samples B through F, a main peak with a retention time close to that of the active pharmaceutical ingredient was observed, accompanied by degradation impurity peaks of varying numbers and abundances. A summary of impurity formation and chromatographic separation parameters under various stress conditions is shown in Table 1.

[0034] Table 1. Impurity distribution and chromatographic resolution under different forced degradation conditions

[0035] The results showed that Futibatinib exhibited good stability under oxidative, thermal, and photothermal conditions, with only a small amount of degradation products detected. However, under alkaline conditions, degradation was most pronounced, with the main peak content decreasing to 87.46%, and a major degradation impurity with a peak area accounting for 11.77% was generated. This impurity was subsequently isolated, purified, and named Fut-2. Several degradation products were also detected under acidic conditions, but the overall degradation degree was significantly lower than under alkaline conditions. Since alkaline degradation conditions can generate a major degradation impurity with high abundance and easy separation, alkaline-degraded samples were chosen as the subjects for subsequent preparation, separation, and structural identification studies.

[0036] 2. Sample preparation and purification 2.1 Preparation of crude product To obtain a sufficient amount of degradation products for structural characterization studies, the NaOH concentration was further increased during the preparation of the crude product.

[0037] Accurately weigh 100 mg of Futibatinib active pharmaceutical ingredient and dissolve it in 100 mL of methanol to prepare a 1 mg / mL solution. Add 50 mL of 10 mol / L NaOH solution and react at 50 °C for 7 h. After the reaction is complete, add 50 mL of 10 mol / L HCl solution to terminate the reaction and adjust the pH of the reaction solution to neutral. Remove residual methanol by rotary evaporation, and freeze-dry the resulting aqueous solution to obtain a crude product containing salt. To remove salt, take an appropriate amount of the above crude product, dissolve it in pure water with stirring to prepare a 300 mg / mL solution, centrifuge at 10000 r / min for 20 min, discard the supernatant, collect the precipitate, and freeze-dry to obtain the crude product. The above degradation preparation process is repeated 5 times to obtain a sufficient quantity of crude product.

[0038] 2.2 Crude product purification A suitable amount of crude product was dissolved in 60% methanol aqueous solution by ultrasonication to prepare a sample solution with a concentration of 25 mg / mL. The solution was then filtered through a 0.45 μm organic filter membrane for preparative separation. Purification was performed using an Agilent preparative liquid chromatography system equipped with an automated fraction collector. The chromatographic conditions were as follows: Column: Agilent Eclipse XDB-C 18 (250 mm × 9.4 mm, 5 μm); Column temperature: room temperature; Flow rate: 6 mL / min; Detection wavelength: 300 nm; Injection volume: 200 μL; Run time: 25 min; Mobile phase A was ultrapure water, and mobile phase B was methanol. The gradient elution program was as follows: 0–1 min: 48% B → 48% B; 1–12 min: 48% B → 85% B; 12–16 min: 85% B → 85% B; 16–17 min: 85% B → 48% B; 17–25 min: 48% B → 48% B. The eluted fractions with retention times of 2.2–2.6 min and 7.7–9.0 min were collected and named Fut-1a and Fut-2, respectively. After combining the corresponding fractions, methanol was removed by rotary evaporation at 50 °C, followed by freeze-drying to obtain the degradation products Fut-1a and Fut-2.

[0039] Since the chromatographic purity of Fut-1a was insufficient for subsequent HRMS, NMR, and HMBC structure identification, preparative liquid chromatography was used for secondary purification. 144 mg of Fut-1a was dissolved in 12 mL of 50% methanol aqueous solution to prepare a sample solution with a concentration of 12 mg / mL. This solution was then filtered through a 0.45 μm organic filter membrane for further purification. The chromatographic conditions were as follows: Column: Agilent Eclipse XDB-C 18(250 mm × 9.4 mm, 5 μm); Column temperature: room temperature; Flow rate: 5 mL / min; Detection wavelength: 300 nm; Injection volume: 200 μL; Run time: 25 min; Mobile phase A was ultrapure water, and mobile phase B was methanol. The gradient elution program was as follows: 0–1 min: 30% B → 30% B; 1–12 min: 30% B → 60% B; 12–16 min: 60% B → 60% B; 16–17 min: 60% B → 30% B; 17–25 min: 30% B → 30% B. Target fractions with retention times of 7.9–8.6 min were collected, combined, and subjected to rotary evaporation at 50 °C to remove methanol, followed by freeze-drying to obtain purified Fut-1.

[0040] HPLC chromatogram of crude degradation products under enhanced alkaline degradation conditions ( Figure 2 A new high-abundance impurity peak appeared in (A) with a retention time of 14.615 min. This degradation impurity was subsequently named Fut-1. Preparative liquid chromatography was used to separate and purify Fut-1 and Fut-2. Figure 2 (B). Purity analysis revealed that the initial obtained Fut-1a had a chromatographic purity of only 83.29%, which did not meet the purity requirements for subsequent HRMS, NMR, and HMBC structural identification. Therefore, Fut-1a was further purified by preparative liquid chromatography to finally obtain high-purity Fut-1 (B). Figure 2 (C). Both purified Fut-1 and Fut-2 were white amorphous powders. (e.g., [unclear text - possibly a reference to a specific compound or product]). Figure 2 As shown in Figure D, the UV absorption spectra of Fut-1 and Fut-2 are highly similar, both exhibiting a strong absorption band at approximately 210 nm and a secondary absorption peak at approximately 300 nm. This high consistency in UV spectral characteristics indicates that the major chromophore structure of the parent compound is retained during degradation. Considering that the lower wavelength region is more susceptible to background absorption of the mobile phase and baseline fluctuations, the secondary absorption peak at approximately 300 nm was chosen as the detection wavelength for HPLC purity analysis. Simultaneously, 300 nm was also used for monitoring during preparative HPLC purification to ensure accurate detection and collection of the target components. The HPLC chromatograms of the purified products are shown below. Figure 2 The chromatographic purities of Fut-1 and Fut-2 reached 98.90% and 98.82%, respectively, indicating that the obtained samples could meet the requirements of subsequent HRMS, NMR, HMBC, and cytotoxicity evaluation experiments.

[0041] 3. HRMS, NMR, and HMBC analysis Weigh 10 mg each of Fut-1 and Fut-2, dissolve them in 50% methanol aqueous solution, and further dilute to 0.1 μg / mL for high-resolution mass spectrometry (HRMS) analysis. The HRMS test conditions were as follows: ionization method: heated electrospray ionization source (HESI); spray voltage: 3400 V; evaporator temperature: 280 ℃; ion transfer tube temperature: 320 ℃; auxiliary gas flow rate: 5 Arb; sheath gas flow rate: 3 Arb; scavenging gas flow rate: 2 Arb. The precise molecular weight and molecular formula of the degradation impurities were determined by HRMS.

[0042] Fut-1 and Fut-2 were dissolved in DMSO-d6, and their concentrations were recorded using a Bruker AVANCE III HD 400 NMR spectrometer. 1 H NMR and 13 C NMR spectrum. The test temperature was 298 K, and the test frequency was 400 MHz. 1 H) and 101 MHz ( 13 C).

[0043] HMBC experiments were performed on the same instrument platform, using standard pulse sequences and optimized long-range pulses. 1 H– 13 The C coupling constant is used to establish the correlation between the two and three bonds between hydrogen and carbon atoms, providing a basis for the analysis of unknown structures.

[0044] like Figure 3 As shown, protonated molecular ion peaks at m / z 365.1768 and 451.2167 were detected in the HRMS spectra of Fut-1 and Fut-2, respectively. Based on the precise mass determination results and elemental composition analysis, the molecular formulas of Fut-1 and Fut-2 are estimated to be C1 and C2, respectively. 19 H 20 N6O2 and C 23 H 26 N6O4. Compared to Futibatinib, Fut-1 has a molecular weight decrease of approximately 54 Da, while Fut-2 has a molecular weight increase of approximately 32 Da, indicating a significant structural transformation during alkaline degradation.

[0045] To further elucidate the structures of the degradation products, Futibatinib, Fut-1, and Fut-2 were analyzed. 1 H NMR, 13 CNMR and HMBC analyses, the corresponding spectra are shown below. Figures 4-6 Comparative NMR data showed that both degradation products retained the characteristic pyrazolo[3,4-]of the parent compound. dThe pyrimidine skeleton and the 3,5-dimethoxyphenylethynyl structural unit were present. However, the signals associated with the side chains attached to the pyrrolidine ring changed significantly, indicating that the degradation reaction mainly occurred in this region. As shown in Tables 2 and 3, the characteristic olefinic hydrogen signals (δ 5.58–5.43, 5.69, and 6.18 ppm) of the acrylamide side chain in Futibatinib disappeared in both Fut-1 and Fut-2. Furthermore, in Fut-1... 1 No new lower-field proton signals at δ 10.01 and 9.69 ppm were observed in the 1H NMR spectrum, presumably exchangeable hydrogen signals. Simultaneously, several new aliphatic hydrogen and carbon signals were observed in the NMR spectrum of Fut-2. These results indicate that the acrylamide side chain of Futibatinib underwent structural transformation during degradation, while its heterocyclic core structure remained largely intact. Furthermore, the key hydrogen-carbon long-range correlation signals observed in the HMBC spectrum provide further important evidence for the structural analysis of Fut-1 and Fut-2.

[0046] Table 2 Fut-1, Fut-2 1 H NMR spectrum assignment

[0047] Table 3 Fut-1, Fut-2 13 C10 NMR spectrum assignment

[0048] The molecular formula of Futibatinib is C 22 H 22 N6O3 has an exact mass of 418.1753 and a relative molecular mass of 418.4570. Its protonated molecular ion is [M+H]. + The theoretical mass-to-charge ratio (m / z) is 419.1787. Based on the above HRMS, NMR, and HMBC analyses, the chemical structures of Fut-1 and Fut-2 were finally determined, as shown in the following formula. The molecular formula of Fut-1 is C1. 19 H 20 N6O2, the structure of which has been identified as: ( S )-3-[(3,5-dimethoxyphenyl)ethynyl]-1-(pyrrolidine-3-yl)-1 H -pyrazolo[3,4- d Pyrimidine-4-amine, with an exact mass and relative molecular mass of 364.1648 and 364.4090, respectively. The molecular formula of Fut-2 is C2. 23 H 26 N6O4, the structure of which has been identified as: ( S)-1-[3-(4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1 H -pyrazolo[3,4- d [Pyrimidin-1-yl]pyrrolidine-1-yl]-3-methoxyprop-1-one, with an exact mass and relative molecular mass of 450.2016 and 450.4990, respectively.

[0049]

[0050] Analysis of the structural changes reveals that, under alkaline conditions, the α,β-unsaturated amide structure in the Futibatinib molecule is the most reactive site. Fut-1 likely originates from the hydrolytic cleavage of the acrylamide side chain; while Fut-2 may be generated by a nucleophilic Michael addition of methanol to the α,β-unsaturated amide double bond, followed by further rearrangement under alkaline conditions. Notably, both degradation products retain pyrazolo[3,4- d The presence of a pyrimidine core skeleton and a dimethoxyphenylethynyl structural unit indicates that the degradation of Futibatinib is mainly concentrated in its electrophilic "warhead" region, while the core pharmacophore structure is relatively stable. Example 2

[0051] Cytotoxicity evaluation The in vitro cytotoxic activity of futibatinib and its degradation products Fut-1 and Fut-2 against five human tumor cell lines was evaluated using the MTT assay. These cell lines included HepG2 liver cancer cells, MCF-7 breast cancer cells, PANC-1 pancreatic cancer cells, A549 lung cancer cells, and MKN-1 gastric cancer cells. Cells in the logarithmic growth phase were digested, and a cell suspension was prepared, with the cell density adjusted to 1 × 10⁻⁶ cells / year. 5 Cells / mL. Cells were then seeded in 96-well plates and cultured at 37 ℃ in a 5% CO2 incubator for 24 h to allow for full cell adhesion. After cell adhesion, different concentrations of Futibatinib, Fut-1, and Fut-2 sample solutions were added, and the cells were cultured for another 48 h. After drug treatment, the culture medium was discarded, and the cells were washed twice with PBS buffer. 100 μL of freshly prepared MTT solution (1.0 mg / mL) was added to each well, and the cells were incubated for another 4 h to allow the live cells to reduce MTT to formazan crystals. The supernatant was then discarded, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The wells were then incubated at 37 ℃ in the dark for 30 min until the crystals were completely dissolved. The absorbance (OD value) of each well was measured using a microplate reader at 490 nm. Eight parallel wells were set up for each concentration, and all experiments were independently repeated three times. The cell growth inhibition rate was calculated using the following formula: Cell growth inhibition rate (%) = [1 − (OD样品 / OD 对照 [] × 100. Based on the obtained concentration-effect curve, the half-maximal inhibitory concentration (IC50) was calculated using a nonlinear regression method. 50 )value.

[0052] The cytotoxic activity results of Fut-1 and Fut-2 against five human tumor cell lines are shown in the table below. Figure 7 The corresponding IC 50 The values ​​are summarized in Table 4.

[0053] Table 4. IC50 of futibatinib, fut-1, and fut-2 after 48 h of treatment with five types of human tumor cells. 50 Value (μg / mL)

[0054] The results showed that both degradation products could inhibit the proliferation of the tested tumor cells, and the inhibition of cell proliferation was significantly concentration-dependent, meaning that the inhibitory effect on cell proliferation gradually increased with increasing drug concentration.

[0055] Overall, Fut-1 maintained good cytotoxic activity, with its activity levels comparable to, and even showing stronger inhibitory effects than, the parent drug Futibatinib in some cell lines. Compared to Futibatinib, Fut-1 showed slightly lower inhibitory activity against HepG2 cells, but enhanced inhibitory effects against MCF-7, PANC-1, A549, and MKN-1 cells. In contrast, Fut-2 showed significantly lower activity in all tested cell lines, with an IC50 of [missing information]. 50 The value was significantly higher than that of Futibatinib and Fut-1. These results indicate that structural modification of the acrylamide side chain of Futibatinib does not necessarily lead to a complete loss of biological activity. Notably, although Fut-1 lost its original electrophilic "warhead" group, it still maintained strong antitumor activity, suggesting that pyrazolo[3,4- d The pyrimidine core skeleton may play an important role in its antiproliferative effect. Furthermore, the comparable or even superior activity of Fut-1 in some cell lines to the parent drug indicates that this degradation product is worthy of further biological research and can provide new reference for the structure-activity relationship study of Futibatinib-related compounds.

Claims

1. Futibatinib degradation products, selected from any of the following structures: 。 2. The method of preparing Futibatinib degradation products according to claim 1, characterized in that, Includes the following steps: Step 1: Dissolve Futibatinib raw material in methanol, add sodium hydroxide solution to react, after the reaction is completed, add hydrochloric acid solution to terminate the reaction, adjust the pH of the reaction solution to 7.0, remove residual methanol by rotary evaporation, and obtain crude product containing salt. Step 2: Dissolve the crude product in methanol-water solution to prepare a sample solution. After filtration through an organic filter membrane, the solution is used for preparative liquid chromatography separation. Collect the eluent fractions with retention times of 2.2–2.6 min and 7.7–9.0 min. The eluent fraction with a retention time of 2.2–2.6 min is Fut-1, and the eluent fraction with a retention time of 7.7–9.0 min is Fut-2.

3. The preparation method according to claim 2, characterized in that, In step 1, the concentration of Futibatinib raw material is 1 mg / mL, the concentration of sodium hydroxide solution is 10 mol / L, and the concentration of hydrochloric acid solution is 10 mol / L.

4. The preparation method according to claim 3, characterized in that, In step 1, the volume ratio of methanol, sodium hydroxide solution, and hydrochloric acid solution is 100 mL: 50 mL: 50 mL.

5. The preparation method according to claim 2, characterized in that, The reaction conditions in step 1 are 45–55 °C for 6–8 h.

6. The preparation method according to claim 2, characterized in that, In step 2, the concentration of the methanol-water solution is 60%, and the concentration of the sample solution is 25 mg / mL.

7. The preparation method according to claim 2, characterized in that, The chromatographic conditions for preparing the liquid chromatography separation in step 2 are as follows: Column: Agilent Eclipse XDB-C 18 ; Column temperature: room temperature; Flow rate: 6 mL / min; Detection wavelength: 300 nm; Injection volume: 200 μL; Running time: 25 min; Mobile phase A is ultrapure water, and mobile phase B is methanol. The gradient elution program is as follows: 0–1 min: 48% B → 48% B; 1–12 min: 48% B → 85% B; 12–16 min: 85% B → 85% B; 16–17 min: 85% B → 48% B; 17–25 min: 48% B → 48% B.

8. The use of the Futibatinib degradation product according to claim 1 in the preparation of tumor drugs.

9. The application according to claim 8, characterized in that, The tumors were selected from breast cancer, pancreatic cancer, lung cancer, and stomach cancer.

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