Structure Characterization of Degradation Products of Drug Candidate HM30571 by UPLC‐QTOF‐MS and In Silico Toxicity Prediction (2019)

Procedure of forced degradation study, structure elucidation of degradation products by UPLC-QTOF-MS and in silico toxicity prediction. The epidermal growth factor receptor (EGFR) overexpressed promotes cancer cell formation and progression, culminating in the aggressive growth of invasive tumors.1, 2 Meanwhile, the vascular endothelial growth factor receptor 2 (VEGFR-2), which regulates endothelial migration and proliferation, is the principal receptor in angiogenesis.3, 4 Our research and development center is designing a series of novel dual-acting compounds that inhibit EGFR and VEGFR-2. Among these compounds, (11) compound (HM30571) [N-(4-((4-bromo-2-fluorophenyl)amino)-7-(2-(2-(dimethylamino)acetamido) ethoxy)(quinazolin-6-yl)acrylamide] (Figure 1) shows excellent inhibitory activities against kinases. In addition, HM30571 demonstrates an anti-angiogenic effect. Accordingly, HM30571 is considered as a dual tyrosine kinase inhibitor of EGFR and VEGFR-2.5 Forced degradation studies can reveal the degradation pathways and intrinsic stability of a drug molecule. These tests are part of the drug development strategy and are usually performed under more severe conditions than those of accelerated testing.6, 7 If degradation by a certain pathway is not predicted and/or is proven unlikely in the experimental data, minimal effort should be exerted on that condition at the pre-phase 1 stage.8 Identifying and controlling the degradation products of a drug substance are crucial tasks in drug discovery and development, because these products affect the drug quality. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines Q3A(R2) and Q3B(R2) recommend the characterization of all drug impurities/degradation products whose levels exceed the identification threshold.9, 10 In forced degradation studies, high-resolution mass spectrometry (HRMS) can provide the masses, proposed structures, and chemical formulae of all degradation products, so is indispensable in drug discovery. Meanwhile, the ICH M7(R1) guideline prescribes the control of mutagenic impurities in pharmaceuticals. The acceptable criteria for impurities with structure alerts with potential genotoxicity should differ from those of ordinary impurities.11, 12 In this study, the active ingredient, its impurities, and degradation products of the forced degradation study are separated by high performance liquid chromatography/ultra violet (HPLC/UV), and the plausible structures are predicted from the analytical data provided by ultra-high performance liquid chromatography/quadrupole time-of-flight/mass spectrometry (UPLC–QTOF–MS). Finally, the genotoxicities and carcinogenicities of the degradation products are predicted by quantitative structure activity relationship (QSAR), which is an ideal methodology, as it achieves high-throughput in vivo screening of many chemicals with low monetary and labor costs. The Agilent 1200 series HPLC system was provided with a diode array (DAD) detector and a 150 mm × 4.6 mm Shisheido Capcellpak MG C18 column packed with 5 μm-diameter particles. The HPLC system was maintained at 30°C. Chromatographic data were acquired by Chemstation (ver. C.01.05) software. Mobile phases A and B were 10 mM potassium phosphate buffer (adjusted to pH 3.5 with phosphoric acid) and acetonitrile, respectively. The flow rate of the mobile phases was maintained at 1.0 mL/min. The HPLC gradient (in time [min]/% Solution B) was varied as 0/10, 15/45, 25/75, 35/75, 37/10, and 45/10. The column was maintained at 30°C and the wavelength was determined at 254 nm. An Acquity UPLC was coupled to a photo-diode array (PDA) detector and QTOF spectrometry (SYNAPT G2, Waters Corporation, USA) for high-resolution MS analysis. Ionization was conducted in positive electrospray ionization (ESI) mode and accurate mass measurements were obtained at a resolution of 20 000 with a scan time of 0.5 s. The parameters for the source were set as follows: capillary voltage = 2.5 kV, source temperature = 100 °C, desolvation temperature = 500 °C, sampling cone voltage = 25 V, cone gas flow = 0 L/h, desolvation gas flow = 800 L/h. The data were acquired by Masslynx ver. 4.1 (Waters Corporation, Milford MA, USA) software. The UPLC was equipped with a 150 mm × 2.1 mm Waters Acquity BEH C18 column packed with 1.7 μm-diameter particles. The mobile phases A and B were 10 mM ammonium acetate (adjusted to pH 3.5 with formic acid) and acetonitrile, respectively. The flow rate of the mobile phase was maintained at 0.3 mL/min. The UPLC gradient (in time [min]/% Solution B) was varied as 0/10, 7/45, 20/70, 25/70, 26.5/10, and 30/10. The column was maintained at 30 °C and the wavelength was 254 nm. The forced degradation was performed based on previous literature which suggests that the degradation of the drug up to 5–20% is considered as sufficient to validate the chromatographic assays.13, 14 In the forced degradation experiments, HM30571 degraded into six major degradation products denoted as DG-1 to DG-6. The degradation products DG-2 (6.5%) and DG-3 (5.7%) were formed only under acid (1 M HCl [pH 1.0] at 60 °C for 12 h) and alkaline (1 M NaOH [pH 12.0] at room temperature for 12 h) stress, respectively (Figure 2). DG-5 was generated under both acidic and alkaline conditions (at proportions of 1.2 and 2.3%, respectively). Products DG-1 (0.5%), DG-4 (2.4%), and DG-6 (5.4%) were formed only under the oxidative stress condition (3% H2O2 at 60 °C for 12 h). In contrast, HM30571 was stable under thermal (at 60 °C for 1 week), photolytic (exposed to visible light for 1.2 Million lux-hours), and humidity stress conditions (75% RH and at 40 °C for 1 week). The MS2 spectra of HM30571 and its degradation products are given in Figure 1. In the positive ESI-mode mass spectrum, the [M + H]+ and [M + K]+ ions of HM30571 appeared at m/z = 531.1162 and 569.0728. The product ion at m/z 403.0 corresponded to the loss of dimethylamino N-methyl acetamide (128 Da) from its precursor ion (m/z = 531.1). Meanwhile, the fragment ions of m/z = 349.0 might derive from the loss of acryl (54 Da), and the m/z = 385.0 part corresponded to H2O elimination from m/z = 403.0. The most likely molecular formula of the protonated molecular ion was selected as the formula with the most accurate mass value (lowest mass error by imposing rational limits), mass difference, and double bond equivalents (DBE) of each peak (Table 1). The UPLC–QTOF–MS results confirmed the protonated ions [M + H]+ of DG-1, DG-2, DG-3, DG-4, DG-5, and DG-6 at m/z = 360.17, 477.10, 549.13, 581.12, 446.06, and 547.11 Da, respectively. The structures of the unknown degradation products were deduced by comparing their fragmentation pattern with them of HM30571 (Figure 1). The MS2 fragmentation spectra of the product ions IM-1 (process impurity-1), IM-2 (process impurity-2), DG-5, and DG-6 show peaks at m/z = 349.0, 385.0, and 403.0. Therefore, it was assumed that they possess the 4-bromo-2-fluoro-anilinequinazolin-linked acrylamide moiety of HM30571. The structural characterization also supported significantly different product ions, which appear in the fragmentation spectra of DG-1, DG-3, and DG-4. The characteristic product ions [M + H-128]+ at m/z = 232.1, 349.0, and 421.0 in the spectra of DG-1, DG-2, and DG-3 correspond to loss of dimethylamino N-methyl acetamide. The product ions of DG-1 appeared at m/z = 232.1 [M + H-128]+, m/z = 214.1 [M + H-128-18]+, and m/z = 178.1 [M + H-128-54]+ in the MS2 spectrum, likely corresponding to the cleavage of dimethylamino N-methyl acetamide, H2O elimination from m/z = 232.1, and loss of the acryl part from m/z = 232.1, respectively. DG-1 was predicted to be formed as the hydrolyzed alcohol form of the amine after the loss of 4-bromo-2-fluoro benzene from HM30571. This structure was supported by the DBE value of 9.5 (4 units lower than DBE of HM30571). The DBE value determines the number of double bonds in a hydrocarbon molecule. The DBE in the mass-scan data of DG-2 (11.5) was two units lower than that of HM30571 (13.5), confirming fewer double bonds in DG-2 than in HM30571. Moreover, the protonated ion peak at m/z = 477.1 [M + H]+ in the MS2 analysis corresponds to the molecular formula C20H23N6O2FBr, which has 54 fewer m/z units than the HM30571 molecule (C23H25N6O3FBr, m/z = 531.1). Therefore, DG-2 was predicted as the acryl cleavage component from HM30571. The ion spectrum of DG-3 presented a protonated molecular ion [M + H]+ at m/z = 549.1265, 18 Da heavier than HM30571. The product ions of DG-3 at m/z = 403.0 and m/z = 421.0 were also 18 Da heavier than those of the HM30571 fragment ions at m/z = 385.0 and m/z = 403.0 respectively. The DBE in the mass-scan data of DG-3 (12.5) was one unit lower than that of HM30571 (13.5). On the other hand, the product ion at m/z = 129 in the MS2 spectrum of DG-3 also appears in the HM30571 spectrum. In the mass fragmentation spectrum of DG-4, the product ions at m/z = 437.0 and 145.1 represent the N-oxide of quinazoline and the N-oxide of tertiary amine, respectively. The DG-4 product is 32 Da heavier than DG-3. The product ions of DG-3 at m/z = 403.0 and DG-4 at m/z = 419.0 are structurally related. Moreover, the DG-3 product at m/z = 129.1 and the DG-4 product at m/z = 145.1 have a similar relationship. In addition, the DBE in the mass-scan data of DG-4 (12.5) was one unit lower than that of HM30571 (13.5), confirming fewer double bonds in DG-4 than in HM30571. The DG-5 fragmentation product was 85 Da lighter than HM30571. The MS2 spectra of DG-5 showed an intense m/z = 446.1 ion in protonated form and no peak at m/z = 129.1. As mentioned above, the product ions in the MS2 fragmentation spectra of DG-5 appeared at m/z = 349.0, m/z = 385.0, and m/z = 403.0. In addition, the DBE in the mass-scan data of DG-5 (12.5) was one unit lower than that of HM30571 (13.5). Therefore, DG-5 was assumed to possess the 4-bromo-2-fluoro-anilinequinazolin-linked acrylamide moiety of HM30571. The ion spectrum of DG-6 exhibited a protonated molecular ion [M + H]+ at m/z = 547.1098, 16 Da above the m/z of HM30571. Meanwhile, the DG-6 product ion at m/z = 145.1 of DG-6 apparently corresponded to the m/z = 129.1 product ion of HM30571. In addition, the DBE in the mass-scan data of DG-6 (13.5) was same as that of HM30571 (13.5). Based on these data, DG-1 to DG-6 were characterized as shown in Figure 1. The IM-1 product showed a characteristic peak at m/z = 86.1, 43 Da lower than the m/z = 129.1 product ion of HM30571. In addition, the DBE in the mass-scan data of IM-1 (13.5) was same as that of HM30571 (13.5). Therefore, IM-1 was predicted as the trimethylamine-eliminated form of HM30571. In case of IM-2, the molecular weight of that was 11 Da above that of HM30571, and the characteristic peak of the product ion at m/z = 140.0 was 11 Da above the m/z = 129.1 peak of HM30571. Other product ions at m/z = 349.0, m/z = 385.0, and m/z = 403.0 corresponded to the product ions of HM30571, but the plausible structure of IM-2 could not be deduced. From the data in the MS2 fragmentation spectra of each degradation product, the likely fragmentation pathway of HM30571 was established. Meanwhile, genotoxicities and the carcinogenicities of HM30571 and its degradation products were assessed by the expert rule-based (Derek Nexus v5.0.2 [Lhasa Limited, Leeds, UK]) and statistical-based (Sarah Nexus v2.0.1 [Lhasa Limited, Leeds, UK]) methods. HM30571 and its degradation products (DG 1-DG6) to be classified as ICH M7 Class 4 or 5, which indicates the current specifications outlined by the guidance on the control of ordinary impurities are appropriate.10, 15 Therefore, further analytical method development is not necessary. However, according to the two methodologies in the genotoxicity assessment, substructures in the input structures of raw material acryloyl chloride and intermediates with NO2 group are potentially mutagenic in selected bacterial species. Acryloyl chloride tested positive for the “Mutagenicity in vitro” endpoint in the Sarah model. Derek returned “equivocal” for the mutagenicity of acryloyl chloride in bacterial cells in vitro, meaning that the evidences for and against the proposition are equally weighted. On the other hand, some intermediates with NO2 group, were all plausibly genotoxic in Derek, where “plausible” means that the evidence supports the proposition. The aromatic nitro compound raised a structural alert and is potentially mutagenic in the Ames test (notably in Salmonella typhimurium strains TA98 and TA100). Meanwhile, the Sarah prediction results of intermediates with NO2 group were all positive. Therefore, acryloyl chloride and intermediates with NO2 group may reasonably be classified as Class 3 (potentially genotoxic materials) in the ICH M7, whether they belong to Class 2 or Class 5 should be determined by the Ames test. Although Derek showed “highly plausible” result for DG-4 and DG-6 predicted to have the N-oxide moiety, conclusively neither method showed a positive result. Primary aromatic amines and their corresponding hydroxylamines are considered mutagenic in the literature, but tertiary aryl amines or their relevant tertiary N-oxides are non-mutagenic.15